Adhesion strength–based, label-free isolation of human pluripotent stem cells

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We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs), partially reprogrammed cells, somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over 10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free, enriched to 95%–99% purity with >80% survival, and had normal transcriptional profiles, differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells.

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Figure 1: Adhesion of hiPSCs undergoing reprogramming and differentiation.
Figure 2: Adhesion strength–based isolation of pluripotent stem cells in microfluidic devices.
Figure 3: Adhesion strength–based isolation of hiPSCs from a heterogeneous reprogramming culture.
Figure 4: Adhesion strength–based enrichment of hiPSCs from differentiating cultures.


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This work was supported by an American Recovery and Reinvestment Act (ARRA) supplement to the US National Institutes of Health (NIH) grant R01 GM065918 (A.J.G.), NIH R43 NS080407 (J.M.C. and A.J.G.), the Stem Cell Engineering Center at Georgia Institute of Technology (T.C.M.), a Sloan Foundation Fellowship (H.L.), the National Science Foundation (NSF) DBI-0649833 (H.L.) and an ARRA sub-award under RC1CA144825 (H.L.), NSF CMMI-1129611 (J.F.), the Georgia Tech Emory Center for Regenerative Medicine and the Parker H. Petit Institute for Bioengineering and Bioscience at Georgia Institute of Technology. We thank J. Wu (Stanford Univ.) for providing blood cell–derived hiPSCs, C. Xu (Emory Univ.) for providing hiPSC-derived cardiomyocytes, T. Hookway for cardiomyocyte culture, A. Ortiz for teratoma studies, and A. Cheng for her help with focal adhesion assays. We thank J. Phillips, D. Dumbauld, Y. Wang and A. Bratt-Leal for insightful discussions and suggestions.

Author information

A.S. and S.S. conducted all adhesion and microfluidic studies, collected data and performed data analysis. S.S., A.S. and H.L. developed microfluidic methods. J.M.C. and S.L.S. established and provided the IMR90-derived hiPSC cells and neural stem cells. T.L., W.C. and J.F. developed and provided micropatterns. M.T.C. and A.S. conducted microarray and epigenetic analysis. A.J.G. and T.C.M. developed the concept, and together with A.S. contributed to the planning and design of the project. A.S., S.S., T.C.M., and A.J.G. wrote the manuscript, and all authors discussed the results and commented on the manuscript.

Correspondence to Andrés J García.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–20 and Supplementary Table 1 (PDF 22257 kb)

Real-time detachment of hiPSCs cocultured with IMR90 cells by μSHEAR

A shear stress of 125 dynes cm–2 was applied to achieve selective detachment. White arrows denote contaminating fibroblasts. (MOV 31906 kb)

Real-time detachment of blood-derived hiPSCs cocultured with blood cells by μSHEAR

A shear stress of 10 dynes cm–2 was applied for 1 min to deplete blood cells, and then the shear stress was increased to 100 dynes cm–2 to achieve selective hiPSC detachment. (MOV 1111 kb)

Beating cardiomyocytes pre-μSHEAR

Cultured hiPSC-derived cardiomyocytes demonstrate spontaneous contractile activity. (MOV 7992 kb)

Beating cardiomyocytes post-μSHEAR

After μSHEAR, residual hiPSC-derived cardiomyocytes in the devices that were were trypsinized and cultured demonstrate spontaneous contractile activity. (MOV 11720 kb)

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Singh, A., Suri, S., Lee, T. et al. Adhesion strength–based, label-free isolation of human pluripotent stem cells. Nat Methods 10, 438–444 (2013) doi:10.1038/nmeth.2437

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