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CD90- (Thy-1-) high selection enhances reprogramming capacity of murine adipose-derived mesenchymal stem cells.

Kawamoto K, Konno M, Nagano H, Nishikawa S, Tomimaru Y, Akita H, Hama N, Wada H, Kobayashi S, Eguchi H, Tanemura M, Ito T, Doki Y, Mori M, Ishii H - Dis. Markers (2013)

Bottom Line: CD90(Hi) ADSCs showed increased numbers of alkaline phosphatase-positive colonies compared with CD90(Lo) ADSCs.CD90(Hi) ADSCs had greater reprogramming capacity than CD90(Lo) ADSCs, suggesting that ADSCs have heterogeneous subpopulations.Thus, CD90(Hi) selection presents an effective strategy to isolate a highly suppressive subpopulation for stem cell-based tolerance induction therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT

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 CD90(Hi) or CD90(Lo), and then transduced with four standard factors (4F; Oct4, Sox2, Klf4, and c-Myc).

Results: Unsorted, CD90(Hi)-sorted, and CD90(Lo)-sorted murine ADSCs were reprogrammed using standard 4F transduction. CD90(Hi) ADSCs showed increased numbers of alkaline phosphatase-positive colonies compared with CD90(Lo) ADSCs. The relative reprogramming efficiencies of unsorted, CD90(Hi)-sorted, and CD90(Lo)-sorted ADSCs were 100%, 116.5%, and 74.7%, respectively. CD90(Hi) cells were more responsive to reprogramming.

Conclusion: CD90(Hi) ADSCs had greater reprogramming capacity than CD90(Lo) ADSCs, suggesting that ADSCs have heterogeneous subpopulations. Thus, CD90(Hi) selection presents an effective strategy to isolate a highly suppressive subpopulation for stem cell-based tolerance induction therapy.

Show MeSH
Lentiviral-mediated transfer of four iPS cell factor genes in murine ADSCs. A schematic representation of the experiment is shown. Cells were transduced with four factors (4F) after 24 h of incubation. Then, 4F-transduced cells were passaged to MMC-treated MEF feeder cells on day 5. The number of reprogrammed iPS colonies was assessed on posttransduction day 30 by AP staining.
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fig1: Lentiviral-mediated transfer of four iPS cell factor genes in murine ADSCs. A schematic representation of the experiment is shown. Cells were transduced with four factors (4F) after 24 h of incubation. Then, 4F-transduced cells were passaged to MMC-treated MEF feeder cells on day 5. The number of reprogrammed iPS colonies was assessed on posttransduction day 30 by AP staining.

Mentions: Figure 1 describes the experimental protocol used in this study. First, we evaluated the reprogramming capacity of unsorted ADSCs. As shown in Figure 2, parental ADSC showed no colonies and AP staining was negative (negative control). However, 4F transduction resulted in ADSC colony formation. After culturing for 30 days, the cells were stained with AP to test iPS cell pluripotency. AP staining was positive in all colonies. These results clearly demonstrated that murine ADSCs can be reprogrammed using standard 4F. Next we used sorted ADSCs to assess the reprogramming capacities of CD90Hi and CD90Low ADSCs. As shown in Figure 3(a), the CD90 marker was widely expressed on the surfaces of the CD31−CD45−CD34−ADSCs. After 24 h of incubation, sorted CD90Hi or CD90Lo ADSCs became attached to the culture dishes (Figure 3(b)) and both showed similar morphologies. Next, we assessed the effect of in vitro culture. As shown in Figure 4(a), 7-day culture resulted in the reexpression of CD90 even after sorted CD90Lo ADSCs. These results suggest that sorted cells might be transduced as soon as possible.


CD90- (Thy-1-) high selection enhances reprogramming capacity of murine adipose-derived mesenchymal stem cells.

Kawamoto K, Konno M, Nagano H, Nishikawa S, Tomimaru Y, Akita H, Hama N, Wada H, Kobayashi S, Eguchi H, Tanemura M, Ito T, Doki Y, Mori M, Ishii H - Dis. Markers (2013)

Lentiviral-mediated transfer of four iPS cell factor genes in murine ADSCs. A schematic representation of the experiment is shown. Cells were transduced with four factors (4F) after 24 h of incubation. Then, 4F-transduced cells were passaged to MMC-treated MEF feeder cells on day 5. The number of reprogrammed iPS colonies was assessed on posttransduction day 30 by AP staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824355&req=5

fig1: Lentiviral-mediated transfer of four iPS cell factor genes in murine ADSCs. A schematic representation of the experiment is shown. Cells were transduced with four factors (4F) after 24 h of incubation. Then, 4F-transduced cells were passaged to MMC-treated MEF feeder cells on day 5. The number of reprogrammed iPS colonies was assessed on posttransduction day 30 by AP staining.
Mentions: Figure 1 describes the experimental protocol used in this study. First, we evaluated the reprogramming capacity of unsorted ADSCs. As shown in Figure 2, parental ADSC showed no colonies and AP staining was negative (negative control). However, 4F transduction resulted in ADSC colony formation. After culturing for 30 days, the cells were stained with AP to test iPS cell pluripotency. AP staining was positive in all colonies. These results clearly demonstrated that murine ADSCs can be reprogrammed using standard 4F. Next we used sorted ADSCs to assess the reprogramming capacities of CD90Hi and CD90Low ADSCs. As shown in Figure 3(a), the CD90 marker was widely expressed on the surfaces of the CD31−CD45−CD34−ADSCs. After 24 h of incubation, sorted CD90Hi or CD90Lo ADSCs became attached to the culture dishes (Figure 3(b)) and both showed similar morphologies. Next, we assessed the effect of in vitro culture. As shown in Figure 4(a), 7-day culture resulted in the reexpression of CD90 even after sorted CD90Lo ADSCs. These results suggest that sorted cells might be transduced as soon as possible.

Bottom Line: CD90(Hi) ADSCs showed increased numbers of alkaline phosphatase-positive colonies compared with CD90(Lo) ADSCs.CD90(Hi) ADSCs had greater reprogramming capacity than CD90(Lo) ADSCs, suggesting that ADSCs have heterogeneous subpopulations.Thus, CD90(Hi) selection presents an effective strategy to isolate a highly suppressive subpopulation for stem cell-based tolerance induction therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT

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 CD90(Hi) or CD90(Lo), and then transduced with four standard factors (4F; Oct4, Sox2, Klf4, and c-Myc).

Results: Unsorted, CD90(Hi)-sorted, and CD90(Lo)-sorted murine ADSCs were reprogrammed using standard 4F transduction. CD90(Hi) ADSCs showed increased numbers of alkaline phosphatase-positive colonies compared with CD90(Lo) ADSCs. The relative reprogramming efficiencies of unsorted, CD90(Hi)-sorted, and CD90(Lo)-sorted ADSCs were 100%, 116.5%, and 74.7%, respectively. CD90(Hi) cells were more responsive to reprogramming.

Conclusion: CD90(Hi) ADSCs had greater reprogramming capacity than CD90(Lo) ADSCs, suggesting that ADSCs have heterogeneous subpopulations. Thus, CD90(Hi) selection presents an effective strategy to isolate a highly suppressive subpopulation for stem cell-based tolerance induction therapy.

Show MeSH