Isolation of bone marrow and adipose-derived mesenchymal stromal cells

Learning Materials in Biosciences NagwaEl-BadriEditor Regenerative Medicine and Stem Cell Biology Learning Materials in Biosciences Learning Materials in Biosciences textbooks compactly and concisely discuss a specific biological, biomedical, biochemical, bioengineering or cell biologic topic. The textbooks in this series are based on lectures for upper-level undergraduates, master’s and graduate students, presented and written by authoritative figures in the field at leading universities around the globe. The titles are organized to guide the reader to a deeper understanding of the concepts covered. Each textbook provides readers with fundamental insights into the subject and prepares them to independently pursue further thinking and research on the topic. Colored figures, step-by-step protocols and take-home messages offer an accessible approach to learning and understanding. In addition to being designed to benefit students, Learning Materials textbooks represent a valuable tool for lecturers and teachers, helping them to prepare their own respective coursework. More information about this series at http://www.springer.com/series/15430 Nagwa El-Badri Editor Regenerative Medicine and Stem Cell Biology Editor Nagwa El-Badri Center of Excellence of Stem Cells and Regenerative Medicine Zewail City of Science and Technology Giza, Egypt ISSN 2509-6125 ISSN 2509-6133 (electronic) Learning Materials in Biosciences ISBN 978-3-030-55358-6 ISBN 978-3-030-55359-3 (eBook) https://doi.org/10.1007/978-3-030-55359-3 #Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Preface The field of stem cells and regenerative medicine has expanded to include many disciplines in biology, medicine, physics, material science, biomedical engineering, and nanotechnology. This multidisciplinary approach necessitates acquiring knowledge that is contextual, practical, and focused on these new disciplines. This book provides an overview of the basic concepts of stem cell research and the important topics in the field that are of interest to students and also to researchers and physicians. The topics have been selected carefully to fulfill both the theoretical and practical aspects of stem cell research, in an approach that is beneficial to researchers who are interested to specialize in the field or to complement their research in other fields. The introduction provides an overview of stem cells and the facts and hype about their usage in the clinic. Some diseases are prescribed stem cell transplantation as routine therapy, especially those of hematopoietic origin. Other therapeutic approaches are still experimental. At the forefront of diseases treated with stem cells are hematological disorders, leukemia, lymphoma, hemoglobinopathies, and immune deficiencies. Research on hematopoietic stem cells has been pioneering in delivering reliable therapy for blood disorders, and it comes as no surprise that almost all of the FDA-approved stem cell products are also of hematopoietic origin. The chapters on adult stem cells cover hematopoietic stem cells, mesenchymal stromal cells, endothelial progenitor cells, and pericytes and provide the reader with a good basis for understanding the biology and applications of these important cells. The chapters on the epigenetic regulation of stem cells, cancer development and its regulation by cancer stem cells and associated stromal cells cover the molecular mechanisms that govern stem cell development and differentiation in health and disease. The same theme extends into the chapter on the use of stem cell therapy in the treatment of metabolic disorders, which provides a much-needed insight on regenerative therapy in the clinical setting, with a focus on diabetes as the most prevalent metabolic disease. Many landmark experiments, from cloning frogs in the 1960s and mammalian cloning in the 1990s to the current direct cellular reprogramming and gene editing provided a more flexible and broader understanding of stem cell biology and of cell biology in general. v Characterization of the embryonic stem cells and Yamanaka’s pioneering experiments in reprogramming somatic cells into induced pluripotent cells made stem cell therapy more achievable. It is now becoming more possible to manipulate mature cells on the genetic and epigenetic levels, to reverse their development and to regenerate their differentiation potential. This revolution in cell biology has not been matched unfortunately with a comparable clinical revolution, where patients have directly and similarly benefited from these unprecedented advances. Advances in biotechnology, nanotechnology, and bioprinting have opened the doors to unlimited possibilities in regenerative medicine. Using natural and synthetic scaffolds fulfills the structural foundation of any organ on which cells are seeded and coaxed to differentiate and develop into the desired tissues. Bioprinting, 3D culture techniques, organ-on-a-chip, and other technical advances expanded the applications of stem cells well into personalized medicine. In vitro disease modeling and testing drugs on patientspecific tissues undoubtedly present a leap in precision medicine. Chapters 10 and 11 discuss tissue engineering with detailed examples of bioscaffold preparation in the form of the decellularized human amniotic membrane. After its use with success in skin and corneal grafts, its attractive anti-inflammatory and antimicrobial properties and low immunogenicity support its use as a scaffold for stem cell growth and differentiation. Detailed protocol for bioscaffold preparation and other protocols for isolation and culture of mesenchymal stromal cells and induced pluripotent stem cells are also detailed. The book concludes with a reminder for young scientists of following the basics of the scientific method, of adherence to ethical practices in their research, and of frequently questioning the methods and goals of their research. These practices tie directly with the introduction on the benefits of stem cell research and its applications, to maximize the hope and minimize the hype in this promising field. Giza, Egypt Nagwa El-Badri vi Preface Acknowledgement The authors would like to thank Ms. Shimaa E. Elshenawy for her valuable editorial assistance. vii Contents 1 Introduction and Basic Concepts in Stem Cell Research and Therapy: The Facts and the Hype ..................................... 1 Mohamed Essawy, Shaimaa Shouman, Shireen Magdy, Ahmed Abdelfattah-Hassan, and Nagwa El-Badri 2 Embryonic and Pluripotent Stem Cells ......................... 37 Shaimaa Shouman, Alaa E. Hussein, Mohamed Essawy, Ahmed Abdelfattah-Hassan, and Nagwa El-Badri 3 Hematopoietic Stem Cells and Control of Hematopoiesis ............ 67 Mohamed Essawy, Ahmed Abdelfattah-Hassan, Eman Radwan, Mostafa F. Abdelhai, S. Elshaboury, and Nagwa El-Badri 4 Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells, and Pericytes ........................................ 109 Azza M. El-Derby, Toka A. Ahmed, Abeer M. Abd El-Hameed, Hoda Elkhenany, Shams M. Saad, and Nagwa El-Badri 5 Cancer Stem Cells and the Development of Cancer ................ 151 Nehal I. Ghoneim, Rania Hassan Mohamed, Alaa Gamal, Shireen Magdy, and Nagwa El-Badri 6 Stem Cell Applications in Metabolic Disorders: Diabetes Mellitus ..... 193 Sara M. Ahmed, Sara S. Elshaboury, and Nagwa El-Badri 7 Epigenetics in Stem Cell Biology .............................. 221 Mohamed A. Nasr, Tasneem Abed, Azza M. El-Derby, Mohamed Medhat Ali, and Nagwa El-Badri 8 Isolation of Bone Marrow and Adipose-Derived Mesenchymal Stromal Cells .................................................... 243 Nehal I. Ghoneim, Alaa E. Hussein, and Nagwa El-Badri ix 1.2.2 Adult Stem Cells Somatic or adult stem cells are rare populations of undifferentiated cells that are found among their differentiated counterparts throughout the adult body. These cells contribute to tissue homeostasis, as they serve as a source of raw material for repair and/or replacement of injured or dead cells [5]. Adult stem cells have only a limited range of differentiation potential when compared with ESCs. Examples of adult stem cells include the following: •Mesenchymal Stem Cells (MSCs) Table 1.1 General comparison between embryonic stem cells, adult stem cells, and iPSCs Cell type Embryonic stem cells Adult stem cells iPSCs Origin Pluripotent cells derived from the inner cell mass of the blastocysts [10,43] Multipotent cells derived from adult tissues [37,44– 46] Somatic cells reprogrammed into embryonic-like pluripotent stem cells [19,47] Self-renewal capacity High [10,43] Limited [37,44–46] High [19,47] Potency Pluripotent [10,43] Multipotent [37,44–46] Pluripotent [19,47] Differentiation Can differentiate into cells of each of the three germ layers [10,43] Restricted lineage differentiation [37,44–46] Can differentiate into cells of each of the three germ lineages [19,47] Surface markers Pluripotency markers (OCT4, SOX2, NANOG, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 [10,43,48] Specific markers of adult tissue-derived stem cells. For example, MSCs express CD90, CD73, and CD105 along with a negative expression for the hematopoietic markers CD45, CD3, CD19, CD11, CD79α, and human leucocyte antigen-DR (HLA-DR) [46,49] Pluripotency markers (OCT4, SOX2, NANOG, SSEA-4, and KLF4 [19,47, 50] Spontaneous oncogenic transformation Present [10,43] Absent [37,44–46] Present [19,47] Immune response Strong [51,52] Strong for allogeneic, but not for autologous cells [53–55] Strong, but can be minimized for autologous cells [56] Ethical concerns Yes [24,51]No[57] Minimal [58] 6 M. Essawy et al. MSCs are adherent fibroblast-like cells when cultured in vitro. They were first isolated from the bone marrow [26,27], where they are most abundant. They produce colony forming-unit fibroblast (CFU-F), when cultured in vitro and are distinguished by the capacity to differentiate into osteocytes, chondrocytes, and adipocytes. There are numerous sources of MSCs including bone marrow [28], adipose tissue [29], dental pulp [30], and synovial membranes [31]. •Hematopoietic Stem Cells (HSCs) HSCs have been isolated from the bone marrow; they have the capacity for self-renewal as well as the ability to differentiate into all blood cell lineages [3]. They are widely used clinically in HSC transplantaion for treating various blood disorders and malignancies. •Neural Stem Cells (NSCs) NSCs are found in the central nervous system; they have the potential to differentiate into both neuronal and non-neuronal glial cells [32]. As such, they have been used clinically in efforts to repair injuries sustained by the nervous system [33,34]. Currently, the use of NSCs for treating neurodegenerative diseases is under investigation [35]. 1.2.3 Other Stem Cells The discovery of stem cells in the human umbilical cord blood (UCB) paved a new and useful source of progenitors; notably umbilical cord blood hematopiotic stem cells (UCBHSCs) have become a viable source of autologous bone marrow stem cells. UCB-HSCs are capable of differentiating into multiple hematopoietic lineages, in addition to their capacity for long-term self-renewal [36,37]. Clinically, UCB stem cells have been employed successfully as HSC transplants in 1988 [38]. As such, parents in some countries now routinely bank the UCB of newborns so as to have a source of HSCs in the advent of any childhood hematological disorders or malignancies. Likewise, as noted earlier, MSCs have been identified in extraembryonic tissues, including Wharton’s jelly [39], amniotic membrane and placenta [39,40], and amniotic fluid [41]. 1.2.4 Induced Pluripotent Stem Cells (iPSCs) iPSCs are generated in vitro in an effort to imitate the potential of ESCs by effectively reversing the differentiation of somatic cells (e.g., skin fibroblasts) in order to become pluripotent [19,42]. The discovery of iPSCs was driven at least in part by the need to identify ESC-like pluripotent stem cells for clinical use which could be generated without 1 Introduction and Basic Concepts in Stem Cell Research and Therapy: ... 7 raising strong ethical concerns. Many ongoing efforts are aimed at improving current reprogramming approaches so as to enhance the current clinical applicability of iPSCs. 1.3 Stem Cell Therapies: The Present and the Future The remarkable potential of stem cells, including their capacities for self-renewal and differentiation, has led to their use in numerous clinical applications, including cell-based therapies [59], drug discovery [60], and tissue engineering [61]. The ultimate goal of stem cell-based therapies is to treat, repair, or replace diseased tissues or organs with ones that are new, healthy, and functional [62,63]; numerous applications of this type are presented in Fig. 1.2. Therefore, stem cells are currently featured in several thousand ongoing clinical trials focused on disease treatment. Most of these protocols focus on the use of stem cells for treating hematological disorders, including myeloid leukemia; lymphoma; sickle cell anemia; immune deficiencies; β-thalassemia [64–67]; wound healing and skin injuries [68]; neurological disorders, such as Parkinson’s diseases and spinal cord injury [69,70]; autoimmune disorders, such as multiple sclerosis, rheumatoid arthritis, Crohn’s disease, and type-1 diabetes [71–74]; and cardiac diseases, including ischemic heart disease [75]. Promising trials, which focus on the use of stem cells to treat ocular disorders, including macular degeneration and retinitis pigmentosa [76,77], and bone diseases, including osteosarcoma, Brain Parkinson’s Disease Alzheimer’s Disease Blood Hematopoietic malignancies Immune deficiencies Liver Cirrhosis Hepatic cancer Pancreas Diabetes Heart Heart diseases (myocardial infarction) Skin Burns Wounds / Injuries Kidney Kidney disorders Muscles Muscular dystrophy Bone / Cartilage Arthritis Osteoporosis Injuries Fig. 1.2 Stem cell therapy for chronic diseases 8 M. Essawy et al. osteoporosis, and osteoarthritis, are also in progress [78,79]. So far, only a handful of the U.S. Food and Drug Administration (FDA) approved stem cell products are available for clinical use, including allogeneic cord blood hematopoietic stem/progenitor cells for treating hematological and immunological disorders (https://www.fda.gov/vaccinesblood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapyproducts). Currently approved stem cell-based therapies are listed in Table 1.2. Table 1.2 Approved human stem cell-based products Approved products Used stem cell type Indications Approval status Approved by ALLOCORD Allogeneic cord blood hematopoietic progenitor cell Used in conjunction with an appropriate preparative regimen for hematopoietic and immunologic reconstitution of patients with inherited or acquired disorders of the hematopoietic system or as a result of myeloablative treatment. Approved Office of Tissues and Advanced Therapies of the FDA (USA) CLEVECORD DUCORD HEMACORD HPC, Cord Blood HPC, Cord Blood—MD Anderson Cord Blood Bank HPC, Cord Blood—Life South HPC, Cord Blood—Blood works HOLOCLAR Ex vivo expanded autologous human corneal epithelial cells containing stem cells Treatment of adult patients with moderate to severe unilateral or bilateral limbal stem cell deficiency due to physical or chemical ocular burns. Conditional Approval European Medicines Agency (EU) ZYNTEGLO Autologous CD34 + hematopoietic stem cells transduced with lentiviral vector encoding the human beta A-T87Qglobin gene Treatment of beta thalassemia. Conditional Approval 1 Introduction and Basic Concepts in Stem Cell Research and Therapy: ... 9 1.3.1 Routine Stem Cell Therapy for Hematopoietic Disorders 1.3.1.1 Hematological Malignancies Transplantation of unmodified or genetically modified HSCs derived from different sources offers a promising approach to the reconstitution or replacement of diseased cells. Cell therapies for hematological disorders, such as hemoglobinopathies (e.g., sickle cell anemia) and blood malignancies (e.g., leukemia and lymphoma), have undergone substantial development over the past few decades, as in the examples discussed below [80]. Leukemia Leukemias are a group of white blood cell malignancies classified by the World Health Organization (WHO) based on genetics, morphology, immunophenotype, and clinical features [81,82]. Interestingly, one of the earliest known cases of leukemia was identified based on the findings from an Egyptian skeleton in dating back to 2160–2000 BCE [83]. Leukemias are classified into several major subtypes, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphoblastic leukemia (CLL) [84]. Chemotherapy was an initially effective treatment for childhood ALL when first attempted in 1948; unfortunately, disease typically relapsed ultimately leading to death [85,86]. Currently, the standard treatment includes combination chemotherapy to destroy the defective hematopoietic system followed by hematopoietic stem cell transplantation (HSCT) [87,88]. This approach is particularly indicated for recurrent disease, and can be introduced shortly after first-line treatment with chemotherapy [89,90]. HSCs can be derived from the bone marrow (BM), umbilical cord blood (UCB), or peripheral blood (PB) [91]. The first successful allogeneic human bone marrow transplantation (BMT) performed in patients with leukemia following optimized radiation and chemotherapy doses resulted in a Nobel Prize in Medicine for Dr. E. Donnall in 1990 [92]. However, histocompatibility mismatching and graft rejection resulted in high relapse rates; as such, the disease relapsed and the success rate was low [93]. Among the efforts made to improve these outcomes, donor leukocyte infusions (DLI) were introduced, by providing immune cells pre-collected from the anticipated HSC donor following myeloablation in patients undergoing leukemia treatment; the goal was to establish donor chimerism and thereby preventing graft rejection [94]. Although, DLI was effective in managing disease relapse, it was related to the development of graft versus host disease (GvHD) in treated patients, resulting from the activity of effector donor T-cells [95]. Reduced-intensity conditioning (RIC) was also applied in an effort to control graft versus host disease (GvHD), while enhancing the graft versus leukemia effect (GVL), thereby maintaining engraftment and eradicating malignancy [96]. The use of less aggressive RIC and non-myeloablative conditioning reduces the overall toxicity and mortality associated with conditioning prior to transplantation, especially in older patients [96]. The relatively recent inclusion of UCB as a source for HSCs overcame the challenges associated with an attempt to locate an HLA-matched allogeneic donor [97]. UCB cells 10 M. Essawy et al. were also less immunogenic and also easy to collect; UCB cells cryopreserved for decades still support the efficient recovery of HSCs [98]. However, UCB maintains comparatively fewer HSCs with respect to adult weight; as such, two bags of cord blood are typically required in order to obtain a sufficient yield of HSCs for transplantation into a single patient [99–101]. Nonetheless, a long-term follow-up of the Eurocord–European Group for Blood and Marrow Transplantation study revealed encouraging results. The study evaluated the outcome of UCB transplantation for 147 children, among whom 74% had been diagnosed with acute leukemia. In these patients, the cumulative incidence of neutrophil recovery was 90% at 2 years post-transplantation, the incidences of acute and chronic GvHD were reported to be 12% and 10%, respectively. At 5 years post-transplantation, the cumulative incidences of relapse and non-relapse mortality were 47% and 9%, respectively; the probability of disease-free survival was 44%. These results stand in strong support of UCB banking and the use of cord blood units to facilitate HLA-identical cord blood transplantation (CBT) [102]. PB-HSCs can be collected by noninvasive means; this provides a safe procedure for both the donor and recipient who can then undergo more rapid engraftment [103]. Administration of recombinant granulocyte colony-stimulating factor (G-CSF) stimulates the release of endogenous HSCs from the BM and into the blood. Currently, about 80% of all allogeneic transplantations are performed using stem cells derived from the PB of adult patients [104]. Similarly, recent developments in targeted therapy approaches have resulted in improved outcomes and can eliminate the negative sequelae associated with indiscriminate cytotoxic myeloablation. Genetically modified T-cells that express antigen-specific chimeric antigen receptor (CAR) will target leukemic cells while sparing those that are otherwise normal [105]. The FDA has approved the use of autologous genetically modified CD19-lymphocyte cells (CAR T-cells) for the treatment of relapsed ALL and diffuse large B-cell lymphoma [106]. Sickle Cell Anemia In addition to traditional HSC transplantation, it is now possible to manipulate the diseased cells by removal, addition, or alteration of specific DNA sequences in order to correct defective or mutated genes. High efficiency and precise genetic manipulation or gene editing of the human genome has recently become possible with the use of the method known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 [107]; this procedure is outlined in Fig. 1.3. CRISPR/Cas9 was used to restore the normal blood cell phenotype by repairing CD34 + hematopoietic stem/progenitor cells (HSPCs) from patients diagnosed with sickle cell anemia, a disorder that typically results from a single nucleotide substitution within a β-globin gene [108]. The gene-edited HSPCs were transplanted back into the patient’s BM to function as a source of healthy autologous red blood progenitors; using this method the disease undergoes genetic correction, and graft rejection is evaded [108]. 1 Introduction and Basic Concepts in Stem Cell Research and Therapy: ... 11 1.3.2 Stem Cell Therapy in Clinical Trials 1.3.2.1 Skin Injuries and Wound Healing Skin is the largest organ in the body and a major part of the integumentary system that covers and protects the human body [109]. Physical, chemical, and biological factors can all disrupt skin integrity. Depending on the depth of injury, skin wounds can be epidermal, or they can involve either partial or full skin thickness [110]. The natural healing mechanisms are compromised by thirdand fourth-degree burn injuries; this presents a significant challenge for both the surgeons and patients. Over the past century, the gold standard for treating burns has been grafting of healthy skin. Skin grafting can include splitthickness skin graft (STSG) and full-thickness skin grafts (FTSG) [111,112]. Skin grafting involves the transfer of healthy skin (autograft or allograft) comprised of the epidermis and a portion of the dermis to the site of injury; problems arise when there is not enough healthy skin, a failure to treat deep wounds, a poor cosmetic outcome, and limited strength of grafted skin when compared with the original skin at the affected site [113]. Skin engineering thus represents an attractive alternative. Autologous keratinocytes or fibroblasts are cultured on a scaffold, in some cases, a scaffold alone is implanted into the wound to improve healing [114]. This technique results in the regeneration of both the epidermal and Fig. 1.3 Illustration of CRISPR/Cas9 gene editing 12 M. Essawy et al. dermal layers; however, this method did not facilitate the regeneration of skin appendages, including hair, nails and skin glands. Of note, traditional skin grafting also failed to regenerate skin appendages; however, pigmented melanocytes and neural and vascular tissues were recovered using this method, an outcome that was not achieved using the engineered skin [114]. Skin replacements can be generated using cellular or acellular scaffolds; based on the composition of the skin-substitute [115]. Acellular skin-substitutes are biodegradable scaffolds (e.g., collagen, elastin, and silicon, among others) that facilitate wound healing by recruiting fibrocytes and vascular cells in vivo and by inhibiting granulation and scar formation. The most common acellular skin-substitutes currently approved by the FDA and undergoing review in clinical trials include Integra ® [116], Alloderm™[117], and NovoSorb™BTM (Biodegradable Temporizing Matrix) [118]. Cellular skin-substitutes that contain epidermal cell sheets include Dermagraft ® and Apligraf ® ; these products were approved by the FDA for the treatment of diabetic foot ulcer [118,119]. ReCell ® is an FDA-approved commercial cell spray device that provides autologous keratinocytes designed to heal second-degree burns. ReCell ® works by facilitating enzymatic digestion of the patient’s healthy skin in order to harvest keratinocytes, which are then sprayed over the wound [120,121]. Commercially available skin-substitutes are still far from perfect. The cells frequently fail to integrate; show poor vascularization, weak mechanical integrity, and scar formation; and are subjected to immune-mediated rejection [109]. Indeed, there are no completely functional skinsubstitutes available at this time; of particular note, there is a great need for a functional skin-substitute that can undergo rapid vascularization. Recent advances in stem cell therapy, nanotechnology, tissue engineering, and microfluidics paved the way for improved skin tissue engineering focused on deep wound healing [122]. Bioscaffolds for skin engineering must all be biocompatible, nontoxic, non-immunogenic, biodegradable, and sufficiently porous so that free exchange of gases and nutrients can occur through a neo-vascularized functional skin-substitute [123]. The cell source for the engineered skin also has a significant impact on the outcome. For example, ESCs can be differentiated into both keratinocytes [124] and fibroblasts [125], but direct clinical applications of these cells are hampered by instability and concerns with respect to the functionality of the resultant tissues. Adipose-derived stem cells (ADSCs) can also differentiate into keratinocytes, fibroblasts, and other skin components; ADSCs also produce extracellular matrix (ECM) which is rich in growth factors and cytokines that enhance healing [126–128]. The ADSC secretome contains vascular endothelial growth factor (VEGF), growth differentiation factor (GDF-11), and transforming growth factor (TGF-β); all of these act on macrophages, fibroblasts, and endothelial cells and lead to limiting the immune responses, enhancing cell proliferation, and promoting angiogenesis at the transplantation site [129]. Clinical applications of autologous ADSCs are still under investigation for healing diabetic foot ulcers (NCT02092870, see https://www.clinicaltrials.gov)[130]. Furthermore, methods used to generate three-dimensional skin grafts using iPSC-derived keratinocytes and fibroblasts remain promising [131]. 1 Introduction and Basic Concepts in Stem Cell Research and Therapy: ... 13 1.3.2.2 Osteoarthritis Osteoarthritis (OA) is a chronic degenerative disease characterized by deterioration of joint articular cartilages; this results in exposed subcondylar bones and leads to friction, pain, and synovitis [132]. Globally, OA is currently estimated as the 11 th highest contributor to adult disability; this results largely from pain, stiffness, and impaired mobility due to disease affecting the knees, feet, hands, and spine joints [133]. Non-surgical approaches for treating OA include intra-articular injections of corticosteroids, hyaluronic acid “viscosupplementation,”or autologous platelet-rich plasma into the deteriorating joints [134–136]. These approaches are designed to alleviate pain, but they do not treat the underlying cause of mechanisms associated with OA [137]. Joint surgery for OA varies from whole knee replacement (arthroplasty) to minimally invasive arthroscopic techniques such as microfracture or microdrilling [138–140]. The aforementioned arthroscopic techniques involve the generation of multiple small fractures within the affected joint, promoting the recruitment of progenitor cells from the underlying BM which then undergo differentiation into chondrocytes [139]. The drawbacks of these approaches include the formation of an inferior form of cartilage that lacks mechanical durability [138]. Alternative cell-based approaches have been applied, including osteochondral transplantation and soft tissue grafting [141]. Among the problems associated with these approaches, outcomes have included poor grafting and integration, calcification of the grafts, and limited number of available donor tissues [142,143]. Accordingly, more effort has been directed toward autologous/allogeneic chondrocyte implantation (ACI) [144]. Currently, there are numerous phase III clinical trials involving ACI that include the expansion of autologous or allogeneic chondrocytes, followed by grafting into the deformed lesion [145]. As an example, a phase III clinical product that is now commercialized with the brand name Chondrosphere ® utilizes scaffold-free spheroids of chondrocytes obtained from autologous articular cartilage that are introduced for use to treat cartilage defects associated with hip injuries (NCT01222559) [146]. The challenges currently encountered include increased susceptibility of the donor to OA after tissue sampling in normal joints and an overall insufficient number of harvested chondrocytes. Likewise, expanded chondrocytes may undergo dedifferentiation and lose their ability to generate cartilage matrix [147]. MSCs have also emerged as a promising source of cells for this application owing to their robust capacity for expansion and chondrogenic differentiation [148,149]. In addition, MSCs secrete a variety of cytokines and growth factors with anti-inflammatory effects [150]; these cytokines may function to counteract the inflammatory processes associated with OA. Autologous bone marrow-derived MSCs have been used to repair full-thickness cartilage defects in two cases [151]. In this study, BM was aspirated from the iliac crests and cultured until adherent MSCs had undergone several expansion passages. Cultured MSCs were then collected, embedded in a collagen-gel scaffold, and transplanted onto the surface of the defective articular in the knee joint. Symptoms were relieved at 6 months, and both male and female patients were satisfied with the outcomes during the 4 years following transplantation [151]. MSCs derived from the umbilical cord, placenta, 14 M. Essawy et al. Wharton’s jelly or amniotic membrane all have shown promise with respect to novel treatments for patients diagnosed with OA [152–154]. In particular, UC-MSCs exhibited higher proliferative, clonogenic, anti-inflammatory, and chondrogenic potential compared with MSCs from maternal-derived decidua or BM [155]. CARTISTEM ® is a commercialized product that utilizes UC-MSCs for the treatment of cartilage deterioration in patients with OA; it is currently approved for a phase III clinical trial with the goals of evaluating safety and expanding its indications for use (NCT01041001, NCT01626677). Recently, phase II clinical trials have been initiated to assess the role of ADSCs for the treatment of patients with OA (NCT02838069) [78]. 1.3.3 From Bench to Bedside 1.3.3.1 Diabetes Mellitus (DM) Diabetes mellitus (DM) is a chronic inflammatory metabolic disorder that results in sustained hyperglycemia due to defects in insulin production (Type I), insulin utilization (Type II), or a combination of both [156]. Type I DM (T1DM) is an autoimmune disease, wherein activated immune cells attack insulin-secreting β-cells in the pancreas, resulting in insulin deficiency [157]; contrarily, type II DM (T2DM) is characterized as a chronic inflammation state that ultimately leads to insulin resistance, reduced insulin secretion, β-cells exhaustion, and apoptosis [158–160]. Untreated DM leads to severe complications that can be life-threatening and have significant impact on numerous major organs including the kidneys [161], heart [162,163], eyes [164,165], and nervous system [166]. Patients with diabetes attempt to regulate their blood glucose levels and to maintain values at or near normal limits with dietary control [167], hypoglycemic drugs [168], and lifestyle changes [169]. However, these traditional methods often fail to maintain normoglycemia in the long run [170]. Islet transplantation (also known as Edmonton protocol) was developed in 1999 to provide more β-cells and thus increase insulin production for patients diagnosed with T1DM [171–173]. However, the use of this approach was limited due to the risks associated with the surgical procedure [174], the need for long-term immunosuppressive therapy [175], a shortage of organ donors [176], and only limited impact with respect to achieving insulin independence [177]. Stem cell-based therapy provides a new approach for the management and treatment of DM. First, this approach can create a virtually unlimited supply of insulin-producing cells [178–181]; other applications focus on restoring β-cell function [182], modifying immune dysregulations, and reversing the associated metabolic complications [183]. 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Stem Cells. 2020;38(5):596–605. 1 Introduction and Basic Concepts in Stem Cell Research and Therapy: ... 31 2.4.2 Culture of hESCs ....................................................................... 51 2.4.3 Characterization of hESCs ............................................................. 52 2.4.4 Differentiation of ESCs ................................................................ 54 2.5 Applications of Human Embryonic Stem Cells ........ ..... .............. ... .. .............. 54 2.5.1 Disease Modeling ...................................................................... 55 2.5.2 Regenerative Medicine ................................................................. 55 2.6 Induction of Pluripotency: Needs and Challenges ............................................ 56 References............................................................................................ 57 What You Will Learn in This Chapter This chapter will focus on pluripotency as a key feature in determining the differentiation potential of cells and the importance of embryonic and pluripotent stem cells in research and their promising applications in regenerative medicine. It also includes a brief description of the major findings on embryonic stem cells’derivation, characterization, and differentiation. The differences between naÿve and primed pluripotency will be highlighted, and the in vitro growth conditions contribute to these differences. The chapter will also cover major findings in nuclear reprogramming and the developments in induced pluripotent stem cell technology. Finally, we will conclude with the limitations of embryonic stem cells in clinical applications and areas for future research. 2.1 Introduction 2.1.1 Pluripotency Pluripotency is defined by two main characteristics, self-renewal and potency. Self-renewal describes the ability of cells to divide almost infinitely, or to divide long-term in culture, resulting in two daughter cells with distinct cellular fates, where one of the daughter cells maintains the pool of undifferentiated cells [1]. The ability of a cell to differentiate into different cell types that exhibit different characteristics than the mother cell is called cell plasticity [2]. Pluripotent stem cells show a substantial degree of plasticity as they can give rise to cells of the three embryonic germ layers: ectoderm, mesoderm, and endoderm. However, pluripotent stem cells have limited potential to give rise to extraembryonic tissues, particularly the placenta (Fig. 2.1)[2]. Nonetheless, a newly derived type of pluripotent cell, known as extended potential pluripotent stem cells (EPSCs), has been shown to recapitulate both embryonic and extraembryonic tissues [3]. Multiple types of pluripotent stem cells can be derived from different embryonic stages and tissues, as well as artificially from direct reprogramming of somatic cells [2,4]. Pluripotency is also a highly dynamic process, where interchange between naïve and primed states can occur [4]. The 38 S. Shouman et al. unique characteristics of pluripotent cells have led to exploration of novel therapeutic approaches. Moreover, the advances in reprogramming of somatic cells into a more naïve pluripotent state have paved the way for clinical applications with limited constraints [5]. The molecular hallmark for determining pluripotency is the expression of specific transcription factors that halt the activation of lineage-specification genes, leaving pluripotent cells in a quiescent state. The core transcription factors that control pluripotency include the octamer-binding transcription factor 4 (Oct-4), which is also known as POU5F1 [6], the homeodomain transcription factor (Nanog) [7], and the protein SRY-box transcription factor 2 (Sox2), which is related to high-mobility group (HMG) proteins [8]. Other important characteristics of pluripotency include high expression of telomerase reverse transcriptase, which plays a major role in the regulation of cellular life span [9]. In addition cell-surface antigens, such as stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, and CD9 tetraspanin, together with positive intracellular enzyme alkaline phosphatase activity, also play a role in regulating cellular life span [10–14]. However, the most robust methods to determine pluripotency are functional assays. The principle of these assays depends on the ability of pluripotent cells to recapitulate the three germ layers in vitro or in vivo. Examples include (1) in vitro differentiating cell aggregates, termed embryoid bodies [15], (2) in vivo teratoma formation [16], and (3) in vivo chimera formation [17]. These assays are explained in more detail in this chapter. 2.1.2 Historical Overview of Pluripotency Cell reprogramming is the process of induced cell transformation from one specific cell type to another [18]. For many decades, numerous techniques have been developed in cell Fig. 2.1 Different cell potency abilities 2 Embryonic and Pluripotent Stem Cells 39 reprogramming and induced pluripotency. Cellular differentiation was thought to be unidirectional and irreversible (i.e., from immature pluripotent to more mature differentiated cells), like a ball rolling down from the top of a mountain to the bottom. However, this concept was challenged, and it is now known that reprogramming to obtain pluripotency can be achieved using a variety of approaches, including nuclear transfer, cell fusion, and direct reprogramming. Recent work has identified that cell fate is not irreversible, and that it is a plastic or reversible (i.e., the ball can roll back upwards from the bottom to the top of the mountain). The early work of John B. Gurdon in 1962 demonstrated, in lower animal models, that reprogramming can be achieved by nuclear transplantation [19]. In these experiments, a nucleus of a somatic cell from the intestine of a frog was implanted into an enucleated unfertilized frog egg. This egg then generated an adult tadpole. This process of somatic cell reprogramming to the pluripotent embryonic state led to “rejuvenation”and was termed somatic cell nuclear transfer (SCNT) [19]. These early experiments showed that the nucleus of mature cells could be reprogrammed to generate an entirely new animal without sexual reproduction. These experiments fundamentally changed the perception of biology and reproduction and were the basis for the mammalian cloning experimentation that followed (see, Table 2.1, and Fig. 2.2). For many years it seemed that it was not possible to clone mammals. However, Ian Wilmut’s research group cloned Dolly the sheep, born on July 5, 1996 from a mammary cell of an adult sheep [20]. Later, Gurdon and Yamanaka shared the 2012 Nobel Prize in Physiology and Medicine for their innovative contributions to the field of cellular reprogramming. In 1981, Martin Evans, Matthew Kaufman [26], and Gail Martin [16] established the first self-renewable and pluripotent embryonic stem cell (ESC) lines derived from preimplantation mouse embryos. When immune-deficient mice were injected with these cells, Table 2.1 Therapeutic cloning compared to reproductive cloning Therapeutic cloning Reproductive cloning 1. Cloning by SCNT for reprogramming [20] 1. Cloning either by: –SCNT for reprogramming [20]or –Embryo splitting: The IVF cattle embryo at 4-cell stage is divided into 3 or 4 identical cells, and each cell is then developed into healthy monozygotic calves [21] 2. Intended to isolate patient-derived ESCs from embryos created in vitro without later transfer into the uterus [22] 2. Intended to implant the embryo into a female uterus to obtain a whole organism [21] 3. Offers great promise for regenerative medicine through the production of autologous nuclear transfer embryonic stem cells (ntESC) [22] 3. Offers a great option for cloning of livestock especially the genetically engineered animal (e.g., human coagulation factor IX in the milk of transgenic sheep) [23] 4. Applicable for both animal and humans but with some ethical constraints [24] 4. Banned for humans but applicable to animals [25] 40 S. Shouman et al. Fig. 2.2 Therapeutic cloning and reproductive cloning 2 Embryonic and Pluripotent Stem Cells 41 they generated teratomas or teratocarcinomas, which at that time, became one of the hallmarks of pluripotency. In addition, these ESCs were shown to contribute to the formation of chimeric mice after being injected into mouse blastocysts [17]. In 1998, the research group of James Thomson and Jeffrey Jones isolated the first human ESCs from the inner cell mass of blastocysts produced by in vitro fertilization [15]. In the same year, Austin Smith and colleagues described the culture conditions and factors that are important for the in vitro maintenance of ESC pluripotency [27]. In 2001, cell fusion of ESCs and somatic adult thymocytes produced hybrid cells [28]. And as a result, ESCs could effectively reprogram thymocytes into a more embryonic state. This gave the ability to form chimeras, in addition to the tri-lineage differentiation potential. The pluripotency-associated Oct-4 gene, which is normally suppressed in thymocytes, was up-regulated following fusion with ESCs, and epigenetically, the chromatin was less condensed into a more transcriptionally accessible state. These two factors induced the reprogramming of the heterokaryons into a more pluripotent state [28]. However, these hybrid cells still retained some of their somatic characteristics, which represented a challenge for their use in clinical applications. This study gave clues about the possibility of the existence of reprogramming factors, which could lead to ESC-like cells via direct use on somatic cells, without the need for mammalian embryos. Davis and colleges showed possible direct reprogramming of somatic cells into embryonic-like stem cells. Embryonic mouse fibroblasts treated with 5-azacytidine, an inhibitor of DNA methylation, generated myoblasts as shown by ectopic expression of the muscle-specific gene (MyoD) [29]. In 2006, Yamanaka and Takahashi reported a seminal discovery in which they created induced pluripotent stem cells (iPSCs) from mouse fibroblasts by combisning of four reprogramming factors. These factors included Oct 3/4, Sox2, Klf4, and c-Myc (OSKM, also known as the Yamanaka factors).These factors were used to generate pluripotent cells from somatic fibroblasts using a viral delivery system [30]. One year later, Thomson and colleagues generated human induced pluripotent stem cells (hiPSCs) from human fibroblasts using another set of reprogramming factors, Oct 3/4, Nanog, Sox2, and Lin 28 (ONSL) [31]. Different approaches for reprogramming somatic nuclei are illustrated in (Fig. 2.3). 2.2 Pluripotent Stem Cells 2.2.1 Pluripotent Stem Cell State: Naïve Compared to Primed Pluripotent stem cells (PSCs) can be isolated from vertebrates, including mice and humans, based on their tissue of origin and developmental stage (Fig. 2.4). PSCs are further classified as “naïve”or “primed”(Table 2.2), based on their ability to produce all somatic and germline cells, as well as their in vitro growth conditions [33]. The in vitro growth conditions include colony morphology, growth characteristics, culture requirement for maintenance of the pluripotent state, gene expression, and the global state of DNA 42 S. Shouman et al. methylation. Furthermore, the naïve or primed classification can be based on chimera formation or X chromosome inactivation in female cells [33]. We could resemble chimeras as “a mosaic painting during the Byzantine era”which the body of developing organisms is Fig. 2.3 Different approaches for cellular reprogramming 2 Embryonic and Pluripotent Stem Cells 43 Fig. 2.4 (a) Stages of deriving different types of pluripotent stem cells in mice and humans. (b) Current strategies to obtain expanded potential stem cells (EPSCs) 44 S. Shouman et al. composed of distinct cell populations with different genetic origins, resulting from the fusion of more than one zygote. Chimera studies have been used to assess the developmental potency and fate of different embryonic cell lines based on their ability to participate in embryonic development after injection into a blastocyst [39]. Inactivation of one X chromosome randomly occurs at an early stage of female embryonic development to ensure dosage compensation between both genders regarding sex-linked genes expression [40]. However, female naïve pluripotent cells reactivate both X chromosomes (XaXa), in contrast to primed pluripotent cells which have only one active (XaXi) chromosome [41]. The naïve state represents a cellular state that is similar to the preimplantation inner cell mass. This can be described as PSCs in a “ground state”that are free of any lineage commitment, and therefore not constrained epigenetically. In contrast, the primed state is representative of the post-implantation epiblast cells (EpiSCs) that are more committed toward lineage-specific developmental pathways and are epigenetically restricted [33]. To date, the naïve state has been achieved in mouse ESCs (mESCs), but not in human ESCs (hESCs), even though both were derived from preimplantation embryos [15]. It is still unknown whether the reconversion of PSCs from primed to a more naïve state is direct, or Table 2.2 Naïve and primed pluripotent stem cells Cell type mESCs mEpiSCs and hESCs Origin ICM of an early blastocyst [26] Post-implantation epiblast of the mouse embryo [32], ICM of the human embryo [15] Pluripotency state Naïve [33] Primed [33] Teratoma formation Present [34] Present [34] Blastocyst chimeras Present [35] Absent [35] Epigenetic state Global hypomethylation [36] Global hypermethylation [36] Expressed genes High expression of Oct4 (or POU5F1), Nanog and ESRRβ (or ERR2) [36] Oct4, Sox2, Nanog, Fgf5, Brachyury, and Otx2 [34,37]. X-chromosome inactivation status Both X chromosomes are active [36] One X chromosome is inactive [34] Clonogenicity High [33] Low [33] Oct4 enhancer usage Distal [38] Proximal [38] Response to LIF/STAT3 Self-renewal [34,37] None [34,37] Response to Fgf2 Differentiation [34,37] Self-renewal [34,37] 2 Embryonic and Pluripotent Stem Cells 45 involves a transitional state. Overall, more evidence suggests that human and mouse PSCs are not identical, and that differences in gene expression and culture requirements could affect the pluripotency states in vitro and therefore could lead to different outcomes in terms of PSC-based therapies. 2.2.2 Switching Between Pluripotency States: Naïve to Primed and Back Different states of pluripotency of mESCs (in vitro) correspond to in vivo embryonic development. This means that naïve and primed states are not categorical states, but rather, represent successive molecular snapshots during embryonic development [4]. Therefore, questions have been raised about how these cells could be converted back from the primed state to the naïve one. One crucial factor that is involved in this process is the culture conditions that the ESCs are exposed to after isolation, which has proved to be critical in determining their fate. Since hESCs or EpiSCs are in a near primed state, several attempts have been made to reset the pluripotency of hESCs back to a more naïve state, similar to that of mESCs [33]. These attempts included forced resetting through transgenic induction of Oct4, Klf2 and Klf4 [42], or Nanog and Klf2 [43] in the presence of 2i/ leukemia inhibitory factor (LIF) culturing conditions, or by simply manipulating the culture conditions to reset “genetically unmodified”hESCs into naïve ESCs [44,45]. These attempts have enabled successful direct derivation of naïve hESCs from the inner cell mass (ICM) by adding different growth factors and small molecules such as LIF, FGF2, Activin A, GSK3 inhibitors, STAT3 inhibitors, ROCK inhibitors, and MEK inhibitors [45]. In all of the forced or non-forced previous attempts, the naïve hESCs that were generated met the naïve criteria of mESCs. Epigenetic modifications also determine the pluripotency state as the mESC (naïve) genome is globally hypomethylated, whereas the EpiSCs (primed) genome is hypermethylated [4]. The pattern of histone modification especially on the gene promoter region is different in naïve cells, which prefer to use the distal enhancer for Oct4 gene transcription, while the proximal enhancer is primarily used in the primed cells. This difference suggests that there are histone modifications that change chromatin structure and accessibility in order to regulate transcription [38]. In humans, the epigenetic status of ESCs is considered to be primed, since it is similar to mouse EpiSCs [4]. However, recent work has identified that the primed hESC state can be reversed back to the naïve state through epigenetic resetting via transient histone deacetylase inhibition [46]. Therefore, non-transgenic naïve hESCs can be obtained via either manipulation of the culture conditions or epigenetic resetting. However, it is still debatable whether it is acceptable to unify the definition of naïve/primed pluripotency in human ESCs (and possibly other species) based on ESC characteristics identified in rodents. Instead, identifying speciesspecific characteristics for naïve/primed ESCs may be necessary. 46 S. Shouman et al. 2.3 Types of Pluripotent Stem Cells 2.3.1 Embryonic Stem Cells (ESCs) ESCs are isolated from the ICM of early preimplantation embryos, at E3.5 in mice [26], or from human blastocysts [15]. When mESCs were isolated and cultured under proper conditions, including essential growth factors, feeder cells, or feeder-free medium in addition to proper incubation, they maintained their naïve pluripotency state for a long time. Cultured human ESCs (hESCs) were less naïve (more primed) than mESCs, and differed in their culture requirements (Fig. 2.4, Table 2.2)[36]. To date, it is not clear whether the differences between mESCs and hESCs are only due to the culture conditions or they are also due to other factors. The in vitro maintenance of naïve mESCs requires LIF signaling, while hESCs depend mainly on FGF2 and TGFβ1/Activin2 signaling, and not LIF [36]. Reports suggested that LIF with two inhibitory small molecules, CHIR99021 and PD0325901 (called 2i), inhibited the mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) pathway and the glycogen synthase kinase 3β (GSK3β) pathway. Inhibition of both pathways stabilized the ground state of mESCs. Naïve mESCs express various pluripotency markers, including OCT-4 (Pou5f1), NANOG, and Esrrβ(Err2), in addition, they lack the X-inactivation state in female cells. On the other hand, hESCs express high levels of some naïve pluripotency markers, such as NANOG, PRDM14, REX1 (or ZFP42), and E-cadherin, however, hESCs also show some of the primed cell characteristics, including low expression of KLF17 and DPPA3, lack of exclusive nuclear localization of TFE3, lack of hypomethylation and tendency of a preX-inactivation in female ESC lines [36]. Therefore, using conventional direct derivation and culture approaches, hESCs are less naïve than mESCs but more naïve than mouse epiblast stem cells (a primed state of mESCs). To achieve naïve hESCs, in a similar state to naïve mESCs, scientists adapted the culture conditions to reset isolated hESCs in vitro into a more naïve state. This suggests that the culture conditions do affect the state of pluripotency, which will also affect the outcome of using ESCs in clinical practice. 2.3.2 Epiblast Stem Cells (EpiSCs) EpiSCs are isolated from the epiblasts of post-implantation embryos in mice (between E5.5 and E7.5) [32]. However, due to ethical considerations, no EpiSCs have been obtained from human embryos [37]. Mouse EpiSCs (mEpiSCs) share some similarities with mESCs but are classified as a different type of PSCs, based on several cellular and molecular differences (see Table 2.2). Similar to mESCs, mEpiSCs are pluripotent, as they give rise to all three germ layers and germ cells, and can form teratomas when they are injected into immune-deficient mice. However, similar to hESCs, the pluripotency state of mEpiSCs is primed [34]. The characteristics of mEpiSCs are also similar to those of hESCs in some aspects, including the inability to survive as a single-cell clone after trypsinization, and 2 Embryonic and Pluripotent Stem Cells 47 2.4.4 Differentiation of ESCs As discussed earlier in this chapter, pluripotency is the primarily trait of ESCs [15]. Pluripotency is always linked to a high potential to further differentiate into different cell types [2]. As the cell differentiates, its functional specification prevails and pluripotency diminishes [2,97]. Expression of pluripotency markers, including Oct-4, SOX-2, and Nanog maintains the pluripotent state of ESCs by controlling the expression of differentiation gene cascades. Accordingly, lack or loss of these pluripotency markers initiates the differentiation of ESCs, which is accompanied by the expression of differentiation markers [96,97]. For example, differentiation can be driven by the activation of polycomb repressive complexes and microRNAs that regulate or switch-off pluripotency regulators of ESCs [97,108,109]. In addition, growth factors, epigenetic state, and cell-tocell signals cooperatively determine which lineage specification the differentiated ESCs will go through [110–112]. In vivo, cells of the ICM, which is the origin of ESCs, divide to give rise to all cell types in the body, leading to complete structural and functional body mass. Similarly, ESCs can differentiate in vitro into specialized cells of any of the three germ layers, including the ectoderm, mesoderm, and endoderm, in the presence of the proper stimuli and growth factors [15,113]. In the absence of self-renewal, ESCs cultured in vitro can spontaneously differentiate as aggregates of cells or embryoid bodies [114]. Cells of the embryoid bodies have been shown to adopt in vivo-like temporal and spatial differentiation patterns. Ectodermal-like cells appear first, followed by endodermal cells, and further differentiation and specification of mesodermal cells [115,116]. Germ layer cells further differentiate into more specialized cells, such as cardiomyocytes [117], hepatocytes [118], neurons, astrocytes and oligodendrocytes [119,120], and ovarian follicle-like cells [121]. Factors that affect the differentiation of hESCs in vitro include the seeding density, pH, temperature, and most importantly the components of the culture medium and growth factors. Hepatocyte growth factor (HGF) and nerve growth factor (NGF) can induce differentiation into the three germ layers. While activin A and transforming growth factor (TGF)-βare essential to induce mesodermal differentiation, other factors such as bone morphogenic proteins (BMP)-4, retinoic acid (RA), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) can all promote ESC differentiation into both mesodermal and ectodermal lineages [92]. 2.5 Applications of Human Embryonic Stem Cells The use of hESCs allows the advancement of our understanding of disease etiology and also shows great promise for the development of novel therapeutic approaches. There are currently over 40 clinical trials using hESCs that are registered on the NIH Clinical Trials website (https://clinicaltrials.gov/, as of the 16th of May 2020, using “embryonic stem cells”as the search criteria). These studies include using hESCs to generate retinal pigment 54 S. Shouman et al. epithelium in order to treat ophthalmic diseases, such as age-related macular degeneration and retinitis pigmentosa [122]. These studies also include those targeting cardiac diseases. In particular, severe ischemic heart failure was treated using hESC-derived cardiac progenitor cells that were combined with a fibrin scaffold and grafted onto the epicardium of the infarcted area [123]. Clinical trials have also used hESCs to target neurodegenerative diseases, such as Parkinson’s disease (PD) and spinal cord injury (SCI) [124], as well as type 1 diabetes [125]. However, to date, there are no approved FDA products that are based on hESCs [126]. Research is now focused on using hESCs for disease modeling and regenerative medicine, where animal models have failed or are still inappropriate for these purposes. 2.5.1 Disease Modeling ESCs have been used to model disease through development of disease-specific cells that carry relevant aberrations or mutations. Human ESCs are either modified using gene editing or induced to acquire chromosomal aberrations via manipulatng in vitro cell culture conditions [127,128]. Disease-specific ESCs may also be directly isolated from defective IVF embryos carrying genetic diseases or chromosomal aberrations. Preimplantation genetic diagnosis and genetic screening are two methods used to identify embryos with monogenic disorders or chromosomal abnormalities [129,130]. In 2004, human-derived ESCs were successfully genetically engineered to model Lesch–Nyhan disease through the induction of a mutation in the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene using homologous recombination [128,131]. Development of successful hESC disease models was also performed for Fragile X Syndrome [132] and Turner’s syndrome [127]. Examples of methods for developing disease models include gene editing techniques, where Zinc finger nucleases were used to mediate site-specific modifications in the ESC genome with high efficiency [133–136]. In addition, transcription activator-like effector nucleases (TALENs) were made to induce genomic modifications and exploit the potentials of hESCs in disease modeling [137]. Gene editing has also been performed in ESCs to model X-linked severe combined immunodeficiency (X-SCID) disorder [133]. More recently, clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR-Cas9) technology [134,138], SCNT [139], and iPSCs [30] have been used as practical alternatives to hESCs use for disease modeling. 2.5.2 Regenerative Medicine Human ESCs can generate various types of differentiated cells for cell replacement therapies and can be also used in clinical trials for disease treatment. Below are some examples of their use in the clinic: 2 Embryonic and Pluripotent Stem Cells 55 2.5.2.1 hESCs and Spinal Cord Injury In 2009, FDA approved the first hESC-based phase I clinical trial using hESC-derived oligodendrocyte progenitor cells (GRNOPC1, ClinicalTrials.gov Identifier: NCT01217008, known as Geron’s trial) for the treatment of acute spinal cord injury (SCI) patients. The treatment protocol included the injection of two million GRNOPC1 cells into affected SCI patients within 7–14 days post-injury, followed by the administration of immune-suppressants for 46 days. Both animal studies and preclinical data have shown that GRNOPC1 cells have the potential to regenerate injured cord and promote motor recovery in SCI patients. After enrolling five patients with SCI, no adverse effects were observed. Safety was measured through assessment of the frequency and severity of adverse events occurring within 1 year of injection. However, no improvement was reported in motor or sensory responses in enrolled SCI patients [124]. This trial was terminated in 2011, and another study using hESC-derived oligodendrocyte progenitor cells (now called AST-OPC1) to treat SCI has been initiated (ClinicalTrials.gov Identifier: NCT02302157). 2.5.2.2 hESCs and Diabetes In 2014, a phase 1/2 clinical trial for type 1 diabetic patients was sponsored by ViaCyte and CIRM (ClinicalTrials.gov Identifier: NCT02239354). The study tested a new product (called VC-01) that contains stem cell-derived pancreatic islet replacements in order to treat type 1 diabetes mellitus. Pancreatic endoderm cells derived from hESCs (PEC-01 cells) were encapsulated in an inert biomaterial in order to protect them from attack by the immune system. The encapsulated “islets”were expected to act as an artificial pancreas in order to effectively control blood glucose levels. The capsule was surgically implanted under the patient’s skin and was expected to mature over several months and start producing insulin. The tolerability, therapeutic dose, and safety were evaluated in the first cohort group. After 24 months, the product that was implanted showed promising results and had minimal adverse effects (related to the surgery) and no immunological sensitivity. The cells had prolonged survival, and their ability to differentiate into pancreatic islet cells was determined using immunohistochemical staining for NKX6–1, insulin, and glucagon markers. Importantly, no off-target tumors were observed. This study suggests that the use of ESCs may be a new effective approach to treat chronic autoimmune diseases, such as type 1 diabetes [125]. 2.6 Induction of Pluripotency: Needs and Challenges Human ESCs have a great potential to treat many degenerative diseases [140]. However, translating hESCs for use in the clinic has been challenging for a variety of reasons. Ethical controversies about the derivation and use of hESCs in research, as well as in the clinic, are still a significant obstacle to advances in this field [141]. Additionally, the use of these cells also leads to immune challenges [142] and other issues of safety and functional efficacy 56 S. Shouman et al. [143]. Safe and ethically accepted alternatives to ESCs for therapies in regenerative medicine have been developed by researchers. As such, iPSCs have been established to model diseases and have been used for drug discovery. Newer gene editing technologies and direct differentiation protocols are also less controversial and more effective source of pluripotent cells for regenerative medicine purposes [144]. Take Home Message •Pluripotent stem cells can be differentiated in vitro to all three germline lineages, excluding the extraembryonic tissue. •Embryonic stem cells are obtained from the inner cell mass of the blastocyst, while induced pluripotent stem cells are obtained by reprogramming of adult somatic cells. •New approaches enabled scientists to create extended or expanded potential stem cells (EPSCs), which can form both extraembryonic and intraembryonic tissues. •Approaches to cellular reprogramming; include nuclear transfer or cloning, cell fusion, and direct reprogramming, paved the way for discovering pluripotency. •Pluripotency hallmarks include unregulated expression of pluripotent genes, in vitro embryoid bodies formation, in vivo teratoma formation, and in vivo chimera formation. •Pluripotency exists in two different states, primed and naïve. 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Cell Death Dis. 2017;8(7):e2953. 2 Embryonic and Pluripotent Stem Cells 63 HSCs undergo translocation to the aorta–gonad–mesonephros (AGM) region. During midgestation, hematopoiesis transitions mainly to the liver and, to a lesser extent, to the spleen and thymus. Finally, BM becomes the primary site of hematopoiesis during late gestation and in adulthood [2]. Definitive hematopoiesis includes HSCs and hematopoietic progenitors derived from them; this process leads to the production of enucleated RBCs and the full set of myeloid and lymphoid lineages. HSCs are one of the several cellular components of the BM; their developmental niche includes all hematopoietic cells derived from HSCs and vascular cells and extracellular matrix. To achieve a larger understanding of hematopoiesis, study of this dynamic microenvironment remains critical. Approaches used for the study of hematopoiesis include in vivo imaging and ex-vivo analysis known as “bone marrow (BM)-on-a-chip”; in the latter case, processes that take place within the BM are studied using a three-dimensional (3D) scaffold on a microchip. Unique biomaterialbased 3D scaffolds have been recently used to generate systems that mimic the interaction of HSCs with the 3D structure of the BM microenvironment [3]. One such study featured a scaffold comprised of macroand micro-porous printed β-tricalcium phosphate (β-TCP), a bioceramic used for bone tissue engineering, combined with Matrigel ® (β-TCP/ Matrigel®); this matrix provided an ideal support for the study of hematopoietic cell recruitment, proliferation, and differentiation and for remodeling of the extracellular matrix. In addition, upon transplantation in murine models, this scaffold promoted neovascularization and provided a functional extramedullary BM niche, which recapitulated both osteogenesis and hematopoiesis [4]. 3.1.2 Anatomy of the Bone Marrow In long bones, BM can be found within the diaphysis and the metaphysis. BM fills the medullary cavity of the diaphysis; the shaft of compact bone that provides physical support for the BM and a site for mineral storage and locomotion. BM can also be found inside the cavities of cancellous bone (also known as trabecular or spongy bone) that include primary and connected secondary trabeculae in the metaphysis. The porous structure of the cancellous bone provides strength and flexibility and is comparatively lighter in weight. The BM coexists with a complex vascular and neural network and is tightly associated with the dynamic bone environment at which new bone tissue is added, removed, or remodeled from spongy to compact and vice versa [5]. 3.1.3 Types and Morphology of BM The BM is a soft and gelatinous-like tissue as it contains primarily hematopoietic cells and adipose tissue; the nature of these components defines the type of the marrow. Hematopoietic red marrow is the primary site of active hematopoiesis; it is comprised of abundant progenitors and mature RBCs, white blood cells, platelets, and adipose tissue. 3 Hematopoietic Stem Cells and Control of Hematopoiesis 71 Red marrow can be found inside virtually all bones of neonates but becomes less widespread with increasing age. In adults, red marrow is confined to axial skeletal structures, including the skull, vertebrae, ribs, sternum, pelvic bones, and in the proximal metaphysis of the humerus and femur. The second type of BM is the yellow marrow, which includes primarily adipose cells accompanied by islands of hematopoietic tissue. There is a dynamic balance between the two types of marrow throughout the life span of an individual. The results of several studies suggest that the yellow marrow could, at least in part, revert to red marrow in response to specific erythropoietic stimuli [6]. Yellow marrow has been described as a “buffering tissue,”which facilitates the expansion or regression of hematopoietic cells within the bone [7]. 3.2 Hematopoiesis and the Hematopoietic State As typical stem cells, HSCs have the capacity for self-renewal and the ability to differentiate and give rise to all blood cell lineages. In the mouse embryo, precursors of hemogenic endothelial cells (HECs) go through intermediate stages of development to form the first HSCs in the AGM region [8]. 3.2.1 Tracing Hematopoiesis Throughout Development 3.2.1.1 Primitive and Definitive Hematopoiesis Large nucleated RBCs and macrophages are generated in the yolk sac as a result of a primordial wave of blood formation, known as primitive hematopoiesis [9]. Adult-type hematopoiesis swiftly replaces the primordial wave, which occurs in the AGM region [10]. At this point during embryogenesis, a tube developing into a single aorta is created after the lateral plate mesoderm undergoes migration and comes into contact with the endoderm. This process is followed by the emergence of HSCs in the ventral wall of the dorsal aorta near the AGM region. Subsequently, the fetal liver, thymus, spleen, and, eventually, BM are overtaken by HSCs that are capable of long-term self-renewal to establish definitive hematopoiesis (Fig. 3.1)[11]. 3.2.1.2 Extraembryonic Hematopoiesis Extraembryonic hematopoiesis is among the earliest stages of primitive hematopoiesis. At embryonic day (E) 7.0 in mice, the first hematopoietic progenitors can be identified in the yolk sac [12]. Hematopoietic activity can also be detected in the umbilical arteries and in the allantois, but not in the umbilical veins [13]. These findings support the hypothesis that HSCs originate mainly during arterial development. It remains unclear whether placental HSCs originate de novo or via colonization from earlier sites of hematopoiesis at the time that circulation is initiated or both [14,15]. 72 M. Essawy et al. 3.2.1.3 Mesoderm to Hemangioblast Hematopoietic precursor cells were first discovered nearly 100 years ago from studies of total chick blastoderms cultured on cover slips and from explant cultures of the posterior sections of blastoderms during the gastrulation phase; these cells were designated as angioblasts or hemangioblasts. In both types of experiments, the hemangioblasts were shown to be the precursors of both endothelial and hematopoietic cells [16]. Findings from these early studies carried out in chick embryos are fundamental to our current understanding of the concept of a hemangioblast; these findings remain correct through the present time. In mice, migrating mesoderm is generated by means of gastrulation that takes place at E6.5 [17]. The mesoderm differentiates into distinct populations with different developmental fates. In chick embryos, the mesodermal cells from the posterior primitive streak were the source of the initial blood islands [18]. All developing mesodermal cells are marked by a transcription factor and member of the family of T-box genes known as Brachyury. Detection of Brachyury + cells declines once they are patterned and directed toward the generation of blood, connective tissues, endothelium, and skeletal or cardiac muscles [16, 19]. The hematopoietic potential of individual cells in the mouse epiblast, primitive streak, and early yolk sac was established by Padrón-Barthe et al. [19]. In vivo clonal analysis identified specified independent epiblast populations (before gastrulation) such as early Primitive node Primitive pit Primitive groove in primitive streak Bone marrow Fetal liver Aorta-gonadmesonephros (AGM) C. Maturation and expansion D. Quiescence/self-renewal Yolk sac blood islands A. Specification B. Emergence Primitive Hematopoiesis Yolk sac endoderm Blood islands Definitive Hematopoiesis Fig. 3.1 Initiation of primitive and definitive hematopoiesis during development 3 Hematopoietic Stem Cells and Control of Hematopoiesis 73 yolk sac blood and endothelial lineages, and the hemogenic activity was similar in both the embryonic hemogenic endothelium (HE) and a subpopulation of the yolk sac endothelium. Padrón-Barthe et al. [19] also characterized the appearance of the HE in the yolk sac, which ultimately gave rise to hematopoietic precursors showing markers related to definitive hematopoiesis. 3.2.1.4 Hemangioblast to Hemogenic Endothelium It has been proposed that HSCs may be generated from hemangioblasts via formation of an HE intermediate [20]. This hypothesis was based on observations that localized the HE at a site adjacent to the hemangioblasts. Vogeli et al. [21] exploited advancements in single-cell resolution fate mapping of the late blastula and gastrula of zebrafish and confirmed the existence of hemangioblasts in vivo via the emergence of the bi-potential progenitors, which were capable of generating both hematopoietic and endothelial cells adjacent to the lining of the ventral mesoderm. The in vitro transformation of hemangioblasts/blast colony-forming cells into hematopoietic cells was characterized as a two-step process. Initially, the hemangioblasts generated a tightly adherent cell layer, which primarily expressed endothelial cell markers (thus comprising a transitory HE stage) after 24 h, later, at 36–48 h of culture, these cells became non-adherent, rounded, and initiated the formation of hematopoietic blast colonies [20]. 3.2.1.5 Transition from Hemogenic Endothelium to Definitive Hematopoietic Progenitors or Pre-hematopoietic Stem Cells (Pre-HSCs) Before final differentiation into HSCs, a second intermediate stage of hematopoietic precursor cells (pre-HSCs) arises from the HE. These pre-HSCs are found at various sites within the embryo, including the dorsal aorta, the vitelline and umbilical arteries, the yolk sac, and the placenta [22]. Runt-related transcription factor-1 [Runx1, also known as core-binding factor subunit alpha 2 (Cbfa2) and acute myeloid leukemia 1 protein (AML1)] is a critical factor that promotes differentiation of these hematopoietic progenitors from the HE; mutations in this gene are associated with numerous blood disorders. 3.2.1.6 Development and Differentiation of HSCs Once sites of definitive hematopoiesis have been established, HSCs will maintain themselves and also have the capacity to differentiate into hematopoietic progenitor cells (HPCs); these latter cells ultimately give rise to multipotent progenitors (MPPs) and provide the embryo/fetus with the blood cell lineages, which are essential to support rapid growth and development. While the MPPs gradually lose their self-renewal potential, they maintain their capacity to promote multipotential differentiation into adult hematopoietic [23]. 3.2.1.7 Cell Fate Choice Upon undergoing cell division, HSCs can proceed along two distinct pathways; they can undergo self-renewal to produce new HSCs or they can differentiate and produce daughter 74 M. Essawy et al. cells that have the capacity to mature into committed blood cells and cell lineages [24]. Once HSCs divide, they have the option of proceeding along one of several downstream cell fate pathways; the choice is made during the process of cell division. In this regard, symmetric division, asymmetric division, and symmetric commitment are among the possible patterns resulting from HSC division. Asymmetric division permits HSCs to balance their capacity for self-renewal with commitment and differentiation. A single HSC can give rise to two daughter cells with different functions, cell cycle kinetics, and/ or multilineage capacity using a strategy called clone splitting [25]; this mechanism generates one cell that is committed to differentiation and another that maintains the capacity for self-renewal and HSCs pool. By contrast, symmetric division of HSCs gives rise to two daughter cells of the same type and potential. In other words, symmetric division can generate either two stem cells that remain capable of self-renewal or two progenitor cells that have completed their first step toward commitment and differentiation. These strategies are both tightly controlled to achieve a critical balance between self-renewal and differentiation [26]. Whereas, symmetric commitment is an essential pathway of cell division when rapid regeneration of damaged tissue is required, as both daughter cells can generate committed hematopoietic progenitors [27]. 3.3 The Evolving Concept of the Hematopoietic Stem Cell Concepts focused on our understanding of HSCs have undergone significant evolution; HSCs were the first stem cells to be discovered, and, due to their importance with respect to treatment of blood and neoplastic diseases, these cells were the first to be used clinically through BM transplantation. As such, HSCs have been the subject of substantial interest and remain of critical importance in research programs focused on biomedical sciences and regenerative medicine. 3.3.1 Properties of HSCs 3.3.1.1 Self-Renewal HSCs undergo self-renewal to maintain the pool of undifferentiated cells throughout the life of the organism while preserving their capacity to differentiate [28]. Most of HSCs remain dormant; this serves to preserve balanced hematopoiesis and to protect the pool of HSC from succumbing to exhaustion. Only a finite number of HSCs enter the cell cycle and differentiate and mature into blood cells [29]. Several pathways are involved in promoting HSC self-renewal; we consider here the pathways that are most critical and best characterized. Among these, Notch-mediated signaling plays an important role in supporting HSC-mediated self-renewal. Activation of the Notch pathway by the ligands Delta and Jagged led to increasing HSC pool in vivo via enhancing the capacity for selfrenewal (as evaluated by sequential BM transplantation experiments) and prevented 3 Hematopoietic Stem Cells and Control of Hematopoiesis 75 differentiation in vitro [30]. Importantly, Notch signaling is also a critical mechanism underlying osteoblast-mediated support for HSCs; osteoblasts activated by parathyroid hormone expressed Jagged-1 and promoted increased capacity for self-renewal among HSCs in experiments carried out in vivo [31]. Also important is c-Myc, a transcription factor and an oncogene that has been described as a master regulator of genes involved in protein synthesis, cell cycle, and cancer metabolism [32]. Activation of c-Myc occurs downstream of both Notch and homeobox family member HoxB4 signaling; this pathway supported in vitro self-renewal of murine Lin  Sca-1 + HSCs cultured with stem cell factor (SCF), Fms-related receptor tyrosine kinase 3 (Flt3) ligand, and interleukin (IL)-6 for 28 days via upregulation of cell cycle genes (c-myc, cyclin-D2, cyclin-D3, cyclin-E, and E2F1) and increased telomerase activity [33]. The Wnt signaling pathway is also indispensable for the regulation of HSCs; forced expression of β-catenin, a core component of the Wnt signal transduction pathway, led to a 100-fold increase in the number of cultured HSCs and increased expression of both Notch1 and HoxB4 [34]. Wnt3a is an essential factor promoting self-renewal of HSCs; deficiency of Wnt3a led to irreversibly impaired hematopoiesis due to reduced numbers of HSCs and reductions in their capacity for long-term repopulation [35]. However, there are contradictory data vis à vis Wnt and its role in promoting HSC regulation; it is clear that the role of Wnt pathway in hematopoiesis is complex and will require ongoing and careful exploration. Indeed, Luis et al. [36] recently reported that different levels of Wnt activation led to different outcomes with respect to HSC regulation. Specifically, self-renewal required only limited activation of Wnt signaling, while hematopoietic differentiation resulted from intermediate levels; once levels exceeded those associated with physiologic activation, both self-renewal and differentiation were impaired. Smad-mediated signaling is another important pathway, which regulates hematopoiesis. Ligands associated with this pathway include those of the transforming growth factor-β (TGF-β) family, which includes TGF-βand bone morphogenetic proteins (BMPs) among other factors. TGF-βis a potent inhibitor of HSC growth and is considered to be an important regulator of HSC quiescence in vivo [37]. TGF-β-related inhibition is probably related to altered levels of cytokine receptor expression on HSCs together with the upregulation of cell cycle inhibitors, including p21, p27, and p57 [38–40]. By contrast, BMP-4 promoted self-renewal of cultured HSCs in vitro, while diminished levels of BMP4 levels facilitated their differentiation [41]. Fibroblast growth factor (FGF) signaling has also been implicated in the regulation of HSC development and function. Both FGF-1 and FGF-2 support long-term culture (LTC) and the repopulation potential of HSCs identified in unfractionated BM cells; however, these factors were ineffective in experiments performed with Lin  Sca-1 + c-Kit + HSCs [42]. Deletion of FGF receptor 1 (Fgfr1) had no apparent impact on steady-state hematopoiesis; however, recovery was impaired in these mice in response to BM injury with 5-fluorouracil (5FU) [43]. This research group also reported that deletion of Fgf-2 also had no impact on steady-state hematopoiesis, although this factor proved to be essential for HSC/HPC proliferation and recovery via its capacity to induce the expansion of stromal cells, increase 76 M. Essawy et al. the production of SDF-1, and suppress the expression of CXCL12 in BM [44]. Likewise, FGF-mediated signaling was essential to suppress BMP activity in the AGM region during embryogenesis to establish an HSC niche; these actions were mediated via activation of BMP antagonists noggin2 and germlin1a [45]. Taken together, current findings suggest that FGF regulates hematopoiesis and HSCs indirectly via its role in supporting BM stromal cells. Regulation of hematopoiesis by the insulin-like growth factor (IGF) pathway has also been explored; however, current findings are contradictory in nature. For example, while some studies revealed that IGF-1 functioned as a “silent killer”of pluripotent stem cells upon prolonged exposure [46], others reported that IGF-1 supports the osteoblastic niche and leads to improved levels of long-term HSC engraftment [47]. Moreover, IGF-binding protein 2 (IGFBP2) was described as an important factor serving to promote HSC survival [48]. The involvement of all these pathways provides redundancy in the process of HSCs selfrenewal, probably ensuring that if one pathway has problems, other pathways could compensate/cover up the deficiency in order to maintain lifelong normal hematopoiesis. 3.3.1.2 Asymmetric Division Asymmetric division results in two daughter cells that are not physically, molecularly, and/ or functionally identical. The fact that all mature blood cells originate from HSCs with a single phenotype led to the assumption that both HSCs and HPCs were capable of asymmetric division. This hypothesis was confirmed by the discovery of four distinct segregating proteins, including CD53, CD62L/L-selectin, CD63/lamp-3, and CD71/transferrin receptor, and their roles during mitosis of in vitro cultured CD34 + CD133 + HSCs/ HPCs [49]. Furthermore, HSCs (c-kit + Sca-1 + Lin /lo CD34  ) isolated from transgenic Notch reporter mice (wherein green fluorescent protein is highly expressed in putative HSCs and undergoes downregulation as the cells which begin to differentiate) were capable of both symmetric and asymmetric division [50]. In this context, a first-level asymmetric division occurs when HSCs choose to undergo division into two daughter cells; one of the daughter cells serves to maintain the pool of undifferentiated HSCs and the other generates a progenitor cell that is no longer capable of self-renewal and that has initiated the differentiation process (i.e., an HPC). Given that HSCs have the capacity to generate all hematopoietic lineages, other differentiated progenitors will result from differential activation by cytokines or growth factors (as will be discussed later in this chapter); these observations contribute to the second level of asymmetric division. The differentiating daughter cells will continue to grow and to undergo additional asymmetric divisions so as to generate single-potential progenitor cells; these progenitors then divide symmetrically to generate the appropriate blood cell lineage. 3 Hematopoietic Stem Cells and Control of Hematopoiesis 77 3.3.1.3 HSC Heterogeneity HSCs were the first stem cells to be isolated and characterized; they were initially considered to be a homogeneous population of cells, a perception that persisted for many years. However, due to recent technological advances, including functional assays, immunophenotyping, and genetics, this perception has changed. The HSC pool is now known to be heterogeneous. Interestingly, differences reported with respect to their capacity for in vivo repopulation and transplantation were largely due to the properties of distinct HSC subfractions; among these differences, the distinct HSC subfractions can promote differences in reconstitution kinetics, duration of repopulation, differentiation potential, cell cycle status, and the capacity for self-renewal [51–53]. As but one example, use of a flow-assisted cell sorting technique revealed differential expression of phenotypic markers associated with the signaling lymphocyte activation molecule (SLAM) family, including CD150, CD48, CD229, and CD244, in what was previously assumed to be a highly purified, homogeneous pool of Kit + Sca + lin  HSCs, also known as KSL cells. These findings led to further subdivision of what was then understood to be a heterogeneous population of KSL cells into more homogeneous HSC and HPC populations with different capacities for self-renewal and repopulation [54]. An improved understanding of HSC heterogeneity will promote the discovery of specific markers for appropriate subfractionation of HSCs; this will facilitate an improved understanding of their localization within distinct BM niches and will likewise improve the accuracy of current fate mapping and lineage-tracing approaches. Potential Factors Contributing to HSC Heterogeneity [55]: •Differences with respect to embryonic origin: During early embryonic development, both pre-HSCs and HSCs originate from distinct mesodermal and/or endothelial cells detected within sites associated with primitive hematopoiesis. •Different developmental signals: Different inductive signals could be generated at unique embryonic sites, including the yolk sac, AGM, liver, or developing placenta. Cells may respond to different signals encountered during HSC migration between the multiple embryonic sites and/or from within the circulation. •Intrinsic factors: In the absence of external stimuli, HSCs may have the capacity to control their lineage commitment and heterogeneity by upregulating or downregulating individual or groups of genes and/or receptors, thereby facilitating differential responses to external stimuli. •Microenvironmental and extrinsic factors: HSCs and their progenitors are detected in distinct locations within the adult BM; each location may be capable of activating HSCs in a different fashion, depending on the signals, factors, and stromal cell types present within the tissues. 3.3.1.4 Plasticity Plasticity is a critical feature that defines the nature of HSCs; this term implies that a stem cell can transcend its lineage boundary and give rise to different cells and tissues. The past 78 M. Essawy et al. four decades witnessed many reports of the capacity of HSCs to differentiate into cell types typically associated with other tissues, including those that are not only mesodermal but also ectodermal and endodermal in origin; these cells include the muscle, heart, brain, and liver. As such, HSCs were perceived as a feasible, ethical, and promising source of raw material, which might be used to develop cell-based therapies for various diseases [56–59]. However, the limits of HSC-associated plasticity have recently been challenged. For example, many of these studies featured cells that were not pure populations of HSCs but a mix of different cells, also, many of these studies focused only on phenotypic markers and did not include functional analyses or in vivo tracking of these cells or their progeny [60,61]. Thus, controversies remain as to whether or not HSCs possess this profound degree of flexibility. However, clearly, HSCs maintain intra-hematopoietic and/or hematopoietic lineage plasticity; in other words, it is clear that committed hematopoietic cells are able to be reprogrammed to facilitate production of blood cells from another lineage. As an example of this phenomenon, overexpression of the GATA-1 transcription factor in murine myeloid leukemia cells led to their transformation into erythroid and megakaryocyte-like cells; this is largely understood as proof of myeloid–erythroid plasticity [61], together with various other similar examples [62]. Lineage plasticity may also contribute to HSC heterogeneity as discussed in Sect. 3.3.1.3. 3.3.1.5 Migration As discussed in an earlier section, HSCs migrate from one anatomical site to another during embryogenesis until ultimately reaching sites of adult hematopoiesis; well-regulated and active hematopoiesis was maintained at each site. In mammals, HSCs first appear in the yolk sac and then migrate to the AGM region before reaching the fetal liver; as a final step, these cells take up residence in the BM. Other species feature alternative sites of lifelong active hematopoiesis; while adult hematopoiesis takes place in the long bones and the spleen of mice [63], this process takes place in the liver in frogs [64], and in the kidneys of zebrafish [65]. Even after the HSCs reach sites that maintain adult hematopoiesis, some HSCs and HPCs undergo constant migration from this niche into peripheral circulation and back. Interestingly, peripheral blood and lymph both contain twice as many HSCs/HPCs early in the morning when compared to later hours at night; these results suggest that their release is governed by a circadian rhythm [66–68]. In addition, more circulating HSCs/HPCs were identified during intense exercise [69], and secondary to acute myocardial infarctioninduced inflammation [70] and among patients with cardiovascular disease [71]. Several approaches have been used successfully to induce this migratory behavior in vivo. For example, CXCR4 receptor blockade with the selective agent, AMD 3100, led to deactivation of signaling mediated by CXCL12 (also known as stromal cell-derived factor1 or SDF-1). This blockade promoted mobilization of HSCs and HPCs from their BM niches and ultimately their release into the circulation [72]. Granulocyte colony-stimulating factor (G-CSF) also mobilizes HSCs and HPCs from their BM niche via various means 3 Hematopoietic Stem Cells and Control of Hematopoiesis 79 [73], including activation of c-kit/kit ligand (also known as SCF) and counteracting the impact of very late antigen-4 (VLA-4, also known as α4β1 integrin) and its ligand vascular cell adhesion molecule-1 (VCAM-1). G-CSF also counteracts signaling via the CXCL12/ CXCR4 axis; it serves to suppress osteoblast maturation and expression of CXCL12, leading to a state wherein HSC quiescence is maintained in the BM niche [73]. Furthermore, hypoxia was also implicated in this process; a gradient of hypoxia-inducible factor-1 (HIF-1) promoted the upregulation of CXCL12 (SDF-1) expression and the migration and homing of HSCs/HPCs into ischemic tissues [74]. Accordingly, mobilization of HSCs/ HPCs has been targeted clinically using CXCR4 antagonists, G-CSF, or erythropoietin to generate as much as a 100-fold increased yield of HSCs and HPCs from peripheral circulation to improve stem cell transplantation outcomes in clinical practice [75]. It is thus clear that “quiescent”HSCs actively migrate and return to their original niches; this raises the question as to whether HSCs “choose”their niche and/or whether their niche attracts and calls to them. This question calls for further investigation. 3.3.2 Other Sources of HSCs As HSCs have extensive migratory potential, it was plausible to consider the possibility that they might reside outside their BM niches. Indeed, HSCs and HPCs are found in both peripheral blood (PB) and umbilical cord blood (UCB) as rare populations of cells (typically 1:100,000 when defined as CD34 + CD38  CD45RA  CD90 + CD49f + Rhodamine lo ) that are capable of colony formation in vitro and long-term repopulation in vivo [76,77]. One of the earliest clues regarding the presence of HSCs and HPCs in the peripheral circulation was revealed from an experiment carried out in 1965. In this study, mice tails were shielded during whole body irradiation and the spleen was recolonized by hematopoietic cells from the tail [78]. Several subsequent studies reported successful hematopoietic recovery in response to administration of hematopoietic cells from PB in baboons, dogs, and humans [79–83]. As discussed earlier, mobilization of HSCs into the peripheral circulation is now an approved clinical practice and is used to increase the yield of HSCs for subsequent transplantation. Another important source of HSCs is UCB. The first description of the existence of HSCs at this site was in 1978 in a study that reported that myeloid forming colonies could be generated in vitro from cultured UCB cells [84]. However, important differences were reported that distinguished HSCs/HPCs isolated from UCB from those characterized in BM. Among these differences, UCB HSCs (CD34 + CD38  ) responded more effectively to hematopoietic cytokines and generated seven times as many progeny cells as did BM HSCs [85]. 80 M. Essawy et al. proliferation and also the repopulation potential of LT-HSCs (Kit + Sca + lin  CD150 + ) via activation of IFNγreceptor 1 and STAT1 [137]. Various cytokines and interleukins serve to regulate hematopoiesis [138] by acting on HSCs and other hematopoietic progenitors. In an attempt to determine the most important mediators that promote self-renewal of putative BM HSCs (CD34 + CD38  ), 16 cytokines were tested alone or in combinations, including IL-1, IL-3, IL-6, IL-7, IL-11, IL-12, TNFα, Flt3 ligand (FL), thrombopoietin (TPO), erythropoietin, G-CSF, GM-CSF, SCF, macrophage inflammatory protein lα(MIP-lα), nerve growth factor β(NGF-β), and leukemia inhibitory factor (LIF) [124]. IL-3, SCF, and FL all served to increase the capacity for selfrenewal among putative HSCs when each was used alone (the most effective was FL); the combination of three factors was even more effective. After stimulation of HSC differentiation by TPO, IL-3 was the most effective in this role when used alone or in combination with SCF, FL, and either IL-6, G-CSF, or NGF-β. TNFαhad a negative impact on the capacity of HSCs to undergo self-renewal [124]. BM HSCs (Kit + Sca + lin  Flk2  or Kit + Sca + lin  IL7Ra  ) express the pattern recognition receptors, Toll-like receptors 2 and 4. In vitro activation by their respective ligands (Pam3CSK4 and lipopolysaccharide [LPS], respectively) led to activation of the MyD88 downstream intracellular adapter protein; this ultimately led to myeloid expansion [139]. Moreover, repeated in vivo administration of small doses of LPS resulted in TLR4 activation and defective self-renewal and repopulation potential of HSCs [140]. CD34 + HSCs/HPCs isolated from human BM expressed TLR4, TLR7, TLR8, and TLR9 [141], and human UCB cells expressed TLR1, TLR2, TLR3, TLR4, and TLR6 [142]. The activation of these TLRs on isolated progenitor cells promoted myeloid differentiation. Table 3.2 Impact of various cytokines on maintaining quiescence and/or capacity for self-renewal of HSCs Hematopoietic cytokine Function on HSCs References Stem cell factor (SCF, steel factor, mast growth factor or kit ligand) Maintains and stimulates selfrenewal [124] Thrombopoietin (TPO) Maintains and stimulates selfrenewal [125] Chemokine receptor type 4 (CXCR4) Self-renewal inhibition and quiescence induction [126] Granulocyte-colony stimulating factor (G-CSF) Quiescence induction and stimulation [127,128] Angipoietin-1 (Ang-1) Self-renewal induction [129,130] Interleukin-3 (IL-3) Self-renewal and survival maintenance [131,132] Interleukin-6 (IL-6) Enhances the proliferation and differentiation [133] Fms-related receptor tyrosine kinase 3 ligand (Flt3 ligand) Stimulates the proliferation and differentiation of HSCs [134] 3 Hematopoietic Stem Cells and Control of Hematopoiesis 87 G-CSF is essential for normal granulopoiesis and functions via stimulation of the common myeloid progenitors. The absence of G-CSF limited the repopulation potential of BM cells and reduced their contributions to the myeloid lineage [143]. By contrast, enhanced G-CSF signaling promoted by a mutant G-CSF receptor was associated with higher levels of HSC proliferation via upregulation of the transcription factor, STAT5 [144]. Administration of G-CSF also led to an increased number of HSC (Kit + Sca + lin  CD34  Flk2  CD41  or Kit + Sca + lin  CD150 + CD48  CD41  cells) both in the circulation and in the BM, although it resulted in a reduced potential for repopulation. These effects were achieved via activating both TLR and G-CSF receptors; as noted earlier, TLR2, TLR4, and MyD88 signal adapter contribute to HSC expansion, loss of repopulation activity, and quiescence [145]. The role of TNF with respect to the regulation of HSCs is complex and not yet wellunderstood. In vitro, the administration of TNFαresulted in decreased proliferation and repopulation potential of putative HSCs (CD34 + CD38 /low ); these findings resulted from the activation of the p55 TNF receptor [146]. In contrast, in vivo findings remain somewhat contradictory. Interestingly, deletion of two TNF receptors (Tnfrsf1a and Tnfrsf1b, also known as p55 and p75, respectively) resulted in no changes in the numbers of HSCs (Kit + Sca + lin  Flk2  ) but yielded improved long-term repopulation potential [147]. In contrast, older mice devoid of the Tnfrsf1aor p55 receptor (but not of Tnfrsf1b or p75) showed increased numbers of erythroid and myeloid progenitors and a four-fold reduction in the repopulation potential of HSCs [148]. As such, the complex pleiotropic functions of TNF and its role in host immunity might be extended to the regulation of HSCs as well. In addition to the direct effects of these inflammatory mediators, many of them have an indirect impact on HSC regulation via actions targeting the BM environment. G-CSF acts indirectly on HSCs by suppressing CXCL12 expression in BM niche stromal cells; this leads to mobilization of HSCs into the circulation [149]. Likewise, TLR-mediated activation of freshly isolated BM CD34 + progenitors in vitro via ligands including immunestimulating siRNAs or the TLR7/8 ligand R848 led to the production of many cytokines (IL1-β, IL-6, IL8, TNFα, GM-CSF) and induced myeloid differentiation [141]. This differentiation pathway may be promoted by indirect means, via the actions of newly released cytokines in coordination with direct TLR immune-mediated signaling. Finally, the duration of exposure to inflammatory mediators and the chronicity of the associated inflammatory pathology should also be considered. Short-term inflammatory signals may be beneficial with respect to activating hematopoiesis; however, chronic inflammation can exhaust the BM and the HSC pool [135]. Prolonged inflammation may thus result in BM failure [150] and potentially malignant transformation [151]. Compelling new evidence suggests that HSCs can escape inflammatory exhaustion by re-establishing quiescence [152]. In the case of IFNs, this response involves the transcription factor IRF2 [136] and immunity-related GTPase family M protein-1 or Irgm-1 [153]; however, the full mechanisms underlying this response have yet to be identified. 88 M. Essawy et al. 3.4.3 Role of Oxygen/Hypoxia Oxygen tension has been recently proposed as a regulator of HSCs and HPCs; the BM niche wherein HSCs and HPCs reside has been described as hypoxic [154,155]. Recent studies revealed that HIF-1α, a factor that undergoes upregulation in response to hypoxic conditions, promotes the differential expression of cell proliferation and survival genes; these include IGF, cathepsin D, matrix metalloproteinase-2, urokinase plasminogen activator receptor, fibronectin-1, cytokeratin (CK)-14, CK-18, CK-19, vimentin, transforming growth factor α[156,157], vascular endothelial growth factor (VEGF) [158], and erythropoietin [159]. Administration of G-CSF resulted in stabilization of HIF-1αand increased production of VEGF in the BM [160]. HIF-1αresulted in increased levels of CXCL12 [74] and elevated levels of CXCR4 receptor expression [161]; it also protects HSCs/HPCs from damage caused by overproduction of mitochondrial reactive oxygen species [162]. 3.4.4 Role of the Nervous System The BM environment is heavily enriched with neuronal connections; as such, it has long been proposed that the nervous system may also contribute to the regulation of the HSC niche and likewise of hematopoiesis. Several β 2 -adrenergic signals were found to be essential for G-CSF-induced mobilization of HSCs and HPCs; blockade of these signals by 6-hydroxydopamine (i.e., via chemical sympathectomy) or by β-blockers such as propranolol served to reduce G-CSF-induced HSC mobilization [68]. Neurotransmitters such as norepinephrine also regulate hematopoietic cell migration via activation of Wnt signaling in CD34 + cells, by increasing Sca-1 + c-Kit + Lin  HSC mobilization [163], and by increasing the expression of both CXCR4 and VCAM-1 [164]. 3.4.5 Role of Apoptosis Apoptosis plays an important role in promoting homeostasis. B-cell lymphoma 2 (BCL-2), an anti-apoptotic protein, was overexpressed in an IL-3-dependent hematopoietic progenitor cell line, the murine hematopoietic nonleukemic factor-dependent cell Paterson (FDCP)-Mix. The transfected FDCP-Mix cells could be maintained in in vitro culture without the need for additional IL-3; cells that had not undergone transfection died via apoptosis in the absence of exogenous IL-3 [165]. Similar in vivo approach using BCL-2overexpressing transgenic mice revealed 2.4 times more HSCs in the BM when compared to HSCs/HPCs from wild-type mice. Furthermore, the HSCs from BCL-2-overexpressing transgenic mice experienced superior in vitro survival and similar in vivo engrafting potential [131]; they were also capable of survival in response to lethal irradiation [166]. Both the in vitro and in vivo approaches suggested a role for apoptosis in regulating the survival of HSCs/HPCs, although conclusive evidence is still needed. 3 Hematopoietic Stem Cells and Control of Hematopoiesis 89 3.5 The Hematopoietic Hierarchy The differentiation of HSCs to mature myeloid and lymphoid cells occurs in a stepwise fashion beginning with multipotent, oligopotent, and bipotent cells and ending with fully differentiated cells; this pathway forms the classical hierarchical tree of hematopoiesis [167]. LT-HSCs are at the top of this hierarchy and represent a very small percent (up to 0.2%) of the entire BM cell pool [168], HSCs gradually lose their capacity for self-renewal (ST-HSCs) and become more and more restricted with respect to their differentiation potential. This tree eventually ends with functionally mature blood cells, as shown in Fig. 3.3. However, current thinking suggests that the hematopoietic system developed in association with mammalian evolution; as such, it will be difficult to constrict our current understanding within the classical organization or hierarchical framework. Moreover, the fact that self-renewing HSCs along with other committed progenitors comprise a large part of the hematopoietic cell pool defies the idea of a simple hematopoietic hierarchy. Importantly, recent evidence suggests that several committed single-lineage progenitors were derived directly from multipotent HSCs; these observations highlight the fact that HSCs have the capacity to produce blood cells in a flexible yet efficient manner [169,170]. In newer hierarchical models, HSCs do not remain at the top of the hierarchy, but play an overall more dynamic roles toward the goal of supporting normal lifelong hematopoiesis [171]. 3.6 Epigenetic Control Over HSCs Epigenetics does not only play an important role during early development, but is also essential for tissue homeostasis. The self-renewal or differentiation of HSCs depends on different gene expression patterns, which are, in part, the result of epigenetic changes that expose or conceal different genomic regions. Consequently, different chromatin-modifying proteins, such as Polycomb-group (PcG) and Trithorax-group (TrxG) proteins, were recently considered critical epigenetic regulators of HSC self-renewal and differentiation. Of the PcGgroup, Polycomb complex protein 1 (Bmi-1) [116], Enhancer of zeste homolog 1 (Ezh1) [172] and Ezh2 [173] were shown to promote self-renewal of HSCs by suppressing cell cycle inhibitors; and thus preventing cell cycle arrest, senescence, and apoptosis. While Chromobox protein homolog 7 (Cbx7) [174] maintained self-renewal via suppressing the expression of lineage-specific genes. Of the TrxG proteins, Mixed Lineage Leukemia (MLL or Histone-lysine N-methyltransferase 2A) was essential for HSC selfrenewal and repopulation potential [175], and SET domain-containing protein 1A (SET1A or Histone-lysine N-methyltransferase SETD1A) was shown to protect HSC self-renewal during stress conditions via activating DNA damage recognition and repair pathways [176]. In addition, marked epigenetic differences were found in aged HSCs contributing 90 M. Essawy et al. to their lower differentiation and repopulation potential [177]. Epigenetic modifiers that play an important role in HSCs self-renewal or differentiation are described in Table 3.3. The applicability of epigenetics was achieved by altering the chromatin structure of in vitro cultured HSCs. A mixture of 5-aza-20-deoxycytidine (5aza, DNA methyltransferases inhibitor) and trichostatin A (TSA, histone deacetylase inhibitor) led to increasing putative BM-HSCs (CD34 + ) self-renewal and repopulation potential [191]. In addition, valproic acid (histone deacetylase inhibitor) enhanced the expansion of in vitro cultured putative HSCs (CD34 + cells) from BM, BP, or UCB [192]. 3.7 Bone Marrow Transplantation (BMT) The first experimental evidence of the stem cell theory was demonstrated by Ernest A. McCulloch and James E. Till when they performed BM transplantation into irradiated mice [97,193]. Myeloid multilineage colonies were produced in the spleen of the transplanted mice from these cells where the number of injected cells being proportional to the number of colonies. The multilineage potential of single bone marrow cells (the so-called CFU-S, Colony-Forming Unit in the Spleen) was confirmed by such experiments [98]. Nevertheless, these cells are not identified as true stem cells with a multipotent potential and selfrenewal capability, which in that case was limited. Henceforth, the first successful stem cell transplantation was performed by E. Donnall Thomas on identical human twins in 1957 [194]. After this transplantation, the long-term repopulation with the production of new blood cells was confirmed to be as a result of intravenous injection of bone marrow cells. Moreover, transplantations were performed on Yugoslavian nuclear workers (whose bone marrows were injured by irradiation) by the oncologist Georges Mathé [195] who also performed successful allogeneic bone marrow transplantation on a leukemic patient [196]. For more than 50 years, patients with blood-related disorders have been treated with such transplantations. Adult HSCs can now be exceedingly enhanced with a mixture of numerous surface markers. Transplantation protocols in the case of many blood-related diseases, such as leukemia, include different sources of HSCs such as bone marrow, cord blood, or mobilized peripheral HSCs. However, major obstacles include the low number of HSCs in these tissues. Furthermore, reproducing the reported in vitro conditions and permitting proficient HSC expansion without prompting cell differentiation are still very complicated [197]. Use of cord blood as a source of HSCs [198,199] and new regimes which allowed haploidentical transplantation [200] further facilitated current therapeutic approaches while limiting the undesired consequence of graft-versus-host disease. These approaches are increasingly making the option of allogeneic transplantation available to patients who otherwise do not have a matched-related or volunteer-unrelated donor source of stem cells as shown in Fig. 3.4. 3 Hematopoietic Stem Cells and Control of Hematopoiesis 91 Table 3.3 Epigenetic modifiers that regulate HSCs self-renewal or differentiation Protein [other names] Gene Effect on HSC References DNA (cytosine-5)- methyltransferase 1 [Dnmt1, DNA methyltransferase HsaI or DNA MTase HsaI] DNMT1 Required for HSCs self-renewal, niche retention and progression from multipotent to myeloid progenitors. Deletion leads to pedigree skewing into myelopoiesis and defective selfrenewal [178,179] DNA (cytosine-5)- methyltransferase 3 (A and B) [Dnmt3a/b, DNA methyltransferase HsaIIIA/B or DNA MTase HsaIII A/B] DNMT3 (A and B) Essential for HSCs self-renewal, Dnmt3a deletion increases HSCs life span [180,181] Methylcytosine dioxygenase TET1 [Ten-eleven translocation 1 gene protein] TET (1 and 2) TET1 deficiency increases HSCs selfrenewal potential TET2 deletion results in improving HSCs self-renewal and improving myelopoiesis [182,183] Isocitrate dehydrogenase [NADP] cytoplasmic (IDH 1) or mitochondrial (IDH2) [Cytosolic or mitochondrial NADP-isocitrate dehydrogenase] IDH (1 and 2) Required for TET2 cofactors [184] Polycomb complex protein 1 (Bmi-1) BMI1 Important for HSCs self-renewal [116] Histone-lysine Nmethyltransferase EZH (1 and 2) [Enhancer of zeste homolog 1 and 2, Ezh1 and 2] EZH (1 and 2) Ezh1 important for HSCs selfrenewal and prevents senescene Ezh2 preserves self-renewal and prevents exhasution of HSCs [172,173] Chromobox protein homolog 7 [Cbx7] CBX7 Imporatant for self-renewal of HSCs [174] Chromobox protein homolog 2, 4 and 8 [Cbx2 Cbx4 and Cbx8] CBX2, CBX4 and CBX8 Overexpression leads to differentiation and exhaustion of HSCs [174] Histone-lysine Nmethyltransferase SETD1A (SET1A or SETD1A) SETD1A Protects HSCs self-renewal during stress [176] Histone-lysine Nmethyltransferase 2A [Mixed Lineage Leukemia, MLL, MLL1 or Trithorax-like protein] KMT2A Essential for HSCs self-renewal and repopulation potential [175,185] (continued) 92 M. Essawy et al. Table 3.3 (continued) Protein [other names] Gene Effect on HSC References Histone-lysine Nmethyltransferase, H3 lysine79 specific [DOT1-like protein, Histone H3-K79 methyltransferase] DOT1L Important for embryonic erythropoiesis and maintenance of adult populations of HSCs and HPCs [186,187] Histone H2A deubiquitinase MYSM1 [Mysm1, 2A-DUB, MPN domain-containing protein 1] MYSM1 Involved in HSCs quiescence and self-renewal [188] Histone-lysine Nmethyltransferase SETDB1 [H3-K9-HMTase 4, ESET, SET domain bifurcated 1] SETDB1 Important for HSCs function [189] Polycomb-group protein ASXL1 ASXL1 Associated with polycomb chromatin-binding protein Loss results in reduced self-renewal and impaired hematopoiesis [190] Ex Vivo Expansion HLAmatched Cord Blood Haploidentical Donor Stem Cells Lin-CD34+CD38+ Fig. 3.4 Human HSC transplantation therapy. HLAmatched adult, cord blood or haploidentical adult donor stem and progenitor cells (usually CD34 + enriched cells) are transplanted intravenously following conditioning therapy to permit engraftment of donor marrow into the recipient 3 Hematopoietic Stem Cells and Control of Hematopoiesis 93 3.7.1 Diseases Currently Treated by HSCs 3.7.1.1 Multiple Myeloma According to the Center for International Blood and Marrow Transplant Research (CIBMTR), the majority of hematopoietic stem cell transplants are autologous. Overall survival and progression free survival were amplified in patients younger than 65 years old on a protocol of initial consolidation therapy with melphalan followed by autologous stem cell transplantation and lenalidomide maintenance therapy [201]. Administration of highdose of melphalan plus stem cell transplantation demonstrated a favorable outcome compared with consolidation therapy with melphalan, prednisone, lenalidomide (MPR), and it also showed a better outcome in patients who received a maintenance therapy with lenalidomide. 3.7.1.2 Hodgkin and Non-Hodgkin Lymphoma In cases of recurrent lymphomas (HL and NHL) that showed no response to initial conventional chemotherapy, using a protocol in which chemotherapy was followed by autologous SCT showed favorable outcome. Schmitz and colleagues demonstrated, in a randomized controlled trial, that a high-dose chemotherapy with autologous SCT resulted in better 3-year outcome compared to aggressive conventional chemotherapy in relapsed chemo-sensitive Hodgkin lymphoma [202]. However, there was not a significant difference between the two groups in overall survival. According to CIBMTR, the number of HSC transplant recipients comes second after multiple myeloma. 3.7.1.3 Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) In patients with AML who fail primary induction therapy and do not achieve complete response, allogeneic SCT could improve outcome and prolong overall survival [203]. The study recommended that early HLA typing for patients with AML could help if they fail induction therapy and are considered for BMT. Allogenic stem cell transplant is considered being curative in cases of disease progression and is only indicated in intermediateor highrisk patients with MDS. 3.7.1.4 Acute Lymphocytic Leukemia (ALL) Allogeneic SCT is indicated in refractory and resistant ALL cases when induction therapy fails for a second time in inducing remission. Some studies suggest an increased benefitof allogeneic HSC transplant in patients with high-risk ALL including patients with Philadelphia chromosome and those with t(4, 11) chromosomal translocation [204]. 3.7.1.5 Chronic Myeloid Leukemia/Chronic Lymphocytic Leukemia Combining hematopoietic SCT with available treatments like tyrosine kinase inhibitors has shown high cure rates with low adverse risk profile. SCT is reserved for patients with the refractory disease to first-line agents in CML. 94 M. Essawy et al. 3.7.1.6 Myelofibrosis, Essential Thrombocytosis, and Polycythemia Vera Allogenic SCT demonstrated an improvement in outcomes in patients with myelofibrosis and those diagnosed with myelofibrosis preceded by essential thrombocytosis and polycythemia vera [205]. 3.7.1.7 Solid Tumors Autologous SCT is considered the standard of care in patients with germ cell tumor (testicular tumors) that are refractory to chemotherapy (after the third recurrence with chemotherapy). HSCT has shown promising outcomes in cases of medulloblastoma, metastatic breast cancer, and other solid tumors [206]. 3.7.2 Complications of HSCT Most of the grafts used for HSCT are either whole bone marrow or sorted CD34 + stem and progenitor cells. In both cases, the contamination of HSCs with other CD34 + nonhematopoietic cells, or even tumor cells, leads to higher incidence of graft-versus-host disease (GVHD) and less graft-versus-leukemia (GVL) effects following allogeneic transplantation [207]. On the other hand, the use of very pure HSCs populations was effective with less GVHD [208,209], however, it is rarely used in clinical practice, as this implies more labor and importantly costs. 3.8 The Future Research defining the nature and regulation of HSCs has permitted the manipulation of hematopoiesis regulators in ways that have revolutionized the current treatment options for blood disorders and the use of stem cell transplants. A deeper understanding of HSCs selfrenewal and differentiation mechanisms, the cell-fate choices, and intrinsic/extrinsic regulators of HSCs is still missing. Novel outcomes from ongoing HSC research continue to redefine and refine our knowledge and provide a venue for endless improvements in HSC based clinical therapeutics. This includes improvements in HSCs isolation, labeling and sorting, in vivo imaging, together with recent microfluidics, organ-on-chip and omics approaches. For example, improved approaches that use gene-editing of HSCs to facilitate the transplantation of “corrected”allogeneic/syngeneic cells, thus achieving personalized therapy/medicine as shown in Fig. 3.5. However, important challenges remain, which include, developing robust methods to maintain HSCs in vitro (mimicking their in vivo niche) both to accelerate ongoing research and to increase cell numbers for large-scale therapeutics [210]. 3 Hematopoietic Stem Cells and Control of Hematopoiesis 95 Take Home Messages •Hematopoietic stem cells (HSCs) are pluripotent cells responsible for producing all blood cell types via the process of hematopoiesis. •Hemangioblast is an embryonic stem cell that gives rise to blood vessels and universal blood stem cells (which give myeloid and lymphoid precursors). –Myeloid precursors form several types of differentiated cells including red blood cells (erythrocytes), platelets (megakaryocytes), mast cells, and myeloblasts (basophils, neutrophils, eosinophils, monocytes). –Lymphoid precursors form natural killer cells and lymphocytes (B and T lymphocytes). •Once HSCs divide, they have the option of entering any downstream cell fate pathway giving different blood cell types as needed. 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Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells, and Pericytes 4 Azza M. El-Derby, Toka A. Ahmed, Abeer M. Abd El-Hameed, Hoda Elkhenany, Shams M. Saad, and Nagwa El-Badri Contents 4.1 Adult Stem Cells (ASCs) ..................................................................... 112 4.2 Mesenchymal Stromal Cells .................................................................. 112 4.2.1 MSCs: Sources and Origin ........................................................... 112 4.2.2 Characterization of MSCs ............................................................ 115 4.2.3 Biological Functions of MSCs ....................................................... 117 4.3 Endothelial Progenitor Stem Cells ........................................................... 121 4.3.1 History, Definition, and Origin ....................................................... 121 4.3.2 EPCs Characterization ................................................................ 122 4.3.3 Action Mechanism .................................................................... 123 4.3.4 Clinical Applications ................................................................. 124 A. M. El-Derby · T. A. Ahmed · S. M. Saad · N. El-Badri (*) Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Helmy Institute of Biomedical Sciences, Zewail City of Science and Technology, Giza, Egypt e-mail: [email protected];[email protected]; [email protected];[email protected] A. M. Abd El-Hameed Faculty of Science, Taibah University, Medina, Saudi Arabia e-mail: [email protected] H. Elkhenany Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Helmy Institute of Biomedical Sciences, Zewail City of Science and Technology, Giza, Egypt Department of Surgery, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt e-mail: [email protected] #Springer Nature Switzerland AG 2020 N. El-Badri (ed.), Regenerative Medicine and Stem Cell Biology, Learning Materials in Biosciences, https://doi.org/10.1007/978-3-030-55359-3_4 109 4.4 Pericytes: Biological Characteristics and Physiological Roles .............................. 126 4.4.1 Pericyte Discovery and Location ..................................................... 126 4.4.2 Pericyte Ultrastructure, Characterization, and Origin ............................... 127 4.4.3 Pericytes Physiological Roles ........................................................ 128 References ........................................................................................... 131 Abbreviations AD-MSCs Adipose-derived MSCs AFP Alpha-fetoprotein ang-1 Angiopoietin 1 ANG-2 Angiopoietin-2 ASCs Adult stem cells BDNF Brain-derived neurotrophic factor bFGF Basic fibroblast growth factor BM Bone marrow BM-MSCs Bone marrow-derived MSCs BMP4 Bone morphogenetic protein 4 BPD Developing bronchopulmonary disease CCL-2 (C-C motif) ligand 2 CFU-F Colony-forming unites-fibroblast CNS Nervous system CVD Cardiovascular disease CXCL12 C-X-C motif chemokine 12 DMEM Dulbecco’s Modified Eagle’s medium ECM Extracellular matrix ECs Endothelial cells ECs Endothelial cells eEPCs Early EPCs EMT Epithelial to mesenchymal transition EOC Endothelial outgrowth cells EPCs Endothelial progenitor cells EPO Erythropoietin ESCs Embryonic stem cells FGF Fibroblast growth factor G-CSF Granulocyte-colony stimulating factor GM-CSF Granulocyte-macrophage–colony-stimulating factor HIF Hypoxia-inducible factor HSCs Hematopoietic stem cells HSCs Hepatic stellate cells IDO) Indoleamine 2,3-dioxygenase 110 A. M. El-Derby et al. IGF-1 Insulin-like growth factor-1 IL-(num.) Interleukin (num.) INFInterferon gamma ISCT Society for cellular therapy KDR Kinase insert domain receptor M-CSF Erythropoietin, macrophage colony-stimulating factor MMPs Metalloproteases MNCs Mononuclear cells MSCs Mesenchymal stromal cells/Mesenchymal stem cells NG2 Neural/glial antigen 2 NO Nitric Oxide OECs Outgrowth endothelial cells OPN Osteopontin P-(num.) Cell passage number PAH Pulmonary arterial hypertension PCs Pericytes PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor PDT Population doubling time PGE2 Paracrine factors such as Prostaglandin E2 PSGL-1 Glycoprotein ligand-1 ROCK Rho Kinases ROS Reactive oxygen species SA-β-Gal Senescence-associated beta-galactosidase SDF-1 Stromal-derived factor-1 TACT Therapeutic angiogenesis by cell transplantation TGF-β1 Transforming growth factor β1 TGF-βTransforming growth factor beta TNF-αTumor necrosis factor alpha TSG-6 TNF-stimulated gene 6 UC-MSC Umbilical cord -derived MSCs VEGF Vascular endothelial growth factor α-SMA α-smooth muscle actin What You Will Learn in This Chapter In this chapter, you will learn the origin, characteristics, and function of adult stem cells, and the difference between adult stem cells and their embryonic counterparts. Adult stem cells play an important role in maintaining homeostasis, tissue repair, healing, and regeneration. They have become a favorite source for extensive experimentations and clinical trials because of their unique biological and functional criteria, and practical isolation and culture methods. The chapter focuses on mesenchymal stromal cells, endothelial progenitor cells, and pericytes in terms of their biology and functional properties. 4 Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells ... 111 4.1 Adult Stem Cells (ASCs) Adult stem cells (ASCs) are multipotent somatic cells in an undifferentiated state, representing a small percentage of cells within adult specialized mammalian tissues [1– 3]. ASCs have been detected in almost all tissues including the bone marrow, liver, teeth, testes, ovaries, gut, heart, brain, and skeletal muscle. ASCs are responsible for tissue repair, regeneration, and homeostasis. ASCs reside quiescently in niches that support them structurally and maintain them in an undifferentiated state [3,4]. When activated by intrinsic or extrinsic signals, such as those elicited by cell injury or cell loss, ASCs become activated and undergo asymmetric division [5]. The first cell of the progeny is lineagecommitted and can proliferate and differentiate into a specialized cell as their native origin. The second daughter cell remains undifferentiated to support the long-term maintenance of the stem cell pool [3,6]. In many tissues, ASCs do not directly differentiate into fully specialized cells, but differentiate into intermediate, partially differentiated progenitor cells. Progenitor cells in turn differentiate into more lineage-committed progenitors, or terminally differentiated, fully specialized cells [7–9]. Compared with embryonic stem cells, ASCs can only give rise to a more limited array of differentiated cell types. Figure 4.1 summarizes the main differences between ASCs and their embryonic counterparts. ASCs can be extracted from most tissues in the body, including bone marrow, fat, and peripheral blood. In this chapter, we will focus on three important types of ASCs: mesenchymal stromal cell, endothelial progenitor cells, and pericytes. 4.2 Mesenchymal Stromal Cells Mesenchymal stromal cells (MSCs) are multipotent mesodermal cells that were first described by Alexander Friedenstein [10], as a sub-population within the bone marrow [11]. MSCs are characterized by their fibroblast-like spindles and adherence to plastic. They form fibroblast-like colonies (colony-forming unites-fibroblasts, CFU-F), when cultured at low seeding density, under standard culture conditions [12,13]. CFU-Fs acquire the characteristics of endothelial cells (ECs) when grown under endothelial culture conditions [14]. MSCs characteristically reside in the perivascular niche, which enables them to be more dynamic and easily migrate within the circulatory system toward injured tissues for maintenance and repair. They also migrate via the lymphatic system and thus play a role in repair during inflammation [15–18]. 4.2.1 MSCs: Sources and Origin MSCs reside in almost all organs and are considered a strategic store for the repair or replacement of degenerated tissues [19] (Fig. 4.2). MSCs are commonly isolated for experimental purposes from the bone marrow [20,21] and adipose tissue [22]. The sternum and the iliac crest are the main sources of bone marrow aspirates for stem cell collection 112 A. M. El-Derby et al. HLA-Class II antigens [128,129]. Moreover, the secretome of the MSCs contains myriad anti-inflammatory factors, such as IL-10, and TGF-β[130–133]. MSCs were also reported to affect the innate and adaptive immune system. For example, the co-culturing of MSCs with T-lymphocytes induced T-lymphocytes apoptosis. This action is regarded as one mechanism by which MSCs exert their immunosuppressive potential [134,135]. The immunosuppressive action of MSCs could be achieved also via many other mechanisms, including the recruitment of immune suppressive cells such as IL-10-producing dendritic cells, B cells, as well as CD4 + CD25 + FOXP3 + T regulatory cells. Furthermore, MSCs can suppress macrophage-released IL-6 and TNF-αvia PGE2 and indoleamine 2,3dioxygenase (IDO) secretion [136,137]. The multilevel immunosuppressive action of MSCs makes them suitable for ameliorating and overcoming the immune rejection that is experienced after solid organ transplantation [138,139]. 4.2.3.5 Multipotency and Differentiation MSCs are multipotent cells that differentiate into lineages such as osteoblasts, chondrocytes, adipocytes, myocytes, as well as other cell lineages. The in vitro differentiation of MSCs into adipogenic, osteogenic, and chondrogenic cells is routinely used for the identification of human multipotent MSCs. Furthermore, the ability of MSCs to differentiate in vitro into ECs [140], vascular smooth muscle [141], and myocytes [142] has also been reported. MSCs could be induced to differentiate in vitro into adipocytes when cultured in a medium supplemented with indomethacin, dexamethasone, insulin, and 1methyl-3isobutylmethylxanthine. The Wnt/B-catenin signaling pathway was found to be highly active to induce the commitment of MSCs toward pre-adipocyte formation during the early stages of differentiation. However, this signaling pathway is turned off later in the differentiation process to allow for the maturation of the adipocytes [1,143]. This differentiation could be assessed by measuring the levels of the resultant adipocyte-specific markers, including enzymes such as PPAR-γand the lipoprotein lipase enzyme [144]. Furthermore, the appearance of fat droplets is a significant indicator of the successful adipogenic differentiation process [145,146]. To induce chondrogenic differentiation, MSCs are cultured in a medium that includes TGF-βIII, linoleic acid, transferrin, insulin, selenium acid, ascorbic phosphate, dexamethasone, and pyruvate [147–149]. BMP-2 and TGF-β1 were also used to enhance chondrogenic differentiation. Chondrogenic differentiation can be assessed by measuring the levels of released collagen type II and other proteoglycans through immunohistochemical staining [150]. Osteogenic differentiation is enhanced by treating MSCs with ascorbic acid, B-glycerophosphate, and dexamethasone, resulting in osteoblast formation. Osteoblasts can be detected by measuring the levels of alkaline phosphatase and mineralized calcium deposits in the cells [151]. FGF, PDGF, and TGF-βare a set of key regulators in MSC differentiation, whose modulation, up-regulation, or inhibition could diminish cell proliferation. For example, the downregulation of TGF-βwas found to be linked to increased adipogenic and osteogenic differentiation, while blocking chondrogenic differentiation. Additionally, PDGF 4 Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells ... 119 inhibition and the diminished expression of FGF receptors were found to be related to lower osteogenic differentiation and the inhibition of osteogenic differentiation potential [147]. The differentiation of MSCs into multiple cell types of mesodermal and endodermal origin has been described. Inducing the differentiation of MSCs into hepatocytes could be achieved in two stages. First, MSCs were cultured in IMDM supplemented with nicotinamide, basic fibroblast growth factor (bFGF), and hepatic growth factor (HGF). Then, transferrin, oncostatin M, insulin, dexamethasone, and selenium were added [152,153]. By the end of the differentiation process, the resultant hepatocytes can be characterized by measuring the release of unique liver proteins such as albumin and alpha-fetoprotein (AFP). The differentiation of MSCs into a cholinergic nerve [154], myocytes [142], pancreatic β-cell-like cells [155], and insulin-producing cells [156] has also been reported. Mesenchymal Stem Cell or Mesenchymal Stromal Cell? In 2005, a statement by the International Society for Cell and Gene Therapy (ISCT) stipulated that the terms “mesenchymal stem cells”and “mesenchymal stromal cells” are not equivalent, and cannot be used interchangeably, as they represent two different cell populations [157]. According to the statement, one of the main differences is that mesenchymal stem cells constitute a population that shows progenitor properties in terms of differentiation and self-renewal [115,158]. However, the stromal counterpart refers to a bulk heterogeneous population that includes fibroblasts, myofibroblasts, and a small population of stem/progenitor cells [159, 160], but does not include hematopoietic or endothelial cells. The heterogeneity of mesenchymal stromal cells makes them demonstrate specific homing [161], secretory, and immunomodulatory criteria [162] that are more relevant to MSC-based clinical therapies [163]. The overlap between the two terms could be attributed to the use of the “MSCs” acronym, which can be expanded to imply mesenchymal stromal cells, mesenchymal stem cells, multipotent stem cells, and medicinal signaling cells. However, the ISCT recommends the use of the MSCs acronym for mesenchymal stem cells because it has been used for decades. The ISCT defines MSCs through the following minimal criteria: their adherence to plastic, the expression of CD73, CD90, and CD105, the lack of expression of the hematopoietic and endothelial markers CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-DR, and in vitro adipogenic, chondrogenic, and osteogenic differentiation potential [164]. Later on, in 2019, the ISCT issued a new statement that the previous minimal MSCs criteria are not definitive (164). For example, the lack of CD34 expression was typically used as one of MSCs’defining criteria; however, various reports demonstrated that CD34 expression widely (continued) 120 A. M. El-Derby et al. depends upon cell source and passage, and they stated that MSCs tend to be more CD34 + under in vivo compared to in vitro conditions [165,166]. The ISCT MSC committee recommended that the MSC acronym remains in use, but it should be coupled with the tissue of origin like BM-MSCs for bone marrow origin, AD-MSCs for adipose tissue origin, and UC-MSCs for cells originating from the umbilical cord, because MSCs from different tissues exhibit varied phenotypes, functions, and secretomes [71,167,168]. The MSC committee also recommended that the use of the MSC acronym should be annotated with functional definitions. Furthermore, the term mesenchymal stem cell should not be used without solid functional in vivo and in vitro evidence to prove the self-renewal and differentiation potential. Indeed, they see that CFU-F progenitor assays and in vitro tri-lineage differentiation assays are indications for the progenitor status but are not sufficient to demonstrate the self-renewal capacity of mesenchymal stem cells in the absence of in vivo data [159]. As for the mesenchymal stromal cell, the committee recommended the evaluation of their trophic factors secretion [113,169], their modulatory effect on immune cells [170–172], and other relevant criteria such as angiogenesis modulation [173–176]toreflect the multimodal properties of the mesenchymal stromal cell heterogeneous population. They have published an article [162] that discusses the immune assays for the assessment of mesenchymal stromal cells, and recommended that assays should include quantitative RNA analyses of selected genes, flow cytometry of cell surface markers, protein analysis of the MSC secretome, and the characterization of exosomes and/or microRNA [177–180]. 4.3 Endothelial Progenitor Stem Cells 4.3.1 History, Definition, and Origin EPCs constitute multiple cell types that can differentiate into mature ECs. Unlike other progenitor cells, EPCs share some common features with stem cells such as clonogenicity, self-renewability, and differentiation potential [181,182]. EPCs were first isolated in 1997 by Asahara et al., from human peripheral blood by a molecular isolation technique in which surface-antigen magnetic beads were used to isolate specific peripheral blood mononuclear cells (PBMC CD34+ or PBMC Flk1+ cells) on fibronectin culture plates [181]. More recently, several studies on harvesting EPCs from different sources used either direct isolation from human bone marrow (HBM), human umbilical cord blood (UCB), or human peripheral blood (PB), or indirectly by transdifferentiation form other somatic cells such as neural, dental, cardiac, or adipose tissue [115,183–188]. 4 Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells ... 121 4.3.2 EPCs Characterization EPCs share many common cell surface markers with HSCs, in addition to numerous common genes affecting both hematopoietic and endothelial cell development. It was thus suggested that HSCs and EPCs originate from a common precursor, the hemangioblast [189–191]. Surface markers used to isolate and characterize EPCs include CD34, CD146, CD45, CD115, CD14, CD133, VEGFR1, VEGFR2 (or KDR) [115]. EPC’s phenotype differs based on its source. For example, CD133 + and CD34 + EPCs cells isolated from UCB were higher in number than those isolated from adult PB [192,193]. Other studies showed that cells expressing CD34 or VEGFR-2 markers generated the most mature ECs [194]. Importantly, EPCs have been described as a population of circulating CD34 + cells that can differentiate ex vivo into cells with endothelial cell-like characteristics [195]. Various studies have reported that EPCs are heterogeneous populations comprising multiple subpopulations. EPCs can differentiate into two different subpopulations; early EPCs (eEPCs) similar to EPCs identified by Asahara and et al. and late EPCs known as outgrowth ECs [196–198]. Both types have different features and biological properties that are summarized in (Table 4.2). Table 4.2 Differences between early and Late EPCs “Early”EPCs “Late”EPCs Nomenclature –Early EPCs (eEPCs) –Pro-angiogenic circulating hematopoietic stem/progenitor cells –Endothelial colony-forming cells (ECFCs) –“Late”EPCs –Endothelial outgrowth cells (EOC) Lifespan [199] Short lifespan up to 3 to 4 weeks [181,200,201] Long lifespan and rapid proliferation [14,196,202] Proliferation [197] Minimal proliferative capacity –Significantly higher proliferative potential reaching 28 population doublings (PDs) in 40 days with a doubling time of approximately 34 hours. Colony formation [198] –Colonies are produced in 4–6 days after the initial seeding of mononuclear cells –Colonies are characterized by discrete cell aggregates –Colonies are produced 3–4 weeks after seeding [197] Immunophenotype [197,199] CD45 (+) CD31 (+) CD105 (+/) CD146 (+/) CD14 (+) CD34 (+) CD117 (+) CD45 () CD31 (+++) CD105 (++) CD146 (++) CD14 ()[203] CD34 (++) CD117 (++) CRLR/RAMP-2 (AM1) [204] (continued) 122 A. M. El-Derby et al. 4.3.3 Action Mechanism The formation of new blood vessels by EPCs necessitates their mobilization, migration, adhesion, and differentiation. In case of vascular occlusion, EPCs have been shown to sense altered (low or oscillatory) shear stress, and as a result increase the expression of prooxidant enzymes, which are mediated principally by the transcription factor, NF-κB[208]. Hypoxia can be sensed by ECs in several ways, most notably by the hypoxia-inducible factor and nitric oxide (NO). They both mediate the activation of several signaling Table 4.2 (continued) “Early”EPCs “Late”EPCs Morphology [198, 205] –Appear within 4 to 7 days of culture with spindle-like morphology; have limited proliferation potential –Develop after 2 to 3 weeks of culture with a cobblestone appearance [158] Differentiation [199] –Heterogeneous cells that are differentiated from hemangioblasts –Early EPC can differentiate into late EPCs [206] –Homogeneous and welldifferentiated cells –Considered to be mature endothelial cells –Differ from mature endothelial cells in terms of proliferation rate and cell senescence –OECs are committed to an endothelial lineage [197] Gene Expression Profile (200) –von Willebrand factor (vWF) is not expressed –VEGFR-2 (+) –Express von Willebrand factor (vWF) –VEGFR-2 (++) In vitro Function [199] –KDR (+) –NO (+) –VE-cadherin (+) –Lack tube-forming capacity [197] –KDR (++) –NO (++) –VE-cadherin (++) –Higher tube formation efficiency –Higher angiogenic properties in vitro [207] In vivo Function [199] –Contribute to neovasculogenesis primarily by secreting the angiogenic cytokines that help recruit resident mature endothelial cells and induce their proliferation and survival –No significant difference in contribution to neovasculogenesis in the ischemic limb –A limited degree of engraftment and incorporation into new vessels from early EPCs [203] –Enhance neovasculogenesis by providing a sufficient number of endothelial cells based on their high proliferation potency –No significant difference in contribution to neovasculogenesis in the ischemic limb –Higher capacity to form de novo vessels in vivo [203] (+) positive, (+/) positive or negative, (++) higher, (+++) significantly high 4 Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells ... 123 pathways, which powerfully orchestrates the cellular response to low oxygen levels when activated. As a result, different growth factors, cytokines, and chemokines are released, mediating EPC mobilization from the BM [209,210]. These factors include (VEGF), fibroblast growth factor (FGF-2), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and granulocyte-colony-stimulating factor (G-CSF), as well as angiopoietins [211]. VEGF appears to induce a fast EPCs mobilization from the BM, a phenomenon which has been described in burn patients [212]. However, EPCs were found to have the ability to release VEGF after homing, and generate a local angiogenic response [213]. There are various isoforms of VEGF including VEGF-B, VEGF-C, VEGF-D, but it remains unclear whether there are differences in their effect on EPC regulation. Other factors such as erythropoietin (EPO) can also mobilize EPCs [214]. Granulocyte-macrophage-colony-stimulating factor (GM-CSF) and its related cytokine, granulocyte-colonystimulating factor (G-CSF), both display mobilizing activity, although they are less potent than VEGF or SDF-1 [215]. The adhesion of EPCs to an injured vessel wall is crucial. This occurs through the interaction of the glycoprotein ligand-1 (PSGL-1) expressed on EPCs with the P-selectin expressed on platelets [213]. EPCs play an important physiological function by acting as the main reservoir of ECs, due to their ability to move into the injury site to preserve the integrity of the endothelium [216]. The contribution of EPCs to vascularization has been demonstrated in animal models and humans [213]. Additionally, the reduction in the number of circulating EPCs and/or alterations in their functions associated with various factors might have a marked impact on endothelium function as well as cardiovascular disease (CVD) onset, complications, and consequently in the survival of individuals with CVD [217]. 4.3.4 Clinical Applications EPCs-based therapy is considered to be a promising endothelial regeneration for several diseases including cardiovascular failure, chronic renal failure, pulmonary diseases, in addition to ischemia related conditions and connective tissue disorders [215,218]. They also play an important role in tissue engineering by their ability to vascularize engineered tissues, which could be useful for personalized medicine [219]. EPCs are utilized for multiple applications because they could differentiate into both continuous and discontinuous capillaries in the liver and skeletal muscles [219–221]. They could also outperform a vascular-derived endothelium in vascular network formation and possess a comparable permeability to the endothelium vessels [222–229]. The contribution of EPCs to vascularization has been demonstrated in animal models and humans [213]. Additionally, the number of circulating EPCs and/or alterations in their functions associated with various factors might have a marked impact on endothelium function and CVD onset, complications, and consequently in the survival of individuals with CVD [217]. 124 A. M. El-Derby et al. 4.3.4.1 EPCs as a Biomarker Studies have shown that the number and function of circulating EPCs can act as biological markers for vascular function and cumulative cardiovascular risk [216,230,231]. The number of EPCs varies depending on the disease. For example, a decrease in the number of EPCs was found to be associated with chronic kidney disease [232], coronary artery disease [233], pulmonary hypertension [234], rheumatoid arthritis [235], and hypertension [236], and a dramatic decrease in EPC proliferation and functional deterioration was found in diabetes mellitus type 1 and type 2 patients [237,238]. On the other hand, patients with acute myocardial infarction [231] and ischemic-related conditions [239] have an increasing number of circulating EPCs due to their mobilization from the bone marrow. This suggests the close relationship between the status of the ECs and EPCs functionality and mobilization. This relation gives EPCs a clinical advantage over the use of other CVD biomarkers that only correlate with end-tissue damage or stress, such as creatine kinase-MB (CK-MB) [240], troponin [241], or the causative agents like oxidized low-density lipoprotein (oxLDL) [242] and CRP [243]. 4.3.4.2 EPC Transplantation BM-derived EPCs have homing signals to the site of ischemia in animal models. Kalka et al. tested the effect of injecting ex vivo-expanded human EPCs in mice with ischemic limbs [244]. After the infusion of the EPCs, a significant number of the infused cells were detected in newly formed vessels in mice, and a corresponding increase in the rates of blood flow recovery and capillary density were also observed [244]. In another experiment, donated human CD34 + cells were injected in rats with myocardial infarction. The cells were also tracked and detected in newly formed capillaries, and significant induction of neoangiogenesis was also reported [245]. Similarly, Schuh et al. injected human BrdUlabeled isolated EPCs directly into the border infarct zone 4 weeks after acute myocardial infarction was induced in a rat model. Their results showed a significant increase in the left ventricle developed pressure, the coronary blood flow rate, and the neovascularization rate of blood vessels [246]. EPC transplantation trials extended rapidly to human patients due to their promising therapeutic potential in improving vascularization and endothelial integrity [247]. Kudo et al. conducted a clinical trial on two patients with critical limb ischemia, in which they were injected with peripheral blood-derived CD34 + EPCs. An increase in the feet oxygen pressure, improvement of symptoms, and formation of new collateral blood vessels were observed in the injected patients [248]. Another trial was conducted on 11 patients with myocardial infarction. Here, the patients were injected with a combination of bone marrow-derived autologous MSCs and EPCs. Most of the cases showed improved myocardial contractility and repair in myocardial scars [249]. Based on the previous studies and many others, it was deduced that EPC transplantation has therapeutic potential to improve vascularization. However, further investigations should be carried out to overcome the limitations related to their isolation, characterization, purity, culturing conditions, and to optimize their route of injection [246,250]. 4 Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells ... 125 4.3.4.3 Pulmonary Diseases Many studies have reported the therapeutic role of EPCs and their role as biomarkers for endothelial tissue injury, especially in pulmonary arterial hypertension and chronic obstructive pulmonary disease [251,252]. Endothelial injury and dysfunction are the major risk factors for the development and progression of both conditions [253,254]. When endothelial tissue is damaged but ECs fail to repair the damage, inflammatory cells migrate to the injury site and the subendothelium is exposed to the effects of growth factors and other mediators, resulting in intimal proliferation and blood coagulation [255]. Therefore, the availability and mobilization of EPCs in the lungs might be an effective mechanism for lung tissue regeneration and protection. For example, Yamada et al. conducted a clinical trial on 23 patients with pneumonia during both acute and convalescent phases. Patients received autologous peripheral blood-derived EPCs. Results demonstrated that a sufficient number of EPCs enabled patients to recover from pneumonia and improved the associated fibrotic damage to the lungs [256]. EPCs play a role not only in lung tissue repair but also in its early development [257]. Impaired EPC mobilization, recruitment, and engraftment were reported in premature murine pups exposed to moderate hyperoxia, resulting in impaired alveolar and vascular growth [258]. In humans, preterm infants who expressed lower numbers of EPCs at birth were reported to have an increased risk of developing bronchopulmonary disease [257]. 4.4 Pericytes: Biological Characteristics and Physiological Roles 4.4.1 Pericyte Discovery and Location Pericytes (PCs) are the third example of ASCs. PCs or perivascular cells were described almost 150 years ago based on their anatomical location surrounding the endothelium of microvascular capillaries [259,260]. PCs are also known as mural cells because of their location within the blood vessel, and as “Rouget cells”Charles Rouget, who first described them [261]. They are distributed throughout the body in different tissues at different densities depending on the location. For example, the ratio of PCs to ECs varies from 1:100 in striated muscles to 1:3 in the central nervous system (CNS), and1:1 in the retina, respectively [260,261]. PCs have acquired different names according to their tissue of residence. For example, they are known as “Ito cells”or hepatic stellate cells, in the liver, they are known as mesangial cells in the kidney, and in the bone marrow, they are called adventitial reticular cells [262,263]. The basement membrane (BM) separates the majority of the pericyte-endothelial interface, although both cell types come in contact at certain points via micro-holes in the BM. The size and number of pericyte-endothelial contacts vary between tissues, but approximately 1000 contacts have been identified for a single endothelial cell. The cells may make contact via peg-socket junctions, in which PC cytoplasmic projections (pegs) are inserted into endothelial invaginations (pockets). Adhesion plaques constitute another contact 126 A. M. El-Derby et al. mechanism, and occur between microfilament bundles attached at the pericyte plasma membrane and electron-dense material in the corresponding endothelial cytoplasm [264, 265]. Adhesion plaques, as the name suggests, function to facilitate pericyte adherence to ECs, while peg-and-socket contacts allow the diffusion of molecules and ions between the cytoplasm of the two cell types [264]. Adhesion plaque contacts include fibronectin deposits, while peg-and-socket contacts are secured via the tight, gap, and adherence junctions that contain N-cadherin and β-catenin [266]. 4.4.2 Pericyte Ultrastructure, Characterization, and Origin Pericytes are fibroblast-like cells with distinguishable nuclei, low cytoplasmic content, and several long processes surrounding the endothelial wall. Mature PCs are embedded within the BM of microvessels, which are formed by both pericytes and ECs. Pericytes located on the outer surface of blood capillaries interact with underlying ECs and are covered in the same BM [267]. Pericyte processes are typically connected with more than one endothelial cell via adhesion plaques as well as with peg-and-socket contacts, which permit direct contact between the two cell types [268,269]. This feature, which was first identified by transmission electron microscopy, differentiates primary and secondary pericyte processes [267]. Based on their location in the blood vessels, PCs are characterized as pre-capillary, mid/ true-capillary, and post-capillary PCs [270]. Mid-capillary PCs are distinguished by a lack of α-smooth muscle actin (α-SMA) within the cell and by their elongated and spindle-like shape. Preand post-capillary PCs are shorter, more stellate in shape, and have varying amounts of α-SMA [271]. Phenotypically, PCs can be characterized by the expression of a combination of antigens including platelet-derived growth factor receptor-b (PDGFR-b), neural/glial antigen 2 (NG2), α-SMA, CD146, CD90, and CD105, and absence of CD56, CD45, and CD31 [272,273]. Since PCs lack a specific marker, tracking their lineage is a challenging process [266]. Studies have reported that PCs originate either from the mesoderm or ectoderm based upon their anatomical location [274,275]. Neural crest fate mapping models have indicated that PCs in the CNS, retina, and thymus originated from differentiated neural crest-derived cells [276,277]. On the other hand, the vascular mural cells in coelomic organs such as the lungs [278], gut [279], and liver [280] derive from the mesothelium [261]. Mesothelial cells were thus proposed to undergo the epithelial to mesenchymal transition (EMT) before migrating to these organs to differentiate into PCs [262]. However, PCs were also proposed to arise directly from ECs and bone marrow [281,282]. Furthermore, it has been suggested that PCs residing in the same tissue are heterogeneous and have different origin [283]. For example, Chen et al. reported that coronary PCs originated from endocardial cells after undergoing EMT, but some retinal PCs may be derived from the bone marrow and the neural crest [284]. 4 Adult Stem Cells: Mesenchymal Stromal Cells, Endothelial Progenitor Cells ... 127 4.4.3 Pericytes Physiological Roles 4.4.3.1 Angiogenesis Angiogenesis refers to the formation of new blood vessels from pre-existing ones and is an important process in tissue repair and healing [285]. Stem cells that stimulate angiogenesis process and enhance the sprouting of new vessels have great potential in the as therapeutics for ischemic diseases. Pericytes are excellent candidates for vascular regeneration based on their contribution to vessel growth and stabilization [286]. Extensive research has shown the vital role that PCs play in angiogenesis [287,288], and their interactions with the ECs to maintain the blood vessel integrity and stability has been elucidated [289–291]. The absence of PCs was shown to be associated with the rupture of blood capillaries [292] and vessel damage [293]. Physiologically, most blood vessels are quiescent in adults; however, angiogenesis can be activated during wound healing [294–297] as well as during tumor growth [298,299]. Consequently, PCs have been targeted for pharmacological therapy. 4.4.3.2 Initiation of Neovascularization During embryogenesis, angiogenesis involves the secretion of PDGF-B from ECs,which attracts PDGF-B receptor (PDGFR-B)-expressing PCs that reside in the newly formed vessels [300]. This process is important in maintaining the vessels’functionality and integrity, as a lack of PDGF-B or PDGFR-βin mice embryos was shown to be associated with hemorrhaging, vasodilation, and embryonic lethality [292,301]. Neovascularization is initiated via the activation of quiescent vessels responding to different chemokines, or angiogenic signals including angiopoietin 2 (ANG-2) and VEGF [302]. Neovascularization comprises vessel formation, stabilization, and maturation [264, 303]. Vessel formation is initiated by the surrounding endothelial cells’secretion (ECs) of angiopoietin-2 (ANG-2), which inhibits Tie-2 receptors which inhibit the ANG/Tie signaling pathway. Inhibition of the ANG/Tie signaling pathway permits the detachment and migration of PCs to reside in the endothelial layer, enhancing new angiogenic activity [304]. Furthermore, both PCs and ECs secrete metalloproteases (MMPs) that degrade the BM to facilitate cell detachment [304]. The detachment of PCs is followed by phenotypic changes to their quiescent state including process shortening, an increase in their volume, and the initiation of proliferation [305]. In parallel, VEGF acts in combination with the ECs that lose their junctions, to increase the endothelial layer permeability and permit the passage of plasma proteins to the extracellular matrix (ECM) [306]. This is followed by EC migration toward the nascent ECM responding to different angiogenic factors. EC migration is directed by the tip cell, which is a single endothelial cell with low proliferation and a high migration rate along the VEGF gradient [307]. VEGF signaling could be enhanced by the expression of VEGF receptor 1 (VEGFR1) on PCs [308]. The tip cell migration is followed by the migration of stalk cells and neighboring ECs, to form the lumen that facilitate the growth of the sprouting vessel [306]. 128 A. M. El-Derby et al. 72. Noël D, Caton D, Roche S, Bony C, Lehmann S, Casteilla L, et al. 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Transplantation of endothelial progenitor cells improves neovascularization and left ventricular function after myocardial infarction in a rat model. Basic Res Cardiol. 2008;103(1):69–77. 247. Strauer B-E, Steinhoff G. 10 years of intracoronary and intramyocardial bone marrow stem cell therapy of the heart: from the methodological origin to clinical practice. J Am Coll Cardiol. 2011;58(11):1095–104. 144 A. M. El-Derby et al. N.B. culture dishes and flasks can be coated with 2% gelatin or type I collagen to obtain better adhesion of the explants [54]. 4. Leave the explant on the tissue culture plate for 5–15 min to adhere. 5. After adhesion, gently add fresh complete culture medium (CCM) (DMEM/LG supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) gently to the culture dish/flask without disturbing the explants. 6. Change the medium every 2–3 days according to the cell culture protocol described below. 7. After 5 to 6 days, gently remove the tissue explants from the cell culture dish/flask, leaving the adherent AD-MSCs . 8. Wash the AD-MSCs with PBS and change the medium before visualizing the cells under an inverted microscope. 9. At 80–90% confluence (80–90% of the surface of the cell culture vessel is covered by adherent cells), the cells are either passaged by splitting them over two or more cell culture vessels to avoid over-confluence (according to the manufacture’s recommendation regarding the minimal seeding density of the used cell culture vessels) as passage one (P1), or cryopreserved as passage zero (P0) [55]. •Troubleshooting of the explant culture method 1. During explant culture, tissue fragments must tightly adhere to the culture dish. Adherence of the cultured fragments was found to be essential for the migration of AD-MSCs, as they fail to migrate from floating fragments [56]. 2. Stainless steel mesh can be used to help fragments to adhere to the plate and prevent their floating. Proper adherence of the explant fragment yields a more efficient AD-MSCs population [56]. 3. Small tissue fragments are preferred during explant cultures to increase the surface area exposed of the explant and avoid central necrosis due to insufficient oxygen supply and nutrients [57]. 4. The seeding density affects the migration and growth of cells from tissue fragments. High density seeding may inhibit the outgrowth of cells from the tissue fragments. Different substrates have been reported to enhance AD-MSCs outgrowth from tissue fragments (i.e., collagen, basement membrane proteins, or fibronectin) [58]. 5. The outgrowth of AD-MSCs in explant cultures usually takes 1–3 days. The explant culture would be considered to have failed if no outgrowth was observed after 4–5 days in culture. 6. Longer maintenance of the explant (4–7 days) might induce adipogenic differentiation of AD-MSCs around the tissue fragments due to the secretion of specific adipogenic-inducible factors by the explant [59]. 7. The explant culture may contain a mixture of adherent cells that are not stem cells, but the limited proliferation and self-renewal of these cells allow their exclusion during the first subcultures [60]. 248 N. I. Ghoneim et al. 8. After 7 days of the explant culturing, cells are maintained in culture for an additional 10–14 days until confluence. The AD-MSCs yield obtained from the explant culture method is approximately 5–810 5 cells/g tissue [57]. •Advantages of explant culture 1. Explant culture method was found to give a higher yield of cells than the digestion method after primary culture [48,60]. 2. The presence of primary tissues in the explant provides the outgrowing cells with some of the required cytokines and growth factors. 3. The isolation of cells using explant culture methods without the involvement of enzymes benefits supports their use in therapeutic applications and limits the safety concerns associated with the use of enzymes (cellular stress and chemical contamination). 4. The explant culture method is more cost-effective and more time-efficient than the enzymatic digestion technique [60]. Enzymatic Digestion Method The most commonly used protocol for AD-MSCs isolation from fat is the enzymatic digestion of the extracellular matrix (ECM) to obtain different cell types, including adipocytes, AD-MSCs, fibroblasts, endothelial cells, hematopoietic cells, and immune cells [1,42,61,62]. AD-MSCs isolation by enzymatic degradation is considered the most conventional protocol despite the variation in cell yield and modest reproducibility [37,47], (Fig. 8.1). 1. Collect the inguinal fat pads, and mince into fine pieces of ~2 mm in size, using sterile scissors as described previously. 2. Wash the sample with PBS supplemented with 1% penicillin/streptomycin to remove contamintaing blood and debris. 3. Transfer the minced pieces into a 50 mL tube, and add collagenase solution (0.2% collagenase type I dissolved in PBS and filtered using a 0.2-μm syringe filter), following the manufacturer’s instructions, to enzymatically digest the ECM. The collagenase solution should completely cover the minced AT. N.B. If digesting less than 2.0 grams AT, use a minimum of 10 mL collagenase solution to ensure complete digestion. If digesting 2 grams tissue, add 5 mL collagenase solution/gram AT to each centrifuge tube [63]. Collagenase solution should be freshly prrepared. 4. Incubate the tube containing the AT and colleagenase in a water bath for 1 hr. at 37 C with agitation [64]. Carefully observe the tissue digestion process, as longer incubations may result in damage to the cells of interest [64]. N.B. (It is important to adjust the temperature to 37 C for the optimal activity of collagenase). 8 Isolation of Bone Marrow and Adipose-Derived Mesenchymal Stromal Cells 249 5. After proper digestion, the tissue will be completely liquefied, with no visible solid tissue. Add an equal volume of complete culture medium (CCM) to the heterogeneous cell mixture. N.B. CCM is essential to neutralize collagenase activity. 6. Filter the cell suspension using 100 μm nylon mesh to remove undigested tissues. Centrifuge the cell filtrate at 1200 xg for 10 min at 37 C[65]. After centrifugation, the solution is separated into two layers with the cell pellet at the bottom of the tube. N.B. adipocytes may be found floating as a fatty yellow layer above the aqueous supernatant. 7. Discard the fatty layer and liquid supernatant to obtain the cell pellet containing the SVF, and then resuspend in PBS. 8. Transfer the cell suspenion into a 50-mL tube, wash the cells with PBS by gently pipetting up and down and re-centrifuge at speed 1200 xg for 10 mins at 37 C. N.B. washing removes any traces of red blood cells, adipocytes, and other contaminants [64]. 9. Repeat step (9) if required (cell pellet appears as red colored mass). 10. Resuspend the pellet that now contains the AD-MSCs in 10 mL of CCM for primary culture. Recent studies showed that combining both the enzymatic and mechanical methods could improve the viability, reproducibility, and yield of the AD-MSCs, as mincing the AT into small pieces facilitates the action of the digesting enzyme [37,54,66,67]. •The stromal vascular fraction AT is composed of different cell types. In the late 90s, scientists isolated what is known as the stromal vascular fraction (SVF) form AT for fat tissue engineering purposes [68]. The AT was dissociated either by enzymatic or non-enzymatic methods, centrifuged and differentiated adipocytes (the floating portion on the aqueous layer) were removed leaving behind a heterogeneous mixture of cells, known as the SVF [36,37]. Morphologic and phenotypic studies of the heterogeneous SVF population [69] showed the presence of different cell types within the mixture including AD-MSCs [70], fibroblasts, endothelial cells, pericytes, erythrocytes [61], and immune cells [62] (i.e. monocytes/macrophages and lymphocytes). Selected Methods: Advanced Non-enzymatic Methods of Isolating AD-MSCs Despite the common use of the enzymatic method for the isolation of AD-MSCs, this approach has several limitations because of the variation in the efficacy of AD-MSCs isolated from different ATs and their heterogeneity [71]. In addition to the safety concerns regarding their clinical use, the enzymes used to isolate AD-MSCs can cause cell damage or incomplete dissociation from the connective tissue, in addition to the safety concerns 250 N. I. Ghoneim et al. about their clinical use [72]. Therefore, many researchers have adopted alternative non-enzymatic approaches to isolate AD-MSCs based on mechanical forces such as centrifugation, shear force, and pressure, besides, radiation to avoid the use of enzymes and facilitate the separation of AD-MSCs aggregates from tissue samples [73]. Non-enzymatic separation techniques range from simple techniques to more advanced ones. For example, the plating of the aspirate, without any enzymes, was found to produce a high yield of AD-MSCs [74]. A considerable number of AD-MSCs were separated in lipoaspirate fluid during liposuction due to mechanical forces [75]. Another study found that vigorous shaking and washing of the adipose lipoaspirate with PBS yielded a substantial amount of AD-MSCs from the lipoaspirate floating portion [76]. Furthermore, an alternative method using mechanical dissociation yielded large quantities of adherent AD-MSCs, by the centrifugation of lipoaspirates at 900 g for 15 mins at room temperature [37]. These results indicated that advanced non-enzymatic methods could save time and provide a rather safe AD-MSCs isolation method for therapeutic applications. Fig. 8.1 Procedure for isolation of adipose stem cells 8 Isolation of Bone Marrow and Adipose-Derived Mesenchymal Stromal Cells 251 Isolation of Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) Sample Collection and Isolation To collect BM, female Sprague–Dawley rats (6–8 weeks old) are euthanized and BM-MSCs will be collected as described in (Fig. 8.2). 1. Disinfect animal skin using 70% ethanol. 2. Make an incision around the perimeter of the hind limbs where they attach to the trunk and remove the skin by pulling it toward the foot, which is cut at the ankle bone. 3. Dissect out the hind limbs from the trunk of the body by carefully cutting along the vertebral column, avoid damaging the femur and tibia (long bones). 4. Wash the hind limbs with PBS supplemented with 1% penicillin/streptomycin. 5. Preserve the limbs in ice-cold DMEM medium supplemented with 1% penicillin/ streptomycin. 6. Hemisect the hind limb at the knee joint. 7. Remove the muscle and tendons from the femur and tibia by pulling the tissue toward the end of the bone, starting from the hip, or the ankle toward the knee joint, and then dislocate the joints at the knee if possible. 8. Use sterile gauze to wipe the remaining tissue, clean the bones, and place them in ice cold DMEM medium supplemented with 1% penicillin/streptomycin until the marrow extraction (preferably performed immediately). 9. Minimize the time between the dissection and BM extraction. 10. Harvest the BM using proper sterile techniques. 11. Cut the ends of the femurs and tibia just below the end of the marrow cavity using a bone cutter to expose the marrow inside. 12. Flush each bone with complete culture medium (CCM) using a 5-mL syringe with 21-gauge needle into a 50 mL conical centrifuge tube. Flush all of the marrow until the bones appear white [77]. 13. Resuspend the marrow using a 25-gauge needle. Pull the cell pellets up and down slowly to break up clumps to obtain a single cell suspension. 14. Filter the cell suspension using a 70 μmfilter placed on top of a 50 mL conical centrifuge tube to remove any bone fragments. 15. Centrifuge the cell suspension at 2000 rpm for 10 mins at room temperature (18ºC– 25ºC) and discard the supernatant. 16. Resuspend the pellet in CCM, transfer to 25-cm 2 tissue culture flasks, and place the flasks in a 5% CO 2 /humid incubator. Cell Culture and Propagation MSCs are cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM/LG) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine 252 N. I. Ghoneim et al. [65]. Unlike cell culture in high glucose medium, MSCs expand rapidly for up to 10 passages in low glucose medium without losing their morphology [52], and FBS provides the required growth factors to support cell growth and proliferation [78]. L-glutamine is an amino acid that serves as a source of nitrogen for high energydemand processes. In vitro, cells can use L-glutamine to synthesize nucleotides (DNA and RNA), vitamins, and proteins needed for different metabolic processes during growth and propagation [79]. Together DMEM, FBS, and L-glutamine constitute the formation the CCM used in in vitro cell culture. Antibiotic/ antimycotic reagents such as penicillin/ streptomycin/amphotericin B solution are used at low concentrations (1%) to prevent bacterial and fungal contamination during primary cell culture, although the use of antibiotics/antimycotics may affect the experimental results [80]. Fig. 8.2 Procedure for isolation of bone marrow mesenchymal stromal cells (BM-MSCs) 8 Isolation of Bone Marrow and Adipose-Derived Mesenchymal Stromal Cells 253 1. Seed the cells at a density of 2.1 10 6 cells in 75-cm 2 tissue culture flasks and incubate them in 5% CO 2 and proper humidity (95%) for 72 h to allow them to adhere to the plastic surface [14]. 2. After 72 h, wash the adherent cells with PBS (PBS is added gently on the walls to avoid cells detachment, loss or death by harsh pipetting), and add fresh CCM is added. 3. Incubate the adherent cells for 7 days and change the culture medium every 2–3 days by discarding the old medium and adding new medium with fresh nutrients until the cells reach confluence (80%–90%). N.B. There are various sizes of the cell culture vessels. The seeding density varies according to the surface area (according to the manufacture’s recommendation in the product sheet). 4. Remove the CCM, wash the cells with PBS to remove any traces of the serum (avoiding enzyme inhibition), preparing the cells for passaging using trypsin enzyme. N.B. There are other cell dissociation reagents other than trypsin, that can be used in passaging the cells such as TrypLE [81]. 5. Add 1–3 mL of trypsin to the adherent cells and incubate the cells at 37 C for 2–3 minutes. It is important to monitor and adjust the time of cell trypsinization to prevent over digestion of the cell proteins, which can compromise cell survival [82]. Trypsin is a protease that breaks down polypeptide chains [83]. It is used in cell culture to breakdown the ECM proteins between adjacent cells and adhesion proteins that bind cells to the plastic, thereby enabling cell collection for further culture or use [83]. 6. Observe the trypsinized cells under an inverted microscope to assure complete dissociation of the cells floating in the trypsin. 7. Add an equal volume of serum-containing CCM to neutralize the trypsin [82]. 8. Suspend and transfer the cells into a 15-mL conical centrifuge tube for centrifugation at 300 g for 10 min to obtain the cell pellet. 9. Resuspend the cell pellet in 10 mL CCM. 10. Count the cells using the Trypan blue exclusion assay using hemocytometer, and culture the cells in cell culture vessels of interest in proper seeding density for propagation [84]. N.B. In Trypan blue exclusion assay, Trypan penetrates and stains dead cells that appear in blue color, while viable cells remain unstained. 11. To obtain a pure population of cells, sorting by cell surface markers can be used. The differences between AD-MSCs and BM-MSCs are described in Table 8.1 [2,94, 95]. 254 N. I. Ghoneim et al. 8.3 Characterization of Mesenchymal Stromal Cells After isolation, it is important to maintain a uniformly pure MSCs population for proper experimental design and reproducible data (Fig. 8.3). 8.3.1 Plastic Adherence and Morphology Plastic adherence and a distinctive spindle-shaped morpology are distinguishing features for cultured MSCs. Surface receptors, mainly integrins, promote cell-matrix adherence properties via downstream gene regulation that occurs between the plastic surface, ECM, and integrin receptors [96]. Upon attachment, cells display a characterstic fibroblast-like spindle shape [14,22]. MSCs from different tissue origins all display similar adherence and morphological characteristics with minimal noticeable differences [97]. Variabilities among MSCs of different origins reveal atypical gene expression patterns, exosome secretion, and differentiation capacity [97]. AD-MSCs isolated from the SVF appear to Table 8.1 Differences between adipose-derived mesenchymal stromal cells (AD-MSCs) and bone marrow mesenchymal stromal cells (BM-MSCs) Adiposederived mesenchymal stem cells (AD-MSCs) Bone Marrow Mesenchymal Stem Cells (BM-MSCs) Amount Abundant cells AD-MSCs yield is approximately 500-fold greater when isolated from an equivalent amount of AT [34] Low yield They constitute about 0.001–0.01% of the total bone marrow nucleated cells [85] Accessibility Easy access for collection during liposuction [2] Difficult as bone marrow harvesting is an invasive procedure [2] Gene expression Express CD34 in early in vitro culture passages [86,87] Do not express CD34 [86] Proliferation capacity High [88] Low [88] Differentiation potential High potential for both angiogenic [89] and adipogenic differentiation [90,91] High potential for osteogenic differentiation [92] Stability in long term culture More genetically and morphologically stable [93] Less genetically and morphologically stable [93] Senescence ratio Low [88] High [88] Resistance to hypoxia and oxidative stress High [94] Low [94] Telomerase activity High [94] Low [94] 8 Isolation of Bone Marrow and Adipose-Derived Mesenchymal Stromal Cells 255 consist of different subpopulations of cells with variable adherence abilities. Late adherent cells show more proliferative and self-renewal capabilities than early adherent cells [98]. In contrast, BM-MSCs cultured at a low density in vitro show the ability to form colonies. Colony-forming unit fibroblast (CFU-F) was adopted as a standard assay to potentially determine the proliferation capacity of MSCs from a single precursor cell [12]. 8.3.2 Phenotypic Characterization Different cells express various surface markers according to their origin, lineage, differentiation state, and function. MSCs can be identified by the presence of a group of clusters of differentiation (CD) surface markers (CD90, CD105, and CD73). In addition, MSCs do not express CD14, CD11b, CD45, CD34, CD19, or human leukocyte antigen (HLA)-DR surface markers [31,99,100]. The surface markers included in Table 8.2 are the most common surface markers used to identify MSCs according to the minimum criteria stated by the ISCT [31,101–103]. 8.3.3 Tri-Lineage Differentiation MSCs differentiate into adipocytes [27,104], osteocytes [105], and chondrocytes [106] upon spontaneous or induced differentiation in vitro. Multilineage differentiation of MSCs depends on specific culture conditions [22]. 8.3.3.1 Adipogenic Differentiation Adipocytes (fat cells) are one of the cell derivatives of MSCs upon culture used to push the cells toward the adipogenic lineage and avoid unspecific cell differentiation [22,107]. MSCs are maintained in CCM (DMEM medium containing low glucose concentration and supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/ streptomycin). To drive adipogenic differentiation, the medium is supplemented with Fig. 8.3 Characterization of mesenchymal stromal cells (MSCs) 256 N. I. Ghoneim et al. 100 μM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine, and 0.1 μM dexamethasone, in addition to, and 10 μg/mL human recombinant insulin powder [22,108, 109]. Adipogenic differentiation of MSCs is aachieved by the release and intracellular deposition of oil droplets that can later be stained and visualized (e.g., Oil Red O staining) [21,31,96,97]. 8.3.3.2 Osteogenic Differentiation Osteogenic differentiation potential is another important functional characteristic of MSCs [107,110,111]. Osteogenic culture medium consists of 0.05 mM ascorbic acid, 1 μM dexamethasone, and 10 mM glycerol-3-phosphate in DMEM/LG [108,112]. Recent studies used bone morphogenetic proteins (BMPs) [105,112,113] and insulin-like growth factor-1 (IGF-1) [105,114] to induce or enhance osteogenic differentiation. Alizarin Red or von Kossa staining is used to detect osteoblast differentiation by staining the extracellular calcium deposits (mineralization) [31,108,114]. 8.3.3.3 Chondrogenic Differentiation MSCs can differentiate in vitro into chondrocytes in the presence of chondrogenic inducing factors. The culture medium used for chondrogenic differentiation consists of DMEM (high glucose) supplemented with 1%–2% FBS [115,116], 40 μg/mL L-proline, 100 nM dexamethasone, 100 μM ascorbic acid, 5.4 μg/mL linoleic acid [115], and a mixture of 10 μg/mL insulin 5.5 μg/mL transferrin, and 6.7 ng/mL selenium [116,117]. Recent Table 8.2 MSCs Surface Markers Surface Marker Alternative Name Expressionon MSCs Notes CD 90 Thy-1 CD90 + Glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD105 Endoglin CD105 + SH2 CD73 Ecto50-nucleotidase CD73 + SH3, SH4 CD45 CD45  Pan-leukocyte marker CD34 Mucosialin CD34  Primitive hematopoietic progenitor and endothelial cell marker N.B. CD34 can show expression in BM-MSC, however, the expression declines with culture CD14 LPS receptor CD14  Monocyte and macrophage marker CD11b Integrin αM chain CD11b  Monocyte and macrophage marker CD19 CD19  B cell marker CD79αIg-αCD79α  B cell marker HLADR HLA-DR  Appear only on MSCs during stimulation 8 Isolation of Bone Marrow and Adipose-Derived Mesenchymal Stromal Cells 257 114. Reible B, Schmidmaier G, Moghaddam A, Westhauser F. Insulin-like growth factor-1 as a possible alternative to bone morphogenetic protein-7 to induce osteogenic differentiation of human mesenchymal stem cells in vitro. Int J Mol Sci. 2018;19(6):1–15. 115. Zhou M, et al. Graphene oxide: a growth factor delivery carrier to enhance chondrogenic differentiation of human mesenchymal stem cells in 3D hydrogels. Acta Biomater. 2019;96:271–80. 116. Tanthaisong P, Imsoonthornruksa S, Ngernsoungnern A, Ngernsoungnern P, Ketudat-Cairns M, Parnpai R. Enhanced chondrogenic differentiation of human umbilical cord wharton’s jelly derived mesenchymal stem cells by GSK-3 inhibitors. PLoS One. 2017;12(1):1–15. 117. Nöth U, et al. Chondrogenic differentiation of human mesenchymal stem cells in collagen type I hydrogels. J Biomed Mater Res Part A. Dec. 2007;83A(3):626–35. 264 N. I. Ghoneim et al. In Vitro Methods for Generating Induced Pluripotent Stem Cells 9 Toka A. Ahmed, Shimaa E. Elshenawy, Mohamed Essawy, Rania Hassan Mohamed, and Nagwa El-Badri Contents 9.1 Introduction ................................................................................... 267 9.2 History of Induced Pluripotent Stem Cells .................................................. 267 9.3 Cell Reprogramming Techniques ............................................................ 270 9.4 Practicum...................................................................................... 271 9.4.1 Protocol for Generation of iPSCs .................................................... 271 References ........................................................................................... 285 Abbreviations AMSCs Adipose mesenchymal stem cells BAC Bacterial artificial chromosome bFGF Recombinant basic fibroblast growth factor BJ Human BJ fibroblasts CCM Complete culture medium DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide T. A. Ahmed · S. E. Elshenawy · M. Essawy · N. El-Badri (*) Center of Excellence for Stem Cells and Regenerative Medicine (CESC), Helmy Institute of Biomedical Sciences, Zewail City of Science and Technology, Giza, Egypt e-mail: [email protected];[email protected];messawy@zewailcity. edu.eg;[email protected] R. H. Mohamed Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt e-mail: [email protected] #Springer Nature Switzerland AG 2020 N. El-Badri (ed.), Regenerative Medicine and Stem Cell Biology, Learning Materials in Biosciences, https://doi.org/10.1007/978-3-030-55359-3_9 265 EBV Epstein–Barr virus ESCs Embryonic stem cells FBS Fetal bovine serum HDF Human dermal fibroblast HFF Human foreskin fibroblasts HUVEC Human umbilical vein endothelial cells iPSCs Induced pluripotent stem cells Klf4 Kruppel-like factor 4 KSR Knockout serum replacement MEF-CM MEF culture medium MEFs Mouse embryonic fibroblasts MHC Major histocompatibility complex MNCs Mononuclear cells mod-mRNA mRNAs with modified nucleobases Myod1 Myogenic differentiation 1 NEAAs Nonessential amino acids NPCs Neural precursor cells NSCs Neural stem cells Oct3/4 Octamer-binding transcription factor 3/4 Opti-MEM Opti-minimum essential medium OSKM Oct3/4, Sox2, Klf4, and c-Myc factors OSLN Oct4, Sox2, Nanog, and Lin28 PBS Phosphate-buffered saline PCs Pericytes PPE Personal protective equipment SCNT Somatic cell nuclear transfer SeV Sendai virus Sox2 Sex-determining region Y-box 2 β-Me β-mercaptoethanol What You Will Learn in This Chapter In this chapter, you will get a brief overview of induced pluripotent stem cells (iPSCs) and the advances in cell reprogramming. The generation of pluripotent cells with higher differentiation potential from somatic cells has opened the door for substantial advances in personalized medicine and regenerative medicine applications. You will also learn the basics of inducing pluripotent stem cells from somatic cells and the different factors affecting the reprogramming efficiency. A step-by-step protocol will guide you to different approaches to generate iPSCs from different types of somatic cells and the advantages and disadvantages of each protocol. 266 T. A. Ahmed et al. 9.1 Introduction Embryonic stem cells (ESCs) hold great promise for regenerative medicine and cell therapy due to their potential for unlimited propagation and generation of all varieties of somatic cells. However, the use of ESCs for clinical applications has met with significant controversy due to ethical issues associated with the manipulation of human preimplantation embryos, problems with inadequate tissue matching, and high tumorigenic potential [1– 3]. By contrast, induced pluripotent stem cells (iPSCs) are generated from somatic cells from an individual patient (i.e., from an autologous source); as such, iPSC therapy may overcome some of these limitations. Autologous iPSCs could be applied as part of a personalized medicine approach and might add a new and individualized dimension to drug discovery, disease modeling, and targeted therapy [4]. IPSCs have been generated from somatic cells of both fetal and adult tissues. Somatic cell reprogramming into a pluripotent, embryonic-like state was induced by the introducing of the Oct4, Sox2, Klf4, c-Myc (OSKM factors), and Nanog transcription factors [5,6]. Integrative and non-integrative methods were used to generate iPSCs [3,7–11]. Effective somatic cell reprogramming was initially accomplished by integrating the genetic material via retroviralor lentiviral-mediated gene transfer; however, this method increased the risk of mutagenesis [3,7,12]. Reprogramming of adult cells into iPSCs can be achieved using non-integrative methods, including gene transfer with bacterial episomal vectors and Sendai virus (SeV), which is an RNA virus that does not integrate into genomic DNA; these methods have a higher safety profile and reduce the risk of genotoxicity and mutagenesis [8,13–15]. Several modifications of Yamanaka’s original protocol have been applied in order to improve the efficiency of somatic cell reprogramming using the OSKM factors [16–20]. To enhance reprogramming efficiency, synthetic capped mRNAs with modified nucleobases (mod-mRNA) have been introduced; this has increased the reprogramming efficiency by as much as 4.4% [21,22]. Unfortunately, this modification was only functional when applied to long-lived fibroblast cell lines such as BJs; no significant enhancement was observed when this modification was applied to freshly isolated cells [16]. To overcome this limitation, Kogut et al. improved the reprogramming efficiency up to 90.7% using a combination of miRNA-367/302s and synthetically-modified specific mRNAs that synergistically enhance reprogramming efficiency and conversion of neonatal fibroblasts into iPSCs [9]. 9.2 History of Induced Pluripotent Stem Cells IPSCs are generated by the reprogramming of adult somatic cells (e.g., epithelial cells, fibroblasts, or multipotent stem cells) into cells with pluripotent capabilities [23–25]. The concept of cellular reprogramming and nuclear transfer dates back to experiments performed nearly a century ago (Fig. 9.1)[26–28]. Early, albeit unsuccessful experiments 9 In Vitro Methods for Generating Induced Pluripotent Stem Cells 267 carried out by Spemann (1938) who was attempting to perform nuclear transfer in mouse models nonetheless introduced the concept of cellular reprogramming in order to induce a more embryonic-like state [28,29]. In 1952, Briggs and King pioneered the efforts in somatic cell reprogramming in their experiments that focused on transplantation of blastula cell nuclei into enucleated frog eggs. Unfortunately, they were unable to reproduce their initial findings when targeting other specialized cells [30]. In 1962, John Gurdon successfully generated cloned tadpoles from cells containing the nuclei of the frog’s intestinal cells [31]. In this set of experiments, he demonstrated that differentiated nuclei revert to an undifferentiated state when transplanted into frog’s eggs. Before this breakthrough, differentiation was believed to be uniformly unidirectional, with somatic cells generated from progenitor or immature cells, and not vice versa. “Epigenetic landscape”was a term coined by Conrad Waddington [32] in his efforts to elucidate that factors contributing to somatic cell reprogramming. The concept of the epigenetic landscape provides an explanation of the specific biological paths that can be undertaken by a given cell, including those leading to different developmental stages, including both progenitor and differentiated states. These biological paths have been quantified via construction probability landscapes that define cell developmental and differentiation pathways. This type of analysis works on the principle that the developmental process, the conversion of the cells from an undifferentiated to a differentiated state, as well as the stability of various cell types can be determined by the escape time, a factor that correlates with barrier heights between the differentiated Nucleus removed a b c Nucleus fused with enucleated egg Nucleus fused with enucleated egg Blastocyst Blastocyst Tissue culture Implanted into surrogate mother OSKM cocktail/factors iPSC Somatic cell c-Myc KIf4 Oct3/4Sox2 Clone is bom Morula Morula Nucleus removed Nucleus removed Nucleus removed Enucleated cell Enucleated cell Egg Egg Somatic cell Somatic cell Fig. 9.1 History of somatic cell reprogramming and generation of iPSCs 268 T. A. Ahmed et al. and undifferentiated states. The epigenetic landscape assumes that, as fluctuations increase, the barrier height between these states decreases and the escape time is reduced; these alterations serve to increase the chance of conversion from the undifferentiated to a differentiated state. Consequently, the possibility for deviation from the original developmental paths likewise increases. Accordingly, small fluctuations enhance the process of development and limit the opportunities for deviation from the original paths [33,34]. A key discovery in the field of reprogramming was reported in a landmark set of experiments carried out by Davis and colleagues [35] in 1987, in which it was shown that cell fate could be directed and defined by a transcription factor. In this set of experiments, complementary DNA (cDNA) subtraction probing was performed and three genes were identified, which were expressed primarily in proliferative myoblasts. One of these genes was myogenic differentiation 1 (Myod1) which, when subjected to forced expression in mouse fibroblasts, resulted in their conversion into myosin-expressing myoblasts. These experiments were later considered “the dawn of direct reprogramming.” Another major breakthrough was achieved by Wilmut et al. [36] who generated the cloned sheep, Dolly, from an enucleated oocyte. The researchers postulated that the nuclei of somatic cells and the enucleated egg were capable of generating a complete organism, as the nuclei contain all the genetic information and factors necessary to promote genetic reprogramming. They fused the nucleus from the cell of a mammary gland into an enucleated, unfertilized egg. Dolly was the first cloned animal that was born following this protocol from a total of 13 recipients undergoing embryo implantation into surrogate ewes. In 1997, Tada et al. [27] fused mouse ESCs with female mouse thymocytes, which were reprogrammed into pluripotent hybrids. IPSC technology was pioneered by Shinya Yamanaka in Kyoto, Japan, who demonstrated in 2006 that adult cells can be reprogrammed into iPSCs via the introduction of four specific transcription factors [3]. In November 2007, the first human iPSCs were created from adult cells by two independent research teams. The first report was from the group of James Thomson at the University of Wisconsin in Madison, WI, USA [23], and the second from Shinya Yamanaka and colleagues at the Kyoto University, Japan [7]. Yamanaka has successfully transformed human fibroblasts into pluripotent stem cells by retroviral transduction with four pluripotency genes, including OCT3/4,SOX2,KLF4, and c-Myc [7]. In 2011, the same team generated integration-free human iPSCs via the use of bacterial episomal vectors [8]. Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medicine jointly with John Gurdon for discovering that mature specialized cells could be reprogrammed into immature cells for the purpose of generating differentiated tissue cells [37]. IPSCs generated from adults can overcome many of the limitations of the ESCs; they not only bypass the need for embryos, they can be specifically engineered to match a given patient’s genetic makeup [2,38]. However, reprogramming of adult cells into iPSCs still carries significant risks that could limit their use in clinical settings. For example, viruses used in reprogramming may genetically alter the cells and may enhance the expression of cancer-associated genes [39]. This challenge has resulted in efforts to replace viral vectors 9 In Vitro Methods for Generating Induced Pluripotent Stem Cells 269 [Document text truncated for crawler view.]