induced pluripotent stem ( ips ) cells can be directly generated from fibroblast cultures by expression of four factors ( 4f ) , including octamer - binding transcription factor 4 , sex - determining region y - box 2 , kruppel - like factor 4 , and myelocytomatosis viral oncogene homolog . although these factors present attractive sources for stem cell therapy , the mechanisms by which they are generated are not fully understood . the inefficiency of ips cell generation has prompted the development of two contending models , namely , the stochastic and elite models . a recent multilineage - differentiating stress - enduring cell study suggested the greater utility of the elite model over the stochastic model . in either model , stem cells or progenitor cells isolated for experimental or therapeutic research are usually heterogeneous populations ; therefore , a more favorable subfraction for use in stem cell therapy may exist . indeed , several reports have suggested that the differentiation stage of the starting cell line has a critical influence on the efficiency of reprogramming into ips cells in hematopoiesis . immature cell populations , such as linc - kitsca1cd48cd150cd34 ( lack of lineage / hardy - zuckerman 4 feline sarcoma viral oncogene homolog / spinocerebellar ataxia / cluster of differentiation 48/150/34 ) hematopoietic stem cells , in general give rise to ips cells at higher efficiencies than terminally differentiated cell types . furthermore , a recent study demonstrated that cd90 adipose tissue - derived mesenchymal stem cells ( adscs ) are more capable of forming bone both in vitro and in vivo than in cd105 cells . cd90 ( thy-1 ) is a 2537-kda glycosylphosphatidylinositol - anchored glycoprotein expressed mainly in leukocytes that have been used as murine pan t - cell markers , like cd2 , cd5 , and cd28 . multipotent mesenchymal stem cells ( mscs ) , which represent a nonhematopoietic cell population , can differentiate into mesenchymal tissues ( i.e. , bone , cartilage , or fat ) [ 5 , 6 ] ; they were first isolated from bone marrow and later from nonmarrow tissues , including umbilical cord blood and adipose tissue [ 5 , 6 ] . immunologically , mcss are known to be less immunogenic because of , at least in part , the lack of surface - expressed human leukocyte antigens . moreover , they have been shown to possess anti - inflammatory and immunosuppressive effects both in murine and human models of diabetes [ 7 , 8 ] . thus , the immunoregulatory properties of mscs are appealing , because they inhibit t - cell proliferation and differentiation of monocytes to dendritic cells , modulate b - cell functions , and suppress natural killer cell cytotoxicity . solid organ transplantation is an established and useful treatment option for end - stage organ failure . despite excellent short - term results , long - term survival of transplanted grafts has not improved accordingly because of graft rejection , graft fibrosis , or side effects of immunosuppressants . the aim of stem cell therapy for solid organ transplantation is to prevent or treat acute rejection and to involve autologous or allogeneic stem cell transplantation into patients , either through local delivery or systemic infusion . currently , there are over 100 ongoing clinical trials to evaluate the utility of mscs , especially in kidney transplantation [ 911 ] . adscs have been shown to consist of heterogeneous subpopulations ; therefore , we hypothesized that cd90 adscs might exhibit increased reprogramming capacities and subsequently have better immunoregulatory effects . hence , the aim of the present study was to compare the reprogramming capacity of sorted murine cd90 adscs . our data demonstrated that cd90 selection improved the reprogramming capacity of murine adscs , suggesting that cd90 adscs could help prevent graft rejection . murine adscs were isolated from b6 mice as previously described but with slight modifications . briefly , adipose tissue was obtained from the inguinal fat pads , washed with dulbecco 's phosphate - buffered saline ( d - pbs ; invitrogen , carlsbad , ca , usa ) containing 50  u / ml of penicillin and 50  g / ml of streptomycin ( psm ; invitrogen ) , and then cut into fine pieces , which were incubated with d - pbs containing 1.0  mg / ml of clostridium histolyticum collagenase ( sigma - aldrich , st . louis , mo , usa ) in a 37c shaking incubator for 1  h. the digested tissue was filtered through sterile 70-m nylon mesh , centrifuged at 430  g for 5  min , and resuspended in dulbecco 's modified eagle 's medium ( dmem ) ( nacalai tesque , kyoto , japan ) supplemented with 10% fetal bovine serum ( fbs ; hyclone ; thermo scientific , waltham , ma , usa ) and psm . murine adscs were washed with d - pbs and treated with accutase reagent ( emd millipore , billerica , ma , usa ) in a 37c incubator for 2 - 3  min to dissociate the cells , which were then resuspended in staining media [ d - pbs supplemented with fbs ( 1% ) and edta ( 2  mm ) ] . next , the cells were stained with fluorescein isothiocyanate - conjugated anti - mouse cd34 ( ebioscience , san diego , ca , usa ) , phycoerythrin - conjugated anti - mouse cd31 , efluor450 antimouse cd45 , and allophycocyanin - conjugated anti - mouse cd90.2 for 60  min at 4c . the cells were then washed with staining media , stained with 7-aminoactinomycin d to exclude dead cells , and sorted using the facsaria cell sorter ( bio - rad , hercules , ca , usa ) . the particular phenotypic subsets of cd90 and cd90 adscs , based on cd31 , cd45 , and cd90 expression , were evaluated by multicolor analysis and sorted by adequate gating . to prepare the lentiviruses , human embryonic kidney ( hek)-293ta cells were cultured in dmem supplemented with 10% fbs and psm . then , the cells were seeded at 5.0  10 cells per 100  mm dish 1 day before viral transduction . fugene 6 transfection reagent ( 22.5  l ; promega , madison , wi , usa ) was diluted with 500  l of dmem and incubated for 5  min at room temperature . plasmid dna ( 2.5  g ) was added to the mixture , which was incubated for an additional 15  min at room temperature . then , the culture media was replaced with fresh dmem supplemented 10% fbs and the dna / fugene 6 mixture was added dropwise onto the hek-293ta cells . the medium was replaced after 24  h. after an additional 48  h , virus - containing supernatants , derived from the hek-293ta cultures , were filtered through a 0.22-l cellulose sorted cd90 and cd90 cells were plated onto 6-well culture plates at a density of 1.0  10 cells / well , respectively . after incubation for 24  h , the culture medium was retrieved and viral supernatants were added to each well . mouse embryonic fibroblasts ( mefs ) were mitotically inactivated by the addition of 10  g / ml of mitomycin c ( mmc ; nacalai tesque inc . , kyoto , japan ) for 90  min and then washed with d - pbs three times . the cells were then cultured with complete medium for at least 2  h , trypsinized , counted , and plated on gelatin - coated 60  mm dishes at a density of 5.0  10 cells / well . on posttransduction day 5 , the cells were trypsinized ; suspended in dmem supplemented with 15% ( v / v ) fbs , psm , 1-mm sodium pyruvate ( invitrogen ) , 10 m 2-mercaptoethanol ( nacalai tesque , inc . ) , nonessential amino acid solution , and 1,000  u / ml of leukemia inhibitory factor ; after which they were counted . then , 1000 cells were transferred to mmc - treated mef feeder cells . on post - transduction day 30 , alkaline phosphatase ( ap ) 4f - transduced adscs were stained using an ap staining kit ( muto pure chemicals , tokyo , japan ) following the manufacturer 's recommended protocol . briefly , cultured cells were rinsed twice with d - pbs and then fixed for 5  s in ice - cold methanol . then , the cells were washed with tap water , stained with ap solution at 37 for 120  min , and washed again with running water . as shown in figure 2 , parental adsc showed no colonies and ap staining was negative ( negative control ) . next we used sorted adscs to assess the reprogramming capacities of cd90 and cd90 adscs . as shown in figure 3(a ) , the cd90 marker was widely expressed on the surfaces of the cd31cd45cd34adscs . after 24  h of incubation , sorted cd90 or cd90 adscs became attached to the culture dishes ( figure 3(b ) ) and both showed similar morphologies . as shown in figure 4(a ) , 7-day culture resulted in the reexpression of cd90 even after sorted cd90 adscs . these results suggest that sorted cells might be transduced as soon as possible . to show the successful induction of the undifferentiated state , we employed immunocytochemistry of oct4 and ssea1 . as shown in figure 5 , colonies were positive for oct4 and ssea1 . sorted and 4f - transduced adscs also exhibited colony formation . as shown in figures 6(a ) and 6(b ) , morphologically distinct colonies were visible in both cd90 and cd90 4f - transduced adscs on posttransduction day 14 . there were ap+ colonies in both groups , although the cd90 adscs tended to form larger colonies than cd90 adscs ( figures 4(c ) and 4(d ) ) . as shown in figure 7 , cd90 adscs exhibited more ap+ colonies than  cd90 adscs . the reprogramming efficiencies of unsorted , cd90-sorted , and cd90-sorted adscs were 100% , 116.5% , and 74.7% , respectively . in the present study , we demonstrated that murine adscs can be reprogrammed by standard 4f transduction . furthermore , ips cell formation was also observed in cd90-based sorted cells and the cd90 sorting resulted in enhanced reprogramming capacity of murine adscs compared with cd90 adscs with regard to colony number . moreover , there was a trend in the association between cd90 expression level and individual colony size . these results clearly demonstrated that adscs have heterogeneous subpopulations and that the cd90 adscs present favorable candidates for the application of clinical stem cell therapy . the importance of cd90 in oncogenesis or properties of cancer stem cells has been reported . cd90 expression reportedly has prognostic values in esophageal squamous cell carcinoma ( escc ) patients , because higher cd90 expression was significantly associated with a strong family history of escc and higher incidences of lymph node metastasis . however , the importance of cd90 expression in mscs has not been fully elucidated ; therefore , our next project is to clarify the mechanisms of cd90-mediated immunomodulatory effects . of note , recent study reported that cd90-expressing niche stromal cells would support hematopoiesis . stem cell therapy for autoimmune disease is also well described . in a murine inflammatory bowel disease model , thy-1 is also expressed by thymocytes , peripheral t cells , myoblasts , epidermal cells , and keratinocytes . we and others previously reported that it is possible to reprogram murine and human cells to pluripotency by direct transfection of mature double - stranded micrornas without viral vectors [ 1618 ] . currently , studies are underway to compare the immunoregulatory properties of viral - free strategies both in vitro and in vivo .
background . mesenchymal stem cells ( mscs ) , including adipose tissue - derived mesenchymal stem cells ( adsc ) , are multipotent and can differentiate into various cell types possessing unique immunomodulatory features . 
 several clinical trials have demonstrated the safety and possible efficacy of mscs in organ transplantation . 
 thus , stem cell therapy is promising for tolerance induction . in this study , we assessed the reprogramming capacity of murine adscs and found that cd90 ( thy-1 ) , originally discovered as a thymocyte antigen , could be a useful marker for cell therapy . 
 method . 
 murine adscs were isolated from b6 mice , sorted using a facsaria cell sorter by selection of cd90hi or cd90lo , and then transduced with four standard factors ( 4f ; oct4 , sox2 , klf4 , and c - myc ) . 
 results . 
 unsorted , cd90hi - sorted , and cd90lo - sorted murine adscs were reprogrammed using standard 4f transduction . 
 cd90hi adscs showed increased numbers of alkaline phosphatase - positive colonies compared with cd90lo adscs . 
 the relative reprogramming efficiencies of unsorted , cd90hi - sorted , and cd90lo - sorted adscs were 100% , 116.5% , and 74.7% , respectively . 
 cd90hi cells were more responsive to reprogramming . 
 conclusion . 
 cd90hi adscs had greater reprogramming capacity than cd90lo adscs , suggesting that adscs have heterogeneous subpopulations . 
 thus , cd90hi selection presents an effective strategy to isolate a highly suppressive subpopulation for stem cell - based tolerance induction therapy .