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Generation of human induced pluripotent stem cells from urine samples

Abstract

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet, acquiring donor cells is, in most instances, an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances, as the isolation of urinary cells is simple (30 ml of urine are sufficient), cost-effective and universal (can be applied to any age, gender and race). Moreover, the entire procedure is reasonably quick—around 2 weeks for the urinary cell culture and 3–4 weeks for the reprogramming—and the yield of iPSC colonies is generally high—up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential, and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.

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Figure 1: Morphology of urinary cells at different time points after collection.
Figure 2: Retrovirus infection and UiPSC generation.
Figure 3: UiPSCs show robust differentiation potential.

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Acknowledgements

This work was funded by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01020106), the National Natural Science Foundation of China (31071309), the Ministry of Science and Technology of China 973 program (2011CB965200), the Hong Kong Research Grant Council Collaborative Research Grant (HKU8/CRF/09), Theme Based Research Scheme (T12-705/11), General Research Fund (HKU 780110M), Austrian Science Fund (S93-06) and Genome Research Austria (GEN-AU 820982).

Author information

Affiliations

Authors

Contributions

M.A.E., J.G. and R.G.-V. conceived the original idea. M.A.E., T.Z., C.B. and S.D. devised the protocol. J.G. and R.G.-V. provided advice for urinary cell isolation. T.Z., C.B. and S.D. performed most of the experiments. Y.H. and Y.L. contributed to these experiments. J.C.H., J.Y., Y.W., Y.Z., Q.Z. and X.B. contributed to the more recent adaptations of the protocol and participated in patient-specific UiPSC generation. M.A.E. supervised the experiments. M.A.E., H.-F.T., J.G. and R.G.-V. provided financial support. D.P. provided infrastructure support. M.A.E., T.Z. and C.B. wrote the manuscript and approved the final version.

Corresponding author

Correspondence to Miguel A Esteban.

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Competing interests

J.G. and R.G.V. are cofounders and directors of Evercyte.

Supplementary information

Supplementary Figure 1

Collection and isolation of urinary cells. 30-200 ml of urine can be collected with a disposable container. Urine samples are then transferred to 50 ml tubes and centrifuged. The pellets are resuspended and pooled into a single 50 ml tube. Cells are then washed carefully and transferred to a single well of a 12-well plate in primary medium. Urinary cell colonies routinely appear within the first 4 days of culture. (PDF 2978 kb)

Supplementary Table 1

Urinary cell collection and UiPSC generation in our laboratories. F: Female; M: Male; SCN5A: Sodium channel protein type 5 subunit alpha; LMNA: Lamin A/C; LAMP2: Lysosomal-associated membrane protein 2; LDLR: Low density lipoprotein receptor. (PDF 28 kb)

Supplementary Table 2

Characterization of urinary cells. The expression of all the listed markers was detected by qPCR and/or immunofluorescence, as previously reported1. UC = urinary cells, RPTECs = renal proximal tubular epithelial cells. No expression = -, low expression = +, high expression = ++, very high expression = +++. (PDF 30 kb)

Supplemenatry Table 3

Tools for characterization of urinary cells. NR3C2, L1CAM and SLC2A1 in panel b are renal epithelial markers. (PDF 270 kb)

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Zhou, T., Benda, C., Dunzinger, S. et al. Generation of human induced pluripotent stem cells from urine samples. Nat Protoc 7, 2080–2089 (2012). https://doi.org/10.1038/nprot.2012.115

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