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. 2013;35(5):573-9.
doi: 10.1155/2013/392578. Epub 2013 Oct 27.

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

Koichi Kawamoto  1 , Masamitsu Konno, Hiroaki Nagano, Shimpei Nishikawa, Yoshito Tomimaru, Hirofumi Akita, Naoki Hama, Hiroshi Wada, Shogo Kobayashi, Hidetoshi Eguchi, Masahiro Tanemura, Toshinori Ito, Yuichiro Doki, Masaki Mori, Hideshi Ishii
Affiliations

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

Koichi Kawamoto et al. Dis Markers. 2013.

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.

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Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
4F transduction resulted in iPS colony formation in unsorted murine ADSCs. Phase (a and c) and AP staining (b and d) of pre- and post- (c and d) transduction were shown.
Figure 3
Figure 3
CD90Hi and CD90Lo sorting. (a) Gates for CD90Hi  (P7) and CD90Lo  (P8) are shown. (b) Sorted cells showed similar morphologies 24 h after sorting. Then, the cells were transduced with 4F.
Figure 4
Figure 4
Phenotypical analysis after in vitro culture of sorted ADSCs.
Figure 5
Figure 5
Immunocytochemistry of iPS colonies.
Figure 6
Figure 6
Colony formation of the sorted cells. Phase (a and b) and alkaline phosphatase staining (c and d) of CD90Lo (a and c) and  CD90Hi (b and d) are shown.
Figure 7
Figure 7
Relative reprogramming efficiency of sorted cells compared with unsorted controls.
See this image and copyright information in PMC

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