Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes

Abstract

The utility of induced pluripotent stem (iPS) cells for investigating the molecular logic of pluripotency and for eventual clinical application is limited by the low efficiency of current methods for reprogramming. Here we show that reprogramming of juvenile human primary keratinocytes by retroviral transduction with OCT4, SOX2, KLF4 and c-MYC is at least 100-fold more efficient and twofold faster compared with reprogramming of human fibroblasts. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, global gene expression profiles and differentiation potential in vitro and in vivo. To underscore the efficiency and practicability of this technology, we generated KiPS cells from single adult human hairs. Our findings provide an experimental model for investigating the bases of cellular reprogramming and highlight potential advantages of using keratinocytes to generate patient-specific iPS cells.

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Figure 1: KiPS cell colony formation and cell line characterization.
Figure 2: KiPS cells display retroviral silencing, promoter demethylation and activation of endogenous pluripotency factors.
Figure 5: q-PCR and microarray analysis comparing keratinocytes, fibroblasts, KiPS cells and ES cells.
Figure 3: KiPS cells can differentiate into all three primary germ layers in vitro and in vivo.
Figure 4: The dynamics of efficient keratinocyte reprogramming.
Figure 6: Generation and characterization of KiPS cells from a single plucked hair.

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Acknowledgements

We are grateful to Ignacio Pizá Rodriguez for help and advice with KiPS cell characterization; José Miguel Andrés Vaquero for assistance with FACS analysis; Meritxell Carrió for expert assistance with cell culture techniques; Esther Melo, Lola Mulero Pérez and Mercé Gaudes Martí for bioimaging assistance; Yvonne Richaud and Teresa Lopez Rovira for excellent technical assistance and Luciano Di Croce, Centre for Genomic Regulation, Barcelona, for the gift of c-MYC T58A plasmid. F.G. was partially supported by a fellowship from the Swiss National Science Foundation. M.J.B. and G.T. were partially supported by the Ramón y Cajal program. This work was partially supported by grants from Ministerio de Educación y Ciencia grant BFU2006-12251, European Commission 'Marie-Curie Reintegration Grant' MIRG-CT-2007-046523 the Fondo de Investigaciones Sanitarias (RETIC-RD06/0010/0016, PI061897), Marató de TV3 (063430), the G. Harold and Leila Y. Mathers Charitable Foundation and Fundación Cellex.

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Correspondence to Juan Carlos Izpisúa Belmonte.

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Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Data (PDF 24671 kb)

Supplementary Movie 1

Real-time movie of KiPS4F4 cells differentiated into beating cardiomyocytes. (MOV 3384 kb)

Supplementary Movie 2

Real-time movie of hair-derived iPS cells differentiated into beating cardiomyocytes (Hair sample 1). (MOV 2843 kb)

Supplementary Movie 3

Real-time movie of hair-derived iPS cells differentiated into beating cardiomyocytes (Hair sample 2). (MOV 2678 kb)

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Aasen, T., Raya, A., Barrero, M. et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26, 1276–1284 (2008). https://doi.org/10.1038/nbt.1503

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