Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth.

1 2Mesenchymal stem cell like (MSCl) cells generated from human embryonic 3stem cells support pluripotent cell growth 4Nóra Varga a Q1 , Zoltán Veréb b , Éva Rajnavölgyi b , Katalin Német a , Ferenc Uher a , Balázs Sarkadi a , 5Ágota Apáti a, ⇑ 6 a Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary 7 b Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen, Hungary 8 9 11 article info 12 Article history: 13 Received 12 September 2011 14 Available online xxxx 15 Keywords: 16 Human embryonic stem cells 17 Mesenchymal stem cell like (MSCl) cells 18 Feeder cell 19 Immunosuppressive feature 20 21 abstract 22 Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) 23 through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could 24 be propagated without changes in morphological or functional characteristics for more than 15 passages. 25 These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the 26 growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express 27 the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, 28 HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs 29 (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adi30 pogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, 31 and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly 32 model human MSCs and be applied as efficient feeders in hESC cultures. 33 Ó2011 Published by Elsevier Inc. 34 35 36 37 1. Introduction 38 Mesenchymal stem (stromal) cells (MSCs), originally isolated 39 from bone marrow [1], provide a supportive microenvironment for 40 hematopoietic stem cells. Moreover, MSCs show multipotent stem 41 cell characteristics [2] and special immunological features [3]. MSCs 42 canalsobeobtainedfromvarioustissuesincludingperipheralblood, 43 umbilical cord, placenta, vein wall, muscle, adipose and connective 44 tissues [4]. MSCs can be differentiated into canonical mesodermal 45 tissues,e.g.bone,fatand cartilage,butmayalsoretainawiderdiffer46 entiation capacity [5]. MSCs play a role in tissue repair due to their 47 differentiation potential and immunosuppressive activities [6],as 48 well as by trophic effects mediated by growth factors and cytokines 49 they produce [7]. 50 These features initiated the use of MSCs in various cell-based 51 therapies and by now more than 50 clinical trials related to this cell 52 type have been reported (see http://www.clinicaltrials.gov)[8]. 53 However, there are several limitations of large scale, reproducible, 54 and well characterized production of human MSCs. The aspiration 55 of MSCs from donors is an invasive procedure and several reports 56 have demonstrated donorand tissue-dependent variability of hu57 man bone-marrow-derived MSCs [4,9–13]. Isolation and enrich58 ment of tissue-derived MSCs usually results in heterogeneous 59 cell populations, and a long term ex vivo expansion of human MSCs 60 has been shown to reduce replicative capacity, impair differentia61 tion potential, alter gene expression profiles, and lead to karyotype 62 instability [9,10]. 63 In contrast to MSCs, pluripotent human embryonic stem cell 64 (hESCs) lines have the capacity of unlimited growth and self-re65 newal [14] and can differentiate into all cell types of the human 66 body [15]. There are numerous studies investigating the differenti67 ation of hESCs, especially to yield the clinically most relevant cell 68 types, including cardiomyocytes, hemopoietic cells, neuronal or 69 pancreatic cells (for a recent review see [16]). 70 RecentpublicationshavealsoreportedthederivationofMSC-like 71 cells from hESC lines [17–20], by using various protocols and pro72 ducing MSCs with different characteristics. In the present study 73 we have established a method to derive MSCl cell lines from hESCs 74 with well defined gene and protein expression patterns and immu75 nological features, examined their differentiation potential, sup76 portive role for culturing hESCs, and produced genetically 77 modified MSCl cell clones stably expressing fluorescent marker pro78 teins. This work may significantly help our understanding of human 79 MSC characteristics and the use of these cells in biotechnology 80 applications. 0006-291X/$ - see front matter Ó2011 Published by Elsevier Inc. doi:10.1016/j.bbrc.2011.09.089 Abbreviations: hESC, human embryonic stem cell; MSC, mesenchymal stem (stomal) cells; bmMSC, bone marrow derived MSC. ⇑ Corresponding author. Address: Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Dioszegi u. 64, 1113 Budapest, Hungary. Fax: +36 1 372 4353. E-mail address: [email protected] (Á. Apáti). Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 27406 No. of Pages 7, Model 5G 30 September 2011 Please cite this article in press as: N. Varga et al., Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bbrc.2011.09.089 81 2. Materials and methods 82 2.1. HuES, MSCl cells, HFF-1 and bmMSC cultures 83 The human embryonic stem cell lines (HUES9 and HUES1 orig84 inally provided by Dr. Douglas Melton, Harvard University) were 85 cultured on mitotically inactivated mouse embryonic fibroblasts 86 (MEF) [21]. Spontaneous differentiation of the hESCs were per87 formed via embryoid body (EB) formation as described previously 88 [21]. The cells were trypsinized at day 80 and were further cul89 tured in DMEM supplemented with 10% fetal bovine serum (FBS; 90 Invitrogen) on a gelatinized 10-cm plate. The cells were trypsinized 91 when confluent and split to 1:3 ratio. After two passages most of 92 the cultured cells presented fibroblast-like morphology. The 93 hESC-derived fibroblast-like cells were designated as hESC-derived 94 mesenchymal stem cell like (MSCl) cells. All MSCl cells used in this 95 study were of polyclonal origin. 96 HFF-1 cells were obtained from ATCC and were cultured according 97 to the manufacturer’s instructions (http://www.stemcells.atcc.org). 98 Collection of bone marrow samples from patients with hemato99 logical diseases was approved by the Regional and Institutional 100 ethical review board of the Medical and Health Science Center of 101 the University of Debrecen (protocol No.: UD-MHSC REC/IEC 102 2754-2008). Bone marrow samples were harvested from the iliac 103 crest under medical examination. bmMSC isolated and cultured 104 as described earlier [22]. The cells were used for phenotypic and 105 functional analysis after P5. 106 2.2. Characterization of MSCl, HFF-1 and bmMSC cells 107 Cell surface expression of MSC marker proteins on the surface of 108 MSCl, HFF-1 and bmMSC cells was analyzed by three-color flow 109 cytometry using FITC-, APCor PE-conjugated monoclonal antibod110 ies (mAb) with isotype-matched control mAbs; specific against 111 hematopoietic-, MSC related-, endothelial markers, cell adhesion 112 molecules and integrins as well (see Supplementary Table I for full 113 list). Fluorescence intensities were measured by FACS Calibur flow 114 cytometer and the data were analyzed by using the WinMDI free115 ware (Joseph Trotter, La Jolla, CA). Results were expressed as 116 means of positive cells (%) ± SD. Pluripotency markers of MSCs 117 were tested by immunostaining (Oct4, SSEA4, PODXL) and by real 118 time PCR (Nanog, ABCG2) performed as described previously [21]. 119 The differentiation potential of MSCs was performed by using the 120 Gibco’s StemPro Ò Adipogenesis, Osteogenesis and Chondrogenesis 121 Differentiation Kits according to the manufacturer’s guide. 122 2.3. Lentiviral transduction of MSCl cells 123 For viral-based gene delivery, a third generation lentiviral vec124 tor system was used, as described in [23]. Determination of virus 125 titers and the transduction procedures were performed as de126 scribed previously [24]. The MSCl-2 cells were transduced by a 127 MOI of 2–5, with an eGFP encoding lentiviral vector and further 128 handling was the same as for the parental cell line. 129 2.4. Mitogen-induced cell proliferation and cytokine secretion 130 Peripheral blood mononuclear cells (PBMCs) were isolated by 131 Ficoll gradient centrifugation (Amersham Biosciences, Uppsala, 132 Sweden). Mitogen-activated T lymphocyte proliferation was in133 duced by concanavalin A (ConA) or phytohaemagglutinin (PHA) 134 used at a final concentration of 10 l g/ml (Amersham Pharmacia 135 Biotech) and 1 l g/ml (Sigma–Aldrich), respectively added to 136 110 6 PBMCs. MSCs were added to 1 10 6 PBMCs at 10 3 and 137 10 4 cell numbers and co-cultured for 3 days. On day three prolifer138 ation was detected by the BrDU colorimetric assay directly in the 139 culture plate according to the manufacturer’s instructions (Roche). 140 IL-6, IL-10 and IFNc cytokine secretion was quantitated by en141 zyme linked immunosorbent (ELISA) assay (OptEIA, BD Pharmin142 gen) following the supplier’s instruction. All experiments were 143 performed in triplicates. 144 2.5. Statistical analysis 145 Each experiment was performed at least three times and each 146 sample was tested in triplicates. Data are expressed as mean + SD. 147 Statistically significant differences were determined by two-way 148 ANOVA or paired student-ttests. ⁄ p< 0.05, ⁄⁄ p< 0.01, ⁄⁄⁄ p< 0.001. 149 3. Results and discussion 150 3.1. Generation of HUES9-derived MSCl cells – application as feeder 151 cells 152 Pluripotent HUES9 cells were spontaneously differentiated via 153 EB formation (6 days), and then the differentiation process was Fig. 1. Pluripotency markers of HUES9 cells cultured on different feeder layers, HUES9 cells were grown on MEF (A) or MSCl-2-GFP (B) feeder cells for two days in eight-well chambers for confocal microscopy. Co-culture of HUES and feeder cells were fixed and stained with the antibodies recognizing the following proteins: Oct4, SSEA4, PODXL (red). Antibodies specific for undifferentiated cell markers stained only the HUES9 clumps, while the MEF or MSCl-2-GFP cells did not show any staining with these markers. Nuclei were counterstained with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 2N. Varga et al. / Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx YBBRC 27406 No. of Pages 7, Model 5G 30 September 2011 Please cite this article in press as: N. Varga et al., Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bbrc.2011.09.089 154 completed by further cell culturing on adherent surface in the 155 presence of 10% FBS for 80 days. The differentiated fibroblast-like 156 cells were separated from the other cell types by short trypsiniza157 tion. After 2–3 passages on adherent surface the cells achieved uni158 form, fibroblast like morphology, and these cells (termed MSCl 159 cells) could be propagated at least for 15 passages without mor160 phological or kariotypical changes (Supplementary Fig. 1). By using 161 the same protocol, more than five polyclonal MSCl cultures were 162 independently generated from the HUES9 cell line. 163 In the following experiments we tested the capacity of the MSCl 164 cellsas autogenic feedercellsfor maintainingpluripotency ofhESCs. 165 When undifferentiated HUES1 or HUES9 cells were passaged to 166 mitomycin treated MSCl cultures, each MSCl cell line supported 167 the growth of these pluripotent hESCs without any change in the 168 expression of pluripotency markers or the rate of cell growth, as 169 compared to those seen with cells growing on MEF (Supplementary 170 Fig. 2). Recent publications have already described various methods 171 for the establishment of autogenic feeder cells from hESC [25–27]. 172 Our present method provides means to create applicable feeders 173 for hESC without cell sorting or mechanical separation, and the de174 rived feeder cells demonstrate the capacity to promote hESC 175 expansion. 176 In order to allow a fast separation of hESCs from the human 177 MSCl cells, we have generated eGFP expressing MSCl cells by using 178 a lentiviral gene delivery method. The MSCl-GFP cells were sorted 179 for eGFP positivity, treated with mitomycin and used as feeder for 180 hESCs. As documented in Fig. 1, the MSCl-GFP cells promoted the 181 growth of HUES9 cells in an undifferentiated state. This Figure also 182 documents that the pluripotency markers Oct4, SSEA4 and PODXL 183 were expressed in the pluripotent HUES9 cells, while these mark184 ers were absent in the MSCl-GFP feeder cells. 185 We also extended these studies with RT-PCR measurements of 186 pluripotency markers expression in the undifferentiated hESC 187 and in the MSCl cells. The transcription factors Oct4 and Nanog 188 and the ABCG2 cell surface protein showed high levels of gene 189 expression in the HUES9 cells, while they were close to detection 190 levels in the MSCl cells (data not shown). 191 3.2. Multi-lineage differentiation potential 192 MSCs should fit the functional and phenotypic criteria defined 193 by the Mesenchymal and Tissue Stem Cell Committee of the Inter194 national Society for Cellular Therapy [28]. We first tested whether 195 the MSCl cells could be differentiated toward the canonical meso196 dermal (osteoblastic, chondrogenic, adipogenic) directions. We 197 found that all four established MSCl cell lines exhibited the 198 potential to differentiate to at least one direction, but one of them 199 (MSCl-2) showed robust differentiation potential toward all three 200 directions (Supplementary Fig. 3). To complete a more detailed 201 characterization we next compared the differentiation capacity of 202 MSCl cells with the commercially available human foreskin fibro203 blast cells (HFF-1) widely used as a feeder cell line for hESC cul204 tures, and that of MSCs isolated from human bone marrow. All 205 these MSCs of different origin could be differentiated in vitro to 206 adipogenic, oesteogenic and chondrogenic directions. Followed 207 by a three-week adipogenic induction period, a large number of Fig. 2. Differentiation of MSCl-2, HFF-1 and bmMSC cells. Adipogenic differentiation is demonstrated by intracellular lipid vacuoles stained in red by Oil Red O (red) and with nuclei counterstained with DAPI (blue) (A). Osteogenic differentiation of MSCs is demonstrated by the formation of calcium-hydroxyapatite-positive areas stained in red by Alizarin Red (B). High density cultures showed the development of chondrogenic phenotype when cultured in micromass; pink extracellular matrix staining marks proteoglycans stained with toluidine blue (C). Magnification: 200. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) N. Varga et al. / Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx 3 YBBRC 27406 No. of Pages 7, Model 5G 30 September 2011 Please cite this article in press as: N. Varga et al., Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bbrc.2011.09.089 208 MSCl-2 and bmMSC cells showed oil red positive staining, charac209 teristic for adipocytes (Fig. 2A), while only a small number of HFF-1 210 cells became oil red positive. Similarly, when differentiation was 211 induced by osteogenic induction medium for 2–3 weeks, MSCl-2 212 and bmMSC cells showed dense calcium deposits stained with aliz213 arin red, while the HFF-1 cells exhibited weak osteogenic potential 214 (Fig. 2B). After 3 weeks of differentiation sections made from chon215 drogenic mass cultures showed metachromazy upon toluidin-blue 216 staining indicating chondrocyte formation from all three cell types 217 (Fig. 2C). Based on these data we suggest that MSCl-2 cells 218 generated from HUES9 cells retained their multilineage differenti219 ation potential, similar to that found in bmMSCs [4,29], whereas 220 HFF-1 cells possessed reduced differentiation capacity. 221 3.2.1. Cell surface markers of MSCl cells 222 In the first set of experiments summarized in Fig. 3 we com223 pared the phenotypic characteristics of three different HUES9-de224 rived polyclonal MSCl cell lines: MSCl-2, MSCl-3 and MSCl-4, 225 which could be cultured for a long period of time, for the expres226 sion of the key cell surface molecules of MSCs. As shown by flow Fig. 3. Expression of common MSC markers in different MSCl cell lines. Flow cytometry measurements of cell surface marker expression of HUES9-derived MSCl cells. Single cell suspensions from MSCl-2, MSCl-3 and MSCl-4 cells were obtained by gentle trypsinization. Non-viable cells were excluded by Topro3 or 7AAD staining. Monoclonal antibodies specific for CD44 (conjugated to FITC), CD73 and CD90 (conjugated to PE) were used to detect MSC markers. Anti CD34-PE was used to demonstrate the absence of hematopoietic cells in the MSCl cultures. Dashed lines show staining with the relevant isotype-matched control mAbs. 4N. Varga et al. / Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx YBBRC 27406 No. of Pages 7, Model 5G 30 September 2011 Please cite this article in press as: N. Varga et al., Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bbrc.2011.09.089 227 cytometry, the selected cell lines expressed similar high levels of 228 CD44, CD73, and CD90 with some variability in the magnitude of 229 CD90 expression and none of these cells expressed CD34, an early 230 hematopoietic stem cell marker (Fig. 3). 231 Foradetailedcharacterization,wehavecomparedtheexpression 232 of the cell surface markers in bmMSC, HFF-1 and MSCl-2 cells, by 233 using flow cytometry analysis. As documented in Table 1, all the 234 three cell types showed a uniformly high expression for all well 235 known MSC markers, e.g. CD44, CD73, CD90, and CD105. Because 236 none of these markers are considered as MSC specific, we further 237 analyzed the expression of integrins, cell adhesion molecules and 238 endothel-related surface proteins on MSCl-2 cells as compared to 239 that of the other cell types. No difference in the expression pattern 240 of these cell surface markers could be shown (Supplementary Table 241 II). Importantly, none of the MSCs showed measurable expression of 242 the hematopoietic markers CD14, CD34, CD45, CD117, CD133 or 243 HLA-R. The unique propertyof HFF-1 cells wastheir high cellsurface 244 expressionofCD14,whichwasundetectableinbmMSCor MSClcells 245 (Table 1). Thus in line with previous publications [17,20,30], the de246 tailed phenotypic analysis of HUES9-derived MSCl-2 and adult 247 bmMSCs cells revealed close phenotypic similarity. 248 3.2.2. Immunosuppressive effects of MSCl-2, HFF-1 and bmMSCs 249 Due to their clinical utility, the immunosuppressive properties 250 of MSCs have extensively been studied and MSCs were shown to 251 suppress immune responses both in vitro and in vivo mediated by 252 multiple mechanisms [31–33] validating MSC as a therapeutically 253 relevant cell type. Numerous studies have demonstrated that hu254 man MSCs decrease alloreactive responses, interfere with dendritic 255 cell (DC) and T-cell functions, and are able to generate an immuno256 suppressive microenvironment. Under in vitro conditions bmMSCs 257 are capable to suppress various T-cell effector functions [33,34] 258 and inhibit mitogen-stimulated lymphocyte proliferation [35,36]. 259 In the present study mitogen-induced T-cell proliferation was 260 used to compare the immunosuppressive properties of MSCl-2, 261 HFF-1 and bmMSC cells. Human PBMC of healthy donors was used 262 as responder cells, and ConA or PHA as mitogenic activators. We 263 found that all cell types responded to mitogenic activation and 264 had an effect on lymphocyte-proliferation induced by ConA or 265 PHA (Fig. 4). As shown in Fig. 4A, the presence of MSCls suppressed 266 the mitogenic response of lymphocytes in a dose-dependent man267 ner and the addition of both 10 3 and 10 4 MSCl-2 cells to 1 10 6 268 PBL efficiently reduced ConA-induced lymphocyte proliferation, 269 whereas the PHA-induced mitogenic response was significant in 270 the presence of 10 4 MSCl-2 cells, only (Fig. 4A). A similar effect 271 could be detected when using bmMSCs as suppressors (Fig. 4C). 272 Proliferation of lymphocytes could also be suppressed by HFF-1 273 cells when ConA but not PHA was used for stimulation (Fig. 4B). 274 In order to elicit their immunosuppressive function, MSCs 275 should acquire increased production of certain cytokines such as 276 IL-6, IL-10 and IFN c [37,38]. IL-6 was shown to increases PGE 2 pro277 duction and thus plays a key role in the inhibition of DC maturation 278 and T-cell proliferation [31,39–42]. Dependent on local IFN c levels 279 MSC-mediated immunosuppression can be enhanced [43], and 280 bmMSCs can drive DC differentiation to IL-10 secreting cells [33], 281 which are potent inhibitors of T lymphocyte proliferation [44]. 282 In the next set of experiments we sought to measure the concen283 tration of secreted IL-6, IL-10 and IFN c cytokines in the supernatant 284 of mitogen-activated PBMCs co-cultured with MSCl-2, HFF-1 and 285 bmMSC cells (Supplementary Fig. 4). Our results revealed that 286 bmMSCs induced significant increases in IL-6 (Supplementary 287 Fig. 4C), IFN c (Supplementary Fig. 4F) and IL-10 (Supplementary 288 Fig. 4I) cytokine levels, whereas MSCl-2 cells were unable to do so 289 even though they efficiently inhibited T lymphocyte proliferation 290 (Fig. 4A, D, and G). The presence of HFF-1 cells induced a slight 291 increase in IL-6 levels (Supplementary Fig. 4B), but the concentra292 tion of secreted IFN c and IL-10 did not change (Supplemantary 293 Fig. 4E and H). These results indicate that the production of the 294 functionally relevant cytokines is not an absolute requirement for 295 exerting the immunosuppressive effects of MSCs, and the MSCl-2 296 cells may use different means to inhibit T-lymphocyte associated 297 effector functions. 298 AlthoughthedetailedimmunologicalcharacterizationofHUES9299 derived MSCl-2 needs further investigations, our present results 300 indicate a strong immunosuppressive effect by the HUES9-derived 301 MSCl-2 cells. Our current experiments reinforce previous observa302 tions [20,30,45] demonstrating that MSCs derived from human 303 embryonic stemcells have the capacityto suppressperipheral blood 304 T lymphocyte proliferation and also extend them by demonstrating 305 the lack of MSCs-induced enhancement of cytokine secretion upon 306 their immunosuppressive action. 307 As a summary, we have prepared and characterized human MSCl 308 cells obtained from the human embryonic stem cell line, HUES9. In 309 previousstudiesvariousprotocols havebeenapplied to obtainMSCs 310 from hESC, including co-culture with mouse OP9 cells [46], 311 embryoid body differentiation [47], spontaneous differentiation in 312 monolayer [20], selection of cell populations by FACS [17], using 313 Rho-associatedkinaseinhibitor[30],overexpressionofHOXB4gene 314 [18] and mechanical separation [19]. In the present study we have 315 developed a novel, simplified method to obtain phenotypically and 316 functionally homogeneous hESC-derived MSC populations. Our der317 ivation protocol required neither the use of xenogenic feeder cell, 318 nor selection (manual or FACS), nor chemical inducers. These MSCl 319 cells showed long term stability and could be maintained in culture 320 over 30 passages without detectable phenotypic or functional 321 changes. Cell surface markers, multipotent differentiation potential 322 and immunosuppressive effects of MSCl-2 proved to be similar to 323 bmMSCs but was different from that of HFF-1. Our data suggest that 324 MSCl-2 cells may be successfully applied as a model system for 325 studying human MSC features and may be used in biotechnology 326 applications. 327 4. Disclosures 328 The authors indicate no potential conflicts of interest. Table 1 Expression of common phenotypic markers by MSCs of various origins. MSCl-2 HFF-1 bmMSC CD44/H-CAM 96.97 ± 2.81 93.23 ± 9.44 89.69 ± 10.04 CD105/Endoglin 89.61 ± 3.95 95.73 ± 6.19 86.08 ± 8.85 CD73/NT5E 99.51 ± 0.25 99.21 ± 0.22 93.57 ± 6.44 CD90/Thy-1 96.24 ± 2.41 87.38 ± 7.87 89.78 ± 7.67 CD34 0.00 1.24 ± 2.49 0.00 CD14 1.04 ± 1.88 85.27 ± 10.47 0.09 ± 0.19 CD45/Protein tyrosine phosphatase receptor C 0.00 0.00 0.00 CD117/c-kit 0.00 0.00 0.00 CD133/Prominin 1 0.00 0.00 0.00 HLA-DR 0.00 0.00 0.00 N. Varga et al. / Biochemical and Biophysical Research Communications xxx (2011) xxx–xxx 5 YBBRC 27406 No. of Pages 7, Model 5G 30 September 2011 Please cite this article in press as: N. Varga et al., Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bbrc.2011.09.089 329 Acknowledgments 330 This work has been supported by Grants from OTKA (NK72057), 331 ETT (213-09), STEMKILL (OM00108/2008), KMOP-1.1.2-07/1-2008332 0003 and TÁMOP-4.2.2-08/1-2008-0015. The authors wish to 333 thank Beáta Haraszti for help in cell culturing, for Monika Bátkai 334 for helping with the lentiviral transduction of MSCl cells for György 335 Várady for helping with FACS and Emma Ádám and András Kozma 336 for karyotyping and FISH analysis. 337 Appendix A. Supplementary material 338 Supplementary data associated with this article can be found, in 339 the online version, at doi:10.1016/j.bbrc.2011.09.089. 340 References 341 [1] A.J. Friedenstein, R.K. Chailakhyan, N.V. Latsinik, et al., Stromal cells 342 responsible for transferring the microenvironment of the hemopoietic 343 tissues cloning in vitro and retransplantation in vivo, Transplantation 17 344 (1974) 331–340. 345 [2] M.F. Pittenger, A.M. Mackay, S.C. 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