XB-ART-52692Nat Cell Biol December 1, 2016; 18 (12): 1269-1280.
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Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors.
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.
PubMed ID: 27820600
Article link: Nat Cell Biol
Species referenced: Xenopus
Genes referenced: abcc2 ar arid3a arnt2 ascl1 atp1a1 bcl6 bhlhe40 brca1 cdh1 cdh16 clock crem cux1 ddit3 deaf1 dlx5 elf3 emx2 epas1 esrrb eya1 fosb foxc1 foxj2 foxp1 gabpa gata2 gata3 gata4 ggt1 gli2 glis1 glis2 hic1 hif1a hnf1a hnf1b hnf4a hoxa1 hoxa13 hoxa9 hoxb5 hoxb6 hoxb9 hoxc10 hoxc9 hoxd10 irx3 junb jund lhx1 lrp2 mip myc myt1l nfatc4 nfe2l2 nfib nfic nfil3 npat nr1h4 nr3c1 nr6a1 osr1 otx1 pax2 pax8 pbx1 pou2af1 pou3f3 prss1 rest rfx3 rfx7 rxra sall1 six2 six4 slc23a1 slc6a17 slc6a8l stat2 stat6 tfap2b tfcp2l1 thra tjp1 tp53 trim28 trpv4 vdr vim wt1 zbtb7b
Article Images: [+] show captions
|Figure 1 Systematic selection criteria identify candidate reprogramming factors. (a) A schematic illustrating the strategy and criteria used to identify renal reprogramming factors. TFs, transcription factors. (b) Scatter plots depicting the absolute and relative expression levels of transcription factors in liver, brain and kidney tissue23. Factors used for successful reprogramming to hepatocytes and neurons are highlighted in red. Dashed lines indicate the level of the 50th percentile of absolute expression and the 95th percentile of relative expression. The area boxed in red includes the 55 candidate reprogramming factors for renal cell types. (c) Representative images of in situ hybridizations of the indicated genes on E14 mouse kidney sections and stage 26 (St. 26) Xenopus whole-mount embryos. The full set of images is contained in Supplementary Figs 2 and 3. Scale bars, 500 μm.|
|Figure 7. iRECs integrate into reaggregated renal organoids and repopulate decellularized kidneys. (a) A schematic illustration of the reaggregation assay. GFP+ iRECs and E13 TOM+ kidneys were trypsin-digested, mixed in a 1:10 ratio and grown on an air/liquid interface culture. (b) Confocal live-imaging of renal organoids reaggregated with either Ksp-Cre GFP+ iRECs or GFP- transduced control MEFs. The white boxed area is shown enlarged in the right panel. (c) Immunostaining of kidney organoids reaggregated with MEFs or iRECs for the indicated proteins. (d) Tile-scanned confocal images of entire reaggregates after staining for laminin, and enlargement of the white boxed area. (e) Quantification of GFP+ iRECs and MEFs detected inside or outside the laminin-stained basement membrane of reaggregated tubules. Error bars, s.e.m.; student’s unpaired t -test, n = 9 z -stacks, ∗∗∗ P < 0.001. (f) Schematic illustration of the repopulation assay. Wild-type (WT) kidneys were decellularized and injected with iRECs. Confocal images and 3D reconstructions of decellularized kidneys repopulated with Ksp-Cre GFP+ iRECs and immunostained for E-cadherin. Nuclei were stained with Hoechst; ECM, extracellular matrix; MIP, maximum intensity projection of confocal z -stacks. Scale bars, 50 μm (b,c,f), 200 μm (d).|
|Figure 8 Reprogramming of human fibroblasts. (a) Phase-contrast image and immunostaining for the indicated proteins in untreated and 4TF- treated human fibroblasts. (b) Relative mRNA expression level of the indicated genes as determined by qPCR. Error bars, s.e.m. Significant differences were assessed by Student’s unpaired t -test, n = 3 biologically independent samples, ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05. (c) Untreated human fibroblasts and human induced renal epithelial cells (h-iRECs) cultured in 3D Matrigel. Shown are maximum intensity projections of confocal z-stacks. (d) Matrigel-grown h-iREC spheres stained for actin (phalloidin) and β-catenin. (e) Percentage of PROM1+ /EPCAM+ double-positive cells (top panel) and percentage of CDH16-GFP+ cells (bottom panel) as determined by flow cytometry. 4TF human fibroblasts were treated with SV40. (f) Heatmap of RNA-seq expression analysis in human fibroblasts (0TF), CDH16-GFP+ sorted h-iRECs (4TF) and human kidney. Differentially regulated genes (0TF versus 4TFs, one-way ANOVA, P < 0.0001, n = 3 independent biological samples) were hierarchically clustered. The number of genes in each cluster is shown on the right (Pearson’s correlation coefficient 0.7). Scale bars, 50μm (a), 100 μm (c), 20 μm (d).|
|emx2 (empty spiracles homeobox 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.|
|hnf1a (HNF1 homeobox A) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.|
|hnf1b (HNF1 homeobox B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.|
|hnf4a (hepatocyte nuclear factor 4, alpha) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.|
|pax8 (paired box 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.|
|sall1 (spalt-like transcription factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.|
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