Generation of infant- and pediatric-derived urinary induced pluripotent stem cells competent to form kidney organoids

Article metrics

Abstract

Background

Human induced pluripotent stem cells (iPSCs) are a promising tool to investigate pathogenic mechanisms underlying human genetic conditions, such as congenital anomalies of the kidney and urinary tract (CAKUT). Currently, iPSC-based research in pediatrics is limited by the invasiveness of cell collection.

Methods

Urine cells (UCs) were isolated from pediatric urine specimens, including bag collections, and reprogrammed using episomal vectors into urinary iPSCs (UiPSCs). Following iPSC-quality assessment, human kidney organoids were generated.

Results

UCs were isolated from 71% (12/17) of single, remnant urine samples obtained in an outpatient setting (patients 1 month–17 years, volumes 10–75 ml). Three independent UCs were reprogrammed to UiPSCs with early episome loss, confirmed pluripotency and normal karyotyping. Subsequently, these UiPSCs were successfully differentiated into kidney organoids, closely resembling organoids generated from control fibroblast-derived iPSCs. Importantly, under research conditions with immediate sample processing, UC isolation was successful 100% for target pediatric CAKUT patients and controls (11/11) after at most two urine collections.

Conclusions

Urine in small volumes or collected in bags is a reliable source for reprogrammable somatic cells that can be utilized to generate kidney organoids. This constitutes an attractive approach for patient-specific iPSC research involving infants and children with wide applicability and a low threshold for participation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3

References

  1. 1.

    Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

  2. 2.

    Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

  3. 3.

    Li, M. & Izpisua Belmonte, J. C. Organoids - preclinical models of human disease. N. Engl. J. Med. 380, 569–579 (2019).

  4. 4.

    Durbin, M. D., Cadar, A. G., Chun, Y. W. & Hong, C. C. Investigating pediatric disorders with induced pluripotent stem cells. Pediatr. Res. 84, 499–508 (2018).

  5. 5.

    Musunuru, K. Genome editing of human pluripotent stem cells to generate human cellular disease models. Dis. Model Mech. 6, 896–904 (2013).

  6. 6.

    Nicolaou, N., Renkema, K. Y., Bongers, E. M., Giles, R. H. & Knoers, N. V. Genetic, environmental, and epigenetic factors involved in CAKUT. Nat. Rev. Nephrol. 11, 720–731 (2015).

  7. 7.

    Morizane, R. et al. Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nat. Biotechnol. 33, 1193–1200 (2015).

  8. 8.

    Taguchi, A. et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 14, 53–67 (2014).

  9. 9.

    Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568 (2015).

  10. 10.

    Przepiorski, A. et al. A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells. Stem Cell Rep. 11, 470–484 (2018).

  11. 11.

    Okita, K. et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31, 458–466 (2013).

  12. 12.

    Seki, T. et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7, 11–14 (2010).

  13. 13.

    van Mil, A. et al. Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential. Cardiovasc. Res. 114, 1828–1842 (2018).

  14. 14.

    Zhou, T. et al. Generation of induced pluripotent stem cells from urine. J. Am. Soc. Nephrol. 22, 1221–1228 (2011).

  15. 15.

    Guan, X. et al. Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery. Stem Cell Res 12, 467–480 (2014).

  16. 16.

    Massa, M. G. et al. Multiple sclerosis patient-specific primary neurons differentiated from urinary renal epithelial cells via induced pluripotent stem cells. PLoS ONE 11, e0155274 (2016).

  17. 17.

    Jia, B. et al. Modeling of hemophilia A using patient-specific induced pluripotent stem cells derived from urine cells. Life Sci. 108, 22–29 (2014).

  18. 18.

    Li, G. et al. Generation of retinal organoids with mature rods and cones from urine-derived human induced pluripotent stem cells. Stem Cells Int. 2018, 4968658 (2018).

  19. 19.

    Zhou, T. et al. Generation of human induced pluripotent stem cells from urine samples. Nat. Protoc. 7, 2080–2089 (2012).

  20. 20.

    Takasato, M., Er, P. X., Chiu, H. S. & Little, M. H. Generation of kidney organoids from human pluripotent stem cells. Nat. Protoc. 11, 1681–1692 (2016).

  21. 21.

    Forbes, T. A. et al. Patient-iPSC-derived kidney organoids show functional validation of a ciliopathic renal phenotype and reveal underlying pathogenetic mechanisms. Am. J. Hum. Genet. 102, 816–831 (2018).

  22. 22.

    van den Berg, C. W. et al. Renal subcapsular transplantation of PSC-derived kidney organoids induces neo-vasculogenesis and significant glomerular and tubular maturation in vivo. Stem Cell Rep. 10, 751–765 (2018).

  23. 23.

    Kreitzer, F. R. et al. A robust method to derive functional neural crest cells from human pluripotent stem cells. Am. J. Stem Cells 2, 119–131 (2013).

  24. 24.

    Rowan, C. J. et al. Hedgehog-GLI signaling in Foxd1-positive stromal cells promotes nephrogenesis Via TGFbeta signaling. Development 145, dev159947 (2018).

  25. 25.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  26. 26.

    Phipson, B. et al. Evaluation of variability in human kidney organoids. Nat. Methods 16, 79–87 (2019).

  27. 27.

    Cain, J. E. & Rosenblum, N. D. Control of mammalian kidney development by the Hedgehog signaling pathway. Pediatr. Nephrol. 26, 1365–1371 (2011).

  28. 28.

    Benda, C. et al. Urine as a source of stem cells. Adv. Biochem. Eng. Biotechnol. 129, 19–32 (2013).

  29. 29.

    Cheng, L. et al. Generation of urine cell-derived non-integrative human iPSCs and iNSCs: a step-by-step optimized protocol. Front. Mol. Neurosci. 10, 348 (2017).

  30. 30.

    Li, D. et al. Optimized approaches for generation of integration-free iPSCs from human urine-derived cells with small molecules and autologous feeder. Stem Cell Rep. 6, 717–728 (2016).

  31. 31.

    Sauer, V. et al. Human urinary epithelial cells as a source of engraftable hepatocyte-like cells using stem cell technology. Cell Transpl. 25, 2221–2243 (2016).

  32. 32.

    Wang, L. et al. Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells. Stem Cell Res. Ther. 8, 245 (2017).

  33. 33.

    Xue, Y. et al. Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells. PLoS ONE 8, e70573 (2013).

  34. 34.

    Steichen, C. et al. Human induced pluripotent stem (hiPS) cells from urine samples: a non-integrative and feeder-free reprogramming strategy. Curr. Protoc. Hum. Genet. 92, 21.27.21–21.27.22 (2017).

  35. 35.

    Si-Tayeb, K. et al. Urine-sample-derived human induced pluripotent stem cells as a model to study PCSK9-mediated autosomal dominant hypercholesterolemia. Dis. Model Mech. 9, 81–90 (2016).

  36. 36.

    Afzal, M. Z. & Strande, J. L. Generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells. J. Vis. Exp. 52032 (2015).

  37. 37.

    Lee, K. I., Kim, H. T. & Hwang, D. Y. Footprint- and xeno-free human iPSCs derived from urine cells using extracellular matrix-based culture conditions. Biomaterials 35, 8330–8338 (2014).

  38. 38.

    Ronen, D. & Benvenisty, N. Genomic stability in reprogramming. Curr. Opin. Genet. Dev. 22, 444–449 (2012).

  39. 39.

    Tanigawa, S. et al. Organoids from nephrotic disease-derived iPSCs identify impaired NEPHRIN localization and slit diaphragm formation in kidney podocytes. Stem Cell Rep. 11, 727–740 (2018).

  40. 40.

    Smith, J. M., Stablein, D. M., Munoz, R., Hebert, D. & McDonald, R. A. Contributions of the Transplant Registry: The 2006 Annual Report of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS). Pediatr. Transpl. 11, 366–373 (2007).

Download references

Acknowledgements

We thank Natasha Jawa, Wei Wei, Alina Piekna, Monica Piekut, Michele Reddon, and Josefina Brooks for their support. We thank Dr. Bruce Conklin (Gladstone Institute, San Francisco, CA) for providing the WTC11 iPSCs. This work was supported by Canadian Institute of Health Research and Tier I Canada Research Chair (to N.D.R.), The Hospital for Sick Children/Research Institute/RestraComp (to J.M. and S.S.), and Medicine by Design and McLaughlin Center grants (to J.E.).

Author information

All authors met the Pediatric Research authorship requirements. Conception and design, acquisition of data, or analysis and interpretation of data: J.M., S.S., T.C., D.C.R., M.R.H., R.D.C., N.D.R. Drafting the article or revising it critically for important intellectual content: J.M., D.C.R., M.R.H., I.R., J.E., N.D.R. Final approval of the version to be published: J.M., N.D.R.

Correspondence to Norman D. Rosenblum.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mulder, J., Sharmin, S., Chow, T. et al. Generation of infant- and pediatric-derived urinary induced pluripotent stem cells competent to form kidney organoids. Pediatr Res (2019) doi:10.1038/s41390-019-0618-y

Download citation