Potential use of adipose tissue stem cells in the control of aging

52 www.app oaches oagingcon ol.o g Abs ac Cell he apy wi h adul s em cells is a new ba le on o he con ol o aging. Be o e being used o his pu pose, we need o answe se e al basic ques ions abou he biochemis y and physiology o hese cells. This pape p esen s some aspec s and p elimina y esul s ob ained in ou labo a o y using s em cells om adipose issue. In oduc ion Va ious s a egies a e used o con ol he e ec s o aging on he no mal unc ions o an o ganism. An ioxidan s, die , exe cise and e en ac i a ing enzymes o phase II de oxi ica ion appea o delay he decay unc ion o a g ea e o lesse ex en . Howe e , none o hese s a egies p e en biological aging. Hence, o con ol he e ec s o aging i will be necessa y o emphasize he need o explo e he mechanisms o issue epai and egene a ion as a complemen a y me hodology o o he p e en ion s a egies. In his sense, Regene a i e Medicine may ep esen a ascina ing al e na i e o con ol he aging p ocess. One o he aspec s o Regene a i e Medicine is based on he egene a i e capaci y o adul s em cells a e in mos issues and con ibu e o his homeos asis and epai . A e issue damage se e al in acellula and in e cellula pa hways a e ac i a ed in a coo dina ed a emp o es o e he issue in eg i y. Wi h aging occu s gene al decline in he egene a ion po en ial. Besides his loss o egene a i e capaci y is p oduced by al e a ions o he s em cells wi h aging, i is hough ha s em cells ansplan ed in o damaged o aged issues may ha e he apeu ic and es o a i e capaci y. In ac , s em cells ep esen a huge p omise in he he apy o many aging- ela ed degene a i e diso de s aging some diseases such diabe es, hea disease, s oke, Pa kinson, e c [1-4] Po en ial use o adipose issue s em cells in he con ol o aging Ma io F. Muñoz1, F ancisco Zu i a1, Sand o A güelles1, Rocío Vazquez2, José Se es3, Ma ías Guzmán4, Remedios Guillén4, José An onio Rod íguez5, José Luis Co és6, Jaime Muñoz7, José An onio Pin o 7, Me - cedes Cano8 and An onio Ayala1. 1 Depa amen o de Bioquímica y Biología Molecula , Facul ad de Fa macia. Uni e sidad de Se illa. Spain. 2 Clínica Rocío Vazquez. Se illa. 3 Clínica Se es. Se illa. 4 Depa amen o de B oma ología, Toxicología y Medicina Legal. Facul- ad de Fa macia. Uni e sidad de Se illa. 5Ins i u o de Biomedicina de Se illa. Spain. 6 Cen o de In es igación Biomé- dica, G anada. Spain. 7 Cen o Andaluz de Biología Molecula y Medicina Regene a i a, Se illa. Spain.8 Depa amen o de Fisiología. Facul ad de Fa macia. Uni e sidad de Se illa Co espondence: An onio Ayala ,Dp o. Bioquímica y Biología Molecula . Facul ad de Fa macia. Uni e sidad de Se illa. C/.T amon ana s/n. 41012 - Se illa Keywo ds. S em cells; adipose issue; aging. Acknowledgemen s. This wo k was suppo ed by Spanish Minis e io de Ciencia e Inno ación BFU 2010 20882. 53 Ap p o A c h e s o Ag i n g co n o l . Vo l 16. se p e m b e 2012 S em cells S em cells ha e he abili y o sel - enew h ough symme ic di isions and he po en ial o di e en ia ing in o se e al di e en cell ypes depending on hei deg ee o mul ipo en iali y h ough asymme ic di ision [1-3,5-8]. I is now known ha mos o he issues ha e a e y speci ic popula ion o adul s em cells ha allow hei egula enewal o egene a ion [6,8]. Thus, i has been desc ibed he exis ence o hese SC in issues such as muscle, skin, li e , panc eas, b ain [9], in es ines, a y issue [10,11]. In highe animals, s em cells ha e been classi ied in o wo g oups: emb yonic s em cells and o gan- speci ic s em cells o s em cells om adul issue. The la e a e able o o igina e cells o a pa icula o gan in he adul . Ou wo k ocuses on adul s em cells. The ac i i y o hese s em cells a ies g ea ly om one o gan o ano he : hose o he bone ma ow ha o m blood cells a e e y ac i e and a e con inually di iding, while hose which a e, o example, in he small in es ine a e mo e inac i e. Some adul s em cells a e capable o di e en ia ing in o mo e han one cell ype as mesenchymal s em cells. Adipose issue mesenchymal s em cells (ADSC) Wi hin he adul s em cell g oup a e he mesenchymal s em cells (MSC). The MSC belong o he mesenchyma, which by a di e en ia ion p ocess will lead o he blood essels, smoo h muscle, meso helium, lympha ic sys em and connec i e issue i sel . These cells can be ob ained om di e en o gans including e al li e , umbilical co d, and bone ma ow [12,13]. Ano he impo an sou ce is adipose issue, which con ains p ogeni o cells called adipose issue s em cells (ADSC) [2,10,14,15]. Ad an age o ADSC The easy access o he subcu aneous a by liposuc ion, allows ADSC o be ob ained unde local anes hesia and wi h minimal discom o o he pa ien . In addi ion, i s high abundance wi h no e hical p oblems associa ed wi h i s use, make adipose issue an impo an sou ce o MSC [16- 18]. Ano he ad an age is ha he p opo ion o MSC is 500 imes highe [19,20] in adipose issue han in he bone ma ow, so ha a la ge numbe o cells can be ob ained wi hou a la ge numbe o passes, dec easing he isk o ch omosomal abno mali ies induced senescence in cul u es [21]. An addi ional ad an age is i s po en ial o di e en ia e in o bone, ca ilage, endons, skele al muscle, a , endo helial issue and mac ophages when g own unde speci ic condi ions o each lineage [11,13,22]. Su p isingly, he ADSC no only ha e he po en ial o di e en ia e in o cells o mesode mal o igin and o gans, bu also hey ha e he abili y o di e en ia e in o neu ons, endoc ine cells o he panc eas, hepa ocy es, endo helial cells and ca diomyocy es [10]. ADSCs can epai and egene a e he issues by se e al mechanisms: Fi s , he ADSC ansplan ed in o a damaged o diseased issue can sec e e cy okines and g ow h ac o s ha s imula e he eco e y in a pa ac ine manne . The ADSC could modula e he hos s em cell niche by s imula ing he ec ui men o endogenous s em cells o a pa icula si e and p omo e hei di e en ia ion in o he equi ed lineage. Also, ADSC can p o ide an ioxidan s so ha oxic subs ances eleased in o 54 www.app oaches oagingcon ol.o g he local en i onmen a e elimina ed, he eby p omo ing he eco e y o he su i ing cells. Ano he mechanism is hough hei in i o di e en ia ion in o he desi ed line, p io o hei au ologous ansplan a ion [2]. They can also ac as immune modula o s [23]. Today i is possible o isola e and cul u e adul s em cells o he apeu ic use. Once ansplan ed, hese cells can be used o eju ena e damaged issue by aging o o he causes. Howe e , no one knows o su e he consequences o he s em cells ea men s because he e a e many basic ques ions abou he biology o hese CM ha mus be answe ed be o e i s he apeu ic use. Ob iously, hese cells ha e o su i e and pe o m hei unc ion in un a ou able condi ions as he damaged issue mic oen i onmen , whe e many ac o s needed o s em cells a e lacking. Unde hese ad e se condi ions, he su i al o he s em cells will depend on his “molecula heal h” and i s esponsi eness when implan ed in he issue. The e o e, s em cells mus ha e a obus epai mechanisms and esis ance in o de o pa icipa e in issue egene a ion. I is likely ha his molecula heal h and s ess esponse depends on many biochemical and physiological aspec s ela ed o dono age, li es yle, e c. As hese cells a e con inuously ecei ing “on-o ” signals o di ide, i could be ha old cells a e less sensi i e o hese signals. Consequen ly, he he apeu ic applica ions o s em cells in adul issue epai equi es a be e unde s anding o he biology o hese cells, o he en i onmen o he damaged issue o bo h. The wo k we a e ca ying ou in ou labo a o y y o answe se e al o hese basic ques ions abou ADSC. Fi s o all, we ocused on he isola ion me hods. These me hods a e based on magne ic cell so ing (Mil enyi Bio ech), whe e magne ic mic opa icles linked o an ibodies ecognize su ace an igen in he s em cells. The sepa a ion is pe o med on a column inse ed in an ex emely powe ul magne o e ain he labeled cells. This me hodology allows us o build a cell bank o pa ien s o di e en ages ha mee he c i e ia o “Mesenchymal and Tissue S em Cell Commi ee o he In e na ional Socie y o Cellula The apy.” The i s c i e ia is ha cells mus be adhe en o he plas ic. The pho og aphs o Figu e 1 show ha he human ADSC a e adhe en o he cul u e lask wi hou addi ion o any subs a e. Figu e 1. Human ADSC (42 y s) in cul u e lask. 10x magni ica ion. A p e equisi e o conside ing he cul u ed ADSC as MSC, is o demons a e ha hey ha e he po en ial o di e en ia e in o a leas wo di e en cell ypes [11,13,22,24]. Fo his, cells we e cul u ed in speci ic di e en ia ing media. Figu e 2 shows pho og aphs aken be o e and a e 55 Ap p o A c h e s o Ag i n g co n o l . Vo l 16. se p e m b e 2012 di e en ia ion in o adipocy es, whe e i can be obse ed usi o m cells con aining lipid acuoles. The ed s aining o hese acuoles (Fig. 2 and B) shows he di e en ia ion in o adipocy es ADSCs. Fu he mo e, he p esence o black-pu ple shown by he p esence o alkaline phospha ase in os eoblas s gene a ed by ADSC di e en ia ion (Fig. 2C) In addi ion o he abili y o adhe ence o plas ic and di e en ia ion, he esul s p o ided by low cy ome y se e o con i m he na u e o he cells ob ained. Figu e 3 show he esul s ob ained om a cell popula ion o human ADSC. These g aphs ep esen s side sca e (Side Sca e , SS), which gi es in o ma ion abou he exis ence o di e en cell popula ions. The o wa d sca e ( o wa d sca e , FS), gi es an insigh on cell size. As can be seen, he sample con ains a unique cell popula ion, e lec ed in he g aph as a single cloud. To e i y ha he ob ained cells mee he minimum s anda ds equi ed by he commi ee o mesenchymal s em cells and issues o he in e na ional cell he apy, cells mus ha e he su ace ma ke s CD73, CD90 and CD105. On he con a y, hey should no con ain CD14, CD34, CD45, CD19, CD79α and human leukocy e an igen (HLA)-DR. Figu e 2. Di e en ia ion o human ADSC in o adipocy es and os eoblas s. A) Pic u e o human adipocy es ob ained om ADSC (pa ien o 37 yea s) p io o s aining, 10X magni ica ion. B) Pho og aph a e ixa ion and s aining p ocess. C) Pho og aph o os eoblas s a e ixing and s ai- ning 20X magni ica ion. 56 www.app oaches oagingcon ol.o g The his og ams ep esen he numbe o e en s coun ed on each exposed channel. In ou case, he channel FL1, FL2 and FL4 a e se o exci e he luo opho es FITC, PE, Pe CP, espec i ely. Conside ing he an ibodies used, (CD14-Pe CP, CD20-Pe CP, CD34-Pe CP, CD45-Pe CP, CD105-FITC and CD90-PE), FL1 channel gi es us in o ma ion on he numbe o cells exp essing CD90 ma ke (Fig. 3B); FL2 channel gi es in o ma ion abou he p opo ion o CD105- exp essing cells (Fig. 5C) and FL4 channel indica es he pe cen age o posi i e cells is any o he ollowing ma ke s: CD14, CD20, CD34 and CD45 (Fig. 3D). The i s his og am (Fig. 3B) indica es ha 94.53% o he sample is posi i e o he CD90 ma ke . The second his og am (Fig. 3C) shows ha almos 100% o he sample exp essed he ma ke CD105. Only 9.5% o he cells ha e one o he ollowing ma ke s: CD14, CD20, CD34, CD45. Finally, we ha e used a me hod o labeling and acking ha allow us o moni o s em cells once hey a e injec ed in i o. This me hod is based in ans ec ing he ADSC wi h he luci e ase gene by using len i i us. A e in ape i oneal injec ion o luci e in, cells ca ying he luci e ase gen can be obse ed by using an in i o imaging sys em. Figu e 4A shows a cul u e o ans ec ed ADSC in a 25 cm 2 lask p io o adminis a ion o 3 luci e in used as a con ol o bioluminescence. Figu e 4B shows he same cul u e a e he adminis a ion o D-luci e in. Figu e 3. S udy o he deg ee o cellula homogenei y and pheno yping o human ADSC. Da a we e ob ained om he low cy ome e FC-500 (Beckman-Coul e ). The esul s o posi i e e en s o he luo opho e a e in %. 57 Ap p o A c h e s o Ag i n g co n o l . Vo l 16. se p e m b e 2012 Conclusion In summa y, we can say ha cell he apy open a new ba le on in he con ol and ea men o aging ela ed diseases. Howe e , he e a e many basic ques ions abou he biology o hese s em cells ha should be answe ed o p edic he he apeu ic po en ial o hese cells, some o which a e going o be known a e he comple ion o he he p esen s udy because. The main objec i e o his wo k is o iden i y he ac o s a ec ing he “ obus ness” o he biochemis y and physiology o ADSC. Re e ences [1] Bajada, S., Mazako a, I., Richa dson, J. B., and Ashammakhi, N., Upda es on s em cells and hei applica ions in egene a i e medicine, J.Tissue Eng Regen.Med. 2 (2008) 169-183. [2] Gimble, J. M., Ka z, A. J., and Bunnell, B. A., Adipose-de i ed s em cells o egene a i e medicine, Ci c.Res. 100 (2007) 1249-1260. [3] Ho, A. D., Wagne , W., and Mahlknech , U., S em cells and ageing. The po en ial o s em cells o o e come age- ela ed de e io a ions o he body in egene a i e medicine, EMBO Rep. 6 Spec No (2005) S35-S38. [4] Teo, A. K. and Vallie , L., Eme ging use o s em cells in egene a i e medicine, Biochem.J. 428 (2010) 11-23. 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