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Engineered human induced pluripotent stem cell models reveal altered podocytogenesis in congenital heart disease-associated SMAD2 mutations

Nature Biomedical Engineering (2025)Cite this article

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Abstract

Clinical observations of patients with congenital heart disease carrying SMAD2 genetic variants revealed correlations with multi-organ impairments at the developmental and functional levels. Many patients with congenital heart disease present with glomerulosclerosis, periglomerular fibrosis and albuminuria. It remains largely unknown whether SMAD2 variants associated with congenital heart disease can directly alter kidney cell fate, tissue patterning and organ-level function. Here we investigate the role of pathogenic SMAD2 variants in podocytogenesis, nephrogenic cell lineage specification and glomerular filtration barrier function using a combination of CRISPR-based disease modelling, stem cell and microfluidic organ-on-a-chip technologies. We show that the abrogation of SMAD2 results in altered patterning of the mesoderm and intermediate mesoderm cell lineages, which give rise to nearly all kidney cell types. Following further differentiation of intermediate mesoderm cells, the mutant podocytes failed to develop arborizations and interdigitations. A reconstituted glomerulus-on-a-chip system showed substantial albumin leakage, as observed in glomerulopathies. This study implicates chronic heart disease-associated SMAD2 mutations in kidney tissue malformation that might inform targeted regenerative therapies.

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Fig. 1: Loss of SMAD2 shifts mesoderm lineage specification from medial to posterior fate.
Fig. 2: Loss-of-function mutations in the SMAD2 gene direct aberrant differentiation of IM cells.
Fig. 3: Mutant IM cells fail to specialize into terminally differentiated podocytes.
Fig. 4: Mesenchymal phenotype observed in vascular cells differentiated from SMAD2 mutant iPS cells.
Fig. 5: Engineering an isogenic GFB using microfluidic organs-on-chips devices.

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Data availability

The authors declare that all data supporting the findings of this study are included in the article and Supplementary Information. Raw patient data are available from the corresponding author, subject to approval from the Institutional Review Board of Harvard Medical School. The patient data have been previously published in refs. 12,29,30. Data on the clinical studies listed in the tables were obtained from refs. 3,4,11,16,34,37. Source data for clinicopathological information on patients summarized in Tables 1 and 2 are cited in the tables. The scRNA sequencing data analysis source data for ref. 106 are available at https://cellxgene.cziscience.com/collections/f7cecffa-00b4-4560-a29a-8ad626b8ee08; those for ref. 107 at Gene Expression Omnibus (GEO) accession numbers GSE151302, GSE195460 and GSE131882 (https://cellxgene.cziscience.com/collections/b3e2c6e3-9b05-4da9-8f42-da38a664b45b); those for ref. 108 at https://cellxgene.cziscience.com/collections/120e86b4-1195-48c5-845b-b98054105eec; those for ref. 109 at GEO accession number GSE183279 (https://cellxgene.cziscience.com/collections/bcb61471-2a44-4d00-a0af-ff085512674c) and those for ref. 110 at GEO accession number GSE151302 (https://cellxgene.cziscience.com/collections/9b02383a-9358-4f0f-9795-a891ec523bcc). The bubble plot was generated using the CellxGene online data viewer. Western blot stain-free blots, stain-free gels and chemiluminescent images are presented in Supplementary Figs. 4–8. Source data are provided with this paper.

Change history

  • 20 November 2025

    In the version of this article initially published, in the third paragraph of Results, the current callout to “Extended Data Fig. 2a; Fig. 1d,e” was originally incorrect, while in the seventh paragraph of Results, the current callout to “Extended Data Fig. 3b; Fig. 2c–f” was originally incorrect. The text is updated in the HTML and PDF versions of the paper.

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Acknowledgements

S.M. is a recipient of the Whitehead Scholarship in Biomedical Research, a Chair’s Research Award from the Department of Medicine at Duke University, a MEDx Pilot Grant on Biomechanics in Injury or Injury Repair, a Burroughs Wellcome Fund PDEP Career Transition Ad Hoc Award, a Duke Incubation Fund from the Duke Innovation & Entrepreneurship Initiative, a Genentech Research Award and a George M. O’Brien Kidney Center Pilot Grant (P30 DK081943), and NIH Director’s New Innovator Award (Award Number DP2DK139544), which supported the study. R.B. is a recipient of the Lew’s Predoctoral Fellowship in the Center for Biomolecular and Tissue Engineering (CBTE) at Duke University (T32 Support NIH Grant T32GM800555). We acknowledge the National Heart, Lung, and Blood Institute Pediatrics Cardiac Genomics Consortium (PCGC) investigators (R01 HL151257 (C.E.S.), 1UM1HL098166 (J.G.S.) and others) for their support and expertise. T.W. is a recipient of the Ruth L. Kirschstein National Research Service Award (NRSA) T32 Fellowship (2T32 HL 7208-46 A1). J.G.S. is supported by Foundation Leducq 16 CVD 03, and C.E.S. is supported by the Howard Hughes Medical Institute. We thank D. W. Cain from the Duke Human Vaccine Institute (DHVI) Flow Cytometry Facility for assistance with the flow cytometry experiments and data analyses; J. Faust from Evident and L. Cameron from the Department of Biology, Duke University, for helping with the glomerulus-on-a-chip microscopy; Y. Gao for assistance with the live microscopy setup; X. Mou for plasma treating the microfluidic organ chips; and Y. Roye for technical assistance with endothelial cell differentiation. We also thank all members of the Musah Lab and M. Pachino from the Graduate Communication Center (GCC) at Duke University for helpful comments on the paper.

Author information

Author notes

  1. Rohan Bhattacharya

    Present address: Stem Cell and Regenerative Biology Program, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA

  2. Titilola D. Kalejaiye

    Present address: Department of Cell Biology, Duke University, Durham, NC, USA

Authors and Affiliations

  1. Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, USA

    Rohan Bhattacharya, Titilola D. Kalejaiye, Alekshyander Mishra, Sophia M. Leeman, Hamidreza Arzaghi & Samira Musah

  2. Center for Biomolecular and Tissue Engineering, Duke University, Durham, NC, USA

    Rohan Bhattacharya, Hamidreza Arzaghi & Samira Musah

  3. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Tarsha Ward, Jonathan G. Seidman & Christine E. Seidman

  4. Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    Christine E. Seidman

  5. Howard Hughes Medical Institute (HHMI), Harvard University, Boston, MA, USA

    Christine E. Seidman

  6. Division of Nephrology, Department of Medicine, Duke University School of Medicine, Durham, NC, USA

    Samira Musah

  7. Developmental and Stem Cell Biology Program, Duke University, Durham, NC, USA

    Samira Musah

  8. Department of Cell Biology, Duke University, Durham, NC, USA

    Samira Musah

  9. Integrated Toxicology and Environmental Health Program, Duke University, Durham, NC, USA

    Samira Musah

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  1. Rohan Bhattacharya
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Contributions

S.M. and T.W. conceived the idea for this work. S.M., T.W., J.G.S. and R.B. discussed the research approach and strategy. T.W. performed CRISPR–Cas9 experiments, generated the SMAD2 mutant human iPS cell lines used in this study, analysed relevant data and interpreted the results with input from J.G.S. and C.E.S. R.B. performed stem cell differentiation experiments, analysed the data and interpreted all relevant results with input from T.D.K. and S.M. R.B. and S.M.L. performed qPCR analyses. R.B., T.D.K. and H.A. performed western blot analyses. R.B. performed microscopy and microfluidic organ chip experiments and analysed the data. T.D.K. performed protein quantification, endothelial cell differentiation and EdU analysis. T.D.K. and R.B. performed CCK-8 cell viability assay. A.M. performed cell migration analyses from data generated by R.B. and also provided new strategies for visualizing the data. R.B., T.W., T.D.K. and S.M. interpreted the study results. R.B. wrote the initial draft of the paper with input from the other authors. S.M., T.W. and S.M.L. edited the paper. T.W. was supervised by J.G.S. and C.E.S. R.B., T.D.K., A.M., S.M.L. and H.A. were supervised by S.M.

Corresponding author

Correspondence to Samira Musah.

Ethics declarations

Competing interests

S.M. is an inventor on a patent regarding podocyte differentiation held by Harvard University (US20210338736A1; US Patent App 17/366,827, 2021). S.M. is also an inventor on a pending patent application regarding engineered microphysiological systems with in vivo-like tissue structure and function held by Duke University. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Azhar Mohamad, Ryuji Morizane and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Derivation and characterization of SMAD2 mutant cell lines.

a, Schematic representation of SMAD2 mutation installation in human iPS cells. b, Introduction of SMAD2 variants into human iPS cells was confirmed by Sanger Sequencing. c, Representative phase contrast images of WT, SMAD2−/−, and SMAD2mh1/mh1 iPS cells. Scale bar, 650 µm. d, Representative immunoblots showing loss of SMAD2 protein in the mutant cells. e, Karyotypic analysis of WT and mutant cell lines after 4 passages post-gene edits. n = 20 cells from each cell line. f, Representative phase contrast and immunofluorescent images from 3 independent experiments showing the expression of the pluripotency marker OCT4 (red). DAPI (blue) labels the nuclei, and the scale bar represents 150 µm.

Extended Data Fig. 2 Mesoderm derivation.

a, Immunoblots showing expression of SMAD2, SMAD3, pSMAD3, and SMAD4 in the mesoderm cells. Quantifications represent mean ± SEM; pSMAD3 was increased in SMAD2−/− (P = 0.0396) and SMAD2mh1/mh1 (P = 0.0076) mesoderm cells. SMAD3 was increased in SMAD2−/− (P = 0.0874) and SMAD2mh1/mh1 (P = 0.0164) mesoderm cells. n = 3 independent experiments. b, Schematic representation of the derivation of posterior mesoderm cells. c, Representative phase contrast images of WT, SMAD2−/−, and SMAD2mh1/mh1 iPS cell-derived posterior mesoderm. Scale bar, 275 µm. d, Immunoblots showing expression of mesoderm lineage-specific markers in the differentiated mesoderm derived with or without Activin A pulses. n = 2 independent experiments. e, RNA expression of posterior mesoderm-specific genes. Data are mean values ± SEM; n = 3 independent experiments. f, Quantification of cell-secreted Activin A on Days 1 and 2 during posterior mesoderm induction. Data are mean values ± SEM; n = 2 independent experiments. We performed a P < 0.05 one-way ANOVA with Dunnett’s multiple comparison test (a, e). Color symbols shown in the histograms of Figs. a and e represent a biological replicate. Same colors in Fig. e indicate 3 independent wells of mesoderm cells from each biological replicate. Representative images from 3 independent experiments are shown in Fig. c. Images of all the stain-free gels and stain-free blots can be found in Supplementary Fig. 6a. Source data are available for this figure.

Source Data

Extended Data Fig. 3

IM derivation. a, Immunostaining for Actin and DAPI. b, Representative whole-well scan of differentiated IM cells after 14 d of induction. c, Snapshots of the velocity field obtained from Particle Image Velocimetry analysis of divergence of WT and mutant IM cells. The arrows indicate local divergence to the mean direction of migration. d, Immunoblots showing expression of EMT markers SNAI1 and TWIST in IM cells. n = 2 independent experiments. e, Representative immunoblots showing expression of pSMAD1, SMAD2, SMAD3, and SMAD4 in the IM cells. n = 3 independent experiments. pSMAD1 was increased in SMAD2−/− (P = 0.0019) and SMAD2mh1/mh1 (P = 0.0046) IM cells. SMAD3 was increased in SMAD2−/− (P = 0.0368) and SMAD2mh1/mh1 (P = 0.0659) IM cells. f, Flow cytometry analysis and g, quantitation of PE-SNAI1 and BV421-WT1. n = 3 independent experiments. h, Flow cytometry plot and i, quantitation showing co-expression of necroptosis marker AF488-MLKL and BV421-WT1. n = 3 independent experiments. j, Immunoblots showing expression of MLKL oligomers, and k, αSMA in differentiating IM cells. n = 2 independent experiments. n = 2 independent experiments. l, RNA expression analyses for canonical and noncanonical WNT target genes and m, mesenchymal genes in IM cells. n = 3 independent experiments. Data are presented as mean ± SEM (Figs. e, g, i, l, m). We performed a P < 0.05 one-way ANOVA with Dunnett’s multiple comparison test (e, g, i, l) and a paired two-tailed t-test (m). Color symbols shown in the histograms of Figs. e, g, i, l, and m represent a biological replicate. Same colors in Figs. l and m indicate 3 independent wells of IM cells from each biological replicate. Representative images from 3 independent experiments are shown in Figs. (a, b) Scale bar: 100 µm (a). For immunoblots, see supplementary Figs. 6b, and 7. Source data is available for this figure.

Source Data

Extended Data Fig. 4 Podocyte derivation.

a, Phase contrast images showing podocyte foot processes (WT) and effacement-like phenotype (mutants). b, Fluorescence intensity quantification of podocyte lineage-specific markers, n = 3 independent experiments. c, Immunostaining for Nephrin (green), CD2AP (red), and WT1 (yellow) in differentiated podocytes. DAPI, shown in grey, labels the nuclei. The nuclear intensity of WT1 was significantly downregulated in the mutant podocytes. n = 3 independent experiments. d, Representative immunoblots and e, quantitation of pSMAD1, SMAD2, pSMAD3, SMAD3, SMAD6, and SMAD7 in podocytes across 3 biologically independent experiments. f, Immunostaining for YAP (magenta) and PAX2 (green). The nuclear intensity of PAX2 was significantly increased in the mutant podocytes. n = 3 independent experiments. g, Immunostaining for Actin. Top right: zoomed-in regions indicate actin dissolution and subsequent podocyte effacement. h, CCK-8 cell viability assay. n = 3 independent experiments. i, Bubble plot comparing the expression of markers in healthy and diseased human kidney podocytes. j, SMAD3 knockdown podocytes underwent foot process effacement (indicated with orange arrows) and cell death. k, Cell size was significantly reduced after SMAD3 knockdown. n = 3 independent experiments. l, Cell viability was significantly reduced after SMAD3 knockdown. n = 3 independent experiments. m, Immunoblots showing expression of SMAD3 and Synaptopodin after SMAD3 knockdown. n = 2 biologically independent experiments. Data are presented as mean ± SEM (Figs. b, c, e, f, h, I, k, l). We performed a P < 0.05 one-way ANOVA with Dunnett’s (b, c, e, g, h) and Sidak’s (k, l) multiple comparison tests. Color symbols shown in the histograms of Figs. b, c, e, f, h, k, and l represent a biological replicate. Representative images from 3 independent experiments are shown in Figs. (a, c, f, j) and 2 independent experiments are shown in Fig. g. Scale bar: 150 µm (a), 100 µm (c, f, j). For immunoblots, images of all the stain-free gels and stain-free blots are shown in Supplementary Fig. 8a. Source data are available for this figure.

Source Data

Extended Data Fig. 5 Endothelial cell derivation.

a, RNA expression of mesenchymal markers ICAM1, Smoothelin, and Vimentin in WT and mutant endothelial cells. Data are mean values ± SEM; n = 3 independent experiments. ICAM1 was increased in SMAD2−/− (P = 0.0009) and SMAD2mh1/mh1 (P = 0.0078) endothelial cells. Smoothelin was increased in SMAD2−/− (P < 0.0001) and SMAD2mh1/mh1 (P = 0.0020) endothelial cells. Vimentin was increased in SMAD2−/− (P < 0.0001) and SMAD2mh1/mh1 (P < 0.0001) endothelial cells. We performed a P < 0.05 one-way ANOVA with Dunnett’s multiple comparison test. Same colors indicate 3-4 independent wells of endothelial cells from each biological replicate. b, Representative immunofluorescent images showing the expression of mesenchymal markers Transgelin (cyan) and TWIST (red) in endothelial cells after 2 weeks of maintenance culture. n = 2 independent experiments. Scale bar, 100 µm.

Source Data

Extended Data Fig. 6 Recapitulating the glomerular capillary wall using a glomerulus-on-a-chip device.

a, Schematic representation of the development of glomerulus-on-a-chip cultures. b, Example photograph of the experimental setup for glomerulus-on-a-chip cultures connected to two reservoirs for the cell culture media for the urinary (podocyte induction media) and capillary (CultureBoost-R) channels. These microfluidic glomerulus-on-a-chip were connected to media reservoirs via low-retention inlet and outlet tubing, using a peristaltic pump. Media was recirculated and replenished every alternate day. c, Immunostaining for Nephrin (magenta)- and PECAM1 (cyan)-positive podocytes and endothelial cells, respectively. d, Single-channel images of Nephrin+ podocytes (magenta, urinary channel) and PECAM1+ endothelial cells (cyan, capillary channel) along the z-axis. e, ‘Y’ junction of the chips to visualize the incoming apical and basal channels before they overlay at the interface. f, Immunostaining for Nephrin (red) in WT, SMAD2−/−, and SMAD2mh1/mh1 podocytes on the glomerulus-on-a-chip platform cultured in the absence of the endothelial cells. DAPI, shown in grey, labels the nuclei. Representative images from 3 independent experiments are shown in Figs. (c, d, f). Scale bars: 50 µm (c), 30 µm (d), 100 µm (e, f).

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–8, Tables 1 and 2, and Notes 1–3.

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Supplementary Video 1 (download AVI )

Time-lapse video of differentiating WT intermediate mesoderm cells.

Supplementary Video 2 (download AVI )

Time-lapse video of differentiating SMAD2−/− intermediate mesoderm cells.

Supplementary Video 3 (download AVI )

Time-lapse video of differentiating SMAD2mh1/mh1 intermediate mesoderm cells.

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Bhattacharya, R., Ward, T., Kalejaiye, T.D. et al. Engineered human induced pluripotent stem cell models reveal altered podocytogenesis in congenital heart disease-associated SMAD2 mutations. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01543-0

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