Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul;17(1):130-9.
doi: 10.1016/j.scr.2016.06.001. Epub 2016 Jun 3.

Generation of Functional Podocytes From Human Induced Pluripotent Stem Cells

Affiliations
Free PMC article

Generation of Functional Podocytes From Human Induced Pluripotent Stem Cells

Osele Ciampi et al. Stem Cell Res. .
Free PMC article

Abstract

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.

Keywords: Differentiation; Induced pluripotent stem cells; Nephron progenitors; Podocytes.

Figures

Fig. 1
Fig. 1
Human iPSC induction towards mesoderm by the newly identified GSK3β inhibitor CP21R7. (A) Outline of the three-step differentiation protocol to obtain hiPSC-derived podocytes; (B) CP21R7 chemical structure; (C) immunofluorescence analysis of BRY and OCT4 displaying commitment to mesodermal fate and reduction of pluripotency. Scale bar 50 μm; (D) gene expression analysis confirmed the induction towards mesoderm through an increase in T (BRY) expression and the loss of pluripotency through a decrease in NANOG expression. Data are expressed as mean ± s.e.m.
Fig. 2
Fig. 2
Human iPSC specification into intermediate mesoderm cells. (A) Gene expression analysis of PAX2, OSR1 and LHX1. Data are expressed as mean ± s.e.m.; (B) representative immunofluorescence images of co-staining for OSR1/LHX1 and OSR1/PAX2 up to day 4. Scale bar 50 μm.
Fig. 3
Fig. 3
Intermediate mesoderm cell commitment into nephron progenitors. (A) Schematic representation of the five conditions tested for inducing SIX2 expression; (B) SIX2 gene expression analysis showed condition D as the most efficient for inducing SIX2. Data are expressed as mean ± s.e.m.; (C) representative immunofluorescence images of co-staining for SIX2/PAX2 and WT1 expression on day 6 of condition D. Scale bar 50 μm.
Fig. 4
Fig. 4
Podocyte specification. (A) Representative images of hiPSC-derived podocytes visualized by light microscopy (top, scale bar: 20 μm) or by scanning electron microscopy (bottom, scale bar: 10 μm); (B) proliferation assay showed no proliferation properties of hiPSC-derived podocytes during podocyte specification step as compared to cells not exposed to VRAD medium (undifferentiated cells). Data are expressed as mean ± s.e.m.; (C) immunostaining of the main podocyte markers. Scale bar: 20 μm; (D) gene expression analysis confirmed podocyte marker expression. Data are expressed as mean ± s.e.m.
Fig. 5
Fig. 5
Human iPSC-derived podocyte functionality. (A) Representative F-actin immunostaining showed peripheral localization after 24 h of Ang II exposure. Scale bar 50 μm; (B) percentage of cells showing F-actin rearrangement (*P < 0.05, **P < 0.01). Data are expressed as mean ± s.e.m.; (C) gene expression analysis showed a significant reduction in podocyte marker expression after 3 h of Ang II exposure (*P < 0.05, **P < 0.01). Data are expressed as mean ± s.e.m. (D) representative images of hiPSC-derived podocyte uptake of FITC-BSA, which accumulated in the perinuclear regions Scale bar 50 μm. (E–F) Integration of human iPSC-derived podocytes into the developing chimeric kidney organoid after 2 days in vitro. (E) iPSC-derived podocytes positive for the human marker human nuclear antigen (HNA, green) were homogeneously distributed within the chimeric organoid among ureteric bud epithelia positive for E-cadherin (red). (F) 3D reconstruction images showed iPSC-derived podocytes (HNA-positive, green) integrated into WT1-positive (white) structures (arrows). Inset: HNA-positive cell expressing WT1.

Similar articles

See all similar articles

Cited by 15 articles

See all "Cited by" articles

References

    1. Armstrong J.F. The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo. Mech. Dev. 1993;40(1-2):85–97. - PubMed
    1. Barak H. FGF9 and FGF20 maintain the stemness of nephron progenitors in mice and man. Dev. Cell. 2012;22(6):1191–1207. - PMC - PubMed
    1. Batchelder C.A. Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors. Differentiation. 2009;78(1):45–56. - PMC - PubMed
    1. Bollig F. A highly conserved retinoic acid responsive element controls wt1a expression in the zebrafish pronephros. Development. 2009;136(17):2883–2892. - PubMed
    1. Chittiprol S. Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes. Am. J. Phys. Renal Phys. 2011;301(3):F660–F671. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback