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Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors

Nature Cell Biology volume 18, pages 1269–1280 (2016)Cite this article

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Abstract

Direct reprogramming by forced expression of transcription factors can convert one cell type into another. Thus, desired cell types can be generated bypassing pluripotency. However, direct reprogramming towards renal cells remains an unmet challenge. Here, we identify renal cell fate-inducing factors on the basis of their tissue specificity and evolutionarily conserved expression, and demonstrate that combined expression of Emx2, Hnf1b, Hnf4a and Pax8 converts mouse and human fibroblasts into induced renal tubular epithelial cells (iRECs). iRECs exhibit epithelial features, a global gene expression profile resembling their native counterparts, functional properties of differentiated renal tubule cells and sensitivity to nephrotoxic substances. Furthermore, iRECs integrate into kidney organoids and form tubules in decellularized kidneys. Our approach demonstrates that reprogramming factors can be identified by targeted in silico analysis. Renal tubular epithelial cells generated ex vivo by forced expression of transcription factors may facilitate disease modelling, drug and nephrotoxicity testing, and regenerative approaches.

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Figure 1: Systematic selection criteria identify candidate reprogramming factors.
Figure 2: Four transcription factors can induce renal cell fate in MEFs.
Figure 3: iRECs exhibit epithelial properties.
Figure 4: iRECs resemble primary renal epithelial cells in their global gene expression profile.
Figure 5: Nephron segment identity and stability of iRECs.
Figure 6: iRECs show functional properties of renal tubular epithelial cells.
Figure 7: iRECs integrate into reaggregated renal organoids and repopulate decellularized kidneys.
Figure 8: Reprogramming of human fibroblasts.

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Acknowledgements

We thank A. Heer, A. Sammarco and B. Joch for excellent technical assistance; the staff of the SFB1140 project Z01 and R. Nitschke (Life Imaging Centre (LIC), Albert-Ludwigs-University Freiburg) for help with confocal microscopy; D. Pfeifer for performing microarrays; M. Follo for FACS analysis; O. Wessely (Cleveland, USA), P. Vize (Calgary, Canada), N. Ueno (National Institute for Basic Biology, Japan) and D. Trono (Lausanne, Switzerland) for generously providing plasmids; M. Köttgen for thoughtful comments on the manuscript. This work was supported by the Emmy Noether Programme to S.S.L. (LI1817/2-1) and S.J.A. (AR732/1-1), Projects B07 of the collaborative research initiative KIDGEM (SFB 1140) to S.S.L. and S.J.A. and project A03 of SFB 850 to S.J.A. by the German Research Foundation (DFG), BIOSS Centre of Biological Signalling Studies, and the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School) to S.J.A.

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Authors and Affiliations

  1. Department of Medicine, Renal Division, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany

    Michael M. Kaminski, Jelena Tosic, Catena Kresbach, Hannes Engel, Jonas Klockenbusch, Anna-Lena Müller, Roman Pichler, Florian Grahammer, Oliver Kretz, Tobias B. Huber, Gerd Walz, Sebastian J. Arnold & Soeren S. Lienkamp

  2. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Faculty of Medicine, Albertstraße 25, 79104 Freiburg, Germany

    Jelena Tosic & Sebastian J. Arnold

  3. Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Albertstraße 19a, 79104 Freiburg, Germany

    Jelena Tosic

  4. Faculty of Biology, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany

    Jelena Tosic

  5. Department of Neuroanatomy, University of Freiburg, Albertstraße 17, 79104 Freiburg, Germany

    Oliver Kretz

  6. BIOSS Centre of Biological Signalling Studies, University of Freiburg, Schänzlestraße 18, 79104 Freiburg, Germany

    Tobias B. Huber, Gerd Walz, Sebastian J. Arnold & Soeren S. Lienkamp

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Contributions

M.M.K., J.T., C.K., H.E., J.K., A.-L.M., R.P., F.G., O.K. and S.S.L. performed experiments. M.M.K., J.T., S.J.A. and S.S.L. planned and analysed experiments. G.W. and T.B.H. helped with data analysis and during final stages of the manuscript. M.M.K., J.T., S.J.A. and S.S.L. wrote and edited the manuscript. S.S.L. conceived the study.

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Correspondence to Sebastian J. Arnold or Soeren S. Lienkamp.

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The authors declare that the following competing financial interest exists: patent applications are pending (EPA 16152655.3 and EP16185290.0).

Integrated supplementary information

Supplementary Figure 3 Distribution of known reprogramming factors in quantitative expression data.

(a) Scatter-plots depicting the absolute and relative expression levels of transcription factors (TFs) in brain, liver, heart, testis, thymus and muscle tissue. TFs successfully used for reprogramming into the indicated cell type (blue) are highlighted in red. Dashed lines indicate the level of the 50th percentile of absolute expression and the 95th percentile of relative expression. Combinations of TFs are according to the studies referenced below. (b) Summary of Xenopus whole mount in situ hybridization experiments: Spatial expression pattern at stage 38. White, grey and black colored boxes indicate undetectable, weak and strong gene expression within the different segments of the Xenopus pronephros. PT1, PT2, PT3: proximal tubule segment 1, 2 and 3. IT1, IT2: intermediate tubule segment 1 and 2. DT1, DT2: distal tubule segment 1 and 2. CT: connecting tubule (left panel). Temporal expression pattern at stages 22, 26, 33 and 38 of Xenopus development (right panel). (c) Summary of transcription factor expression in E12-E17 mouse embryonic kidney. E12-E17 embryonic kidneys were serially sectioned and analyzed by in situ hybridization for indicated genes. Spatial expression patterns were determined within the kidney anlagen and sections analyzed for expression within the ureteric bud (ub) and metanephric mesenchyme (mm) at E12, and nephrogenic mesenchyme (nm), stroma/interstitium (str), renal vesicle/glomerulus (rv/g) and tubular epithelium (te) from E13 to E17, -: no expression.

Supplementary Figure 4 Whole mount in situ hybridization of candidate reprogramming factors in Xenopus embryos.

Whole mount in situ hybridization (WISH) of the indicated genes. WISH was performed on Xenopus embryos at stages 22, 26, 33, and 38. Enlarged views show the pronephric region. Scale bars, 500 μm.

Supplementary Figure 5 Expression of candidate reprogramming transcription factors during kidney development in mouse.

mRNA expression of the indicated 20 transcription factors was visualized by in situ hybridization on paraffin sections of mouse nephrogenic anlagen and embryonic kidneys from E12 to E17 on consecutive sections from the same embryos ensuring increased comparability between expression of different transcription factors. Scale bars, 200 μm (E12, E13), 500 μm (E14–E17).

Supplementary Figure 6 iRECs can be induced by a multicistronic lentiviral vector and can be generated from adult tail tip fibroblasts.

(a) Flow cytometry (FC) analysis of control MEFs obtained from membrane-tomato red mice (TOM CTL) and from membrane-GFP mice (GFP CTL) used for FC gate setting. MIX CTL: mixture of membrane-tomato red MEFs and membrane-GFP MEFs. (b) Vector map of the multicistronic construct showing the four reprogramming factors (Pax8, Hnf1b, Emx2, Hnf4a) separated by 2A peptide sequences. pWPXLd was used as lentiviral backbone. (c) Expression of 2A-tagged TFs was controlled using an antibody against the 2A consensus peptide motif. GFP transfected cells served as a control. (d) FC analysis of GFP+ cells in KSP-Cre reporter MEFs treated with a lentivirus harboring the multicistronic vector after 39 days of culture. (e) FC analysis for the presence of GFP+ cells in MEFs, young TTFs (P7) and adult TTFs (P56) 1 and 5 weeks after high titer lentiviral infection with 4TFs (Pax8, Emx2, Hnf4a and Hnf1b); representative FC plots are shown, percentage of GFP+ cells is indicated with standard error for the respective experiment. (f) Immunostaining of 0TF or 4TF treated adult TTFs for the indicated proteins. Scale bar, 50 μm (f).

Supplementary Figure 7 Transcriptome analysis of iRECs.

(a) Median RPKM (Reads per kilobase per million mapped reads) in human (left diagrams) and mouse (middle and right diagrams) global gene expression profiles of 10-fold upregulated and downregulated genes in iRECs compared to MEFs or primary RECs. Error bars, quantile range. (b) Median RPKM in global gene expression across renal tubular segments3 of differentially regulated genes as indicated. Error bars, quantile range. One-way ANOVA was used to determine differential gene expression in the microarray dataset. (c) Gene ontology term analysis of 10-fold upregulated genes in iRECs compared to MEFs.

Supplementary Figure 8 Heterogeneity, stability, proliferation and nephrotoxicity testing in iRECs.

(a) Flow cytometry (FC) analysis of MEFs, iRECs and IMCD-3 cells for expression of GFP and EPCAM, AQP1, and ATP1A1. The percentage of cells within each quadrant is given. (b) Co-immunostaining of iRECs for proteins with segment restricted expression as detected by FC. Proximal tubule cells were detected by LTL (Lotus tetragonolobus) staining. (c) Expression levels of the 13 candidate reprogramming factors in microdissected tubule segments of adult rats39. (d) qPCR analysis for detection of exogenous reprogramming factors. iRECs EX: generated with a LoxP containing provirus for removal of the exogenous reprogramming factors after CRE expression. iRECs: generated with a provirus lacking loxP sites for constitutive expression, n = 3 biological samples. (e) Immunostaining of iRECs and iRECs EX for the indicated proteins. (f) mRNA expression levels of the indicated genes as determined by qPCR in MEFs and iRECs EX, n = 3 biological samples. (g) Relative levels of p16 mRNA as determined by qPCR in P20 and P40 iRECs and MEFs, n = 3 biologically independent samples, ns: not significant, P > 0.05. (h) Quantification of cell death (%) in cisplatin treated MEFs (red line) and iRECs (black line) or untreated controls, n = 3 biologically independent samples. (i) FC analysis for KIM1 expression in iRECs and MEFs treated with gentamicin (1 mg ml−1), n = 3 biologically independent samples. Error bars, s.e.m., Student’s unpaired t-test, ∗∗∗P < 0.001, ∗∗P < 0.01,∗P < 0.05 (d,f–h). Scale bar, 50 μm (e).

Supplementary Figure 9 Improvement of human iREC induction efficiency and specificity.

(a) Percentage of THY-1− or EPCAM + cells in mouse and human fibroblasts after treatment with 4TF as determined by flow cytometry (FC). (b) FC analysis of the percentage of THY-1− and EPCAM+ human fibroblasts after treatment with SV40 (large T antigen), 4TF or SV40 and 4TF. (c) Percentage of CDH16-GFP+ reporter mouse fibroblasts after treatment with 4 TFs as determined by FC. Fibroblasts were transduced with a human CDH16-GFP reporter plasmid and SV40, selected for GFP cells with stable integration of the reporter and infected with 4TFs.

Supplementary Figure 10 Scans of the original Western blots as shown in Supplementary Fig. 4c.

Short (10 s) and long (1 min) exposure times are shown.

Supplementary Table 1 List of oligonucleotides.
Full size table
Supplementary Table 2 List of antibodies.
Full size table

Supplementary information

Supplementary Information

Supplementary Information (PDF 3045 kb)

Dome formation of iRECs.

Confocal 3D-stack of iREC dome formation, as depicted in Fig. 3c. iRECs express membrane GFP (green), the nuclei are stained with Hoechst (blue). (AVI 7579 kb)

Integration of iRECs into kidney organoids.

Confocal 3D-stack of reaggregated kidney organoid as depicted in Fig. 7c. iRECs express membrane GFP (green), LTL staining for renal tubular cells is depicted in magenta. (AVI 2874 kb)

iRECs form tubular structures in decellularized kidneys.

Confocal 3D-stack of tubule forming iRECs guided by the extracellular matrix of decellularized kidneys as depicted in Fig. 7f. iRECs express membrane GFP (green), the nuclei are stained with Hoechst (blue). (AVI 2334 kb)

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Kaminski, M., Tosic, J., Kresbach, C. et al. Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors. Nat Cell Biol 18, 1269–1280 (2016). https://doi.org/10.1038/ncb3437

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