Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells

Abstract

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic.

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Figure 1: Differentiation of human PSCs towards intermediate mesoderm-like cells.
Figure 2: Differentiation of human PSCs into intermediate mesoderm ureteric bud progenitor-like cells.
Figure 3: Maturation and integration of differentiated human PSCs into ureteric bud on 3D organ culture.
Figure 4: Differentiated human PSCs demonstrate a ureteric-bud-committed fate.
Figure 5: Differentiation and integration of polycystic kidney disease patient iPSCs into ureteric bud structures in 3D organ cultures.

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Acknowledgements

We thank M. Schwarz for administrative support. We thank J. Kasuboski from Waitt Advanced Biophotonics Core at Salk for help with imaging processing. We thank B. C. Lu for his suggestions regarding kidney dissection and organ culture. Y.X. and K.S. were partially supported by the California Institute for Regenerative Medicine. I.S-M. was partially supported by a Nomis Foundation postdoctoral fellowship. Work in the laboratory of J.C.I.B. was supported by grants from Fundacion Cellex, the G. Harold and L. Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust, IPSEN Foundation, Fundació La Marató de TV3 (121330), CIBER BBN and ISCIII-TERCEL-MINECO.

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Y.X., I.S-M., E.N. and J.C.I.B. designed all experiments. Y.X., I.S-M., E.N. and J.C.I.B. wrote the manuscript. Y.X., I.S-M., T.G. and E.N. performed and analysed all experiments. D.O., I.D., C.R.E. and Y.X. performed in vivo experiments. N.M. was responsible for cell culture and maintenance of the patient samples related to this work. K.S. and M-Z.W. provided reagents. J.M.C. contributed to the overall design of the project.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 iPS cells derived by non-integrative approaches demonstrated the typical hallmarks of pluripotency.

a) Immunofluorescence analysis demonstrating expression of pluripotency markers. b) Karyotype of iPSCs (Fibro-Epi). (c) mRNA fold change of pluripotency markers and different lineage markers during EB differentiation of iPSC/ESCs. (d) Copy numbers of episomal vectors in iPSCs (Fibro-Epi), human ESCs and fibroblasts infected with episomal vectors. Data are represented as mean +/− SD. *p<0.05, (c,d) n = 6 (3 dishes-containing cells per experiment x 2 independent experiments). Scale bars: 100 μm (a).

Supplementary Figure 2 Differentiation of hPSCs into intermediate mesodermlike cells does not induce other lineages.

a,b) mRNA fold change of endodermal and ectodermal-related genes during the course of differentiation of human iPSCs (a) and ESCs (b). Data are represented as mean +/− SD. *p<0.05, n = 9 (3 dishes-containing cells per experiment×3 independent experiments).

Supplementary Figure 3 Differentiation of hPSCs into intermediate mesoderm UB progenitor-like cells.

a) Immunofluorescence analysis demonstrating specificity of the Human Nuclear antigen antibody used in the presence of pure murine cultures. b,c) Immunofluorescence analysis demonstrating specificity of the in vitro differentiation protocol of human PSCs. Non-renal epithelial cells of human origin failed to induce any chimeric UB-like structure. n = >3. Scale bars: 50 μm (a,b,c).

Supplementary Table 1 List of primers used for real-time PCR experiments.

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Xia, Y., Nivet, E., Sancho-Martinez, I. et al. Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nat Cell Biol 15, 1507–1515 (2013). https://doi.org/10.1038/ncb2872

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