YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells

Abstract

Stem cell-derived insulin-producing beta cells (SC-β) offer an inexhaustible supply of functional β cells for cell replacement therapies and disease modeling for diabetes. While successful directed differentiation protocols for this cell type have been described, the mechanisms controlling its differentiation and function are not fully understood. Here we report that the Hippo pathway controls the proliferation and specification of pancreatic progenitors into the endocrine lineage. Downregulation of YAP, an effector of the pathway, enhances endocrine progenitor differentiation and the generation of SC-β cells with improved insulin secretion. A chemical inhibitor of YAP acts as an inducer of endocrine differentiation and reduces the presence of proliferative progenitor cells. Conversely, sustained activation of YAP results in impaired differentiation, blunted glucose-stimulated insulin secretion, and increased proliferation of SC-β cells. Together these results support a role for YAP in controlling the self-renewal and differentiation balance of pancreatic progenitors and limiting endocrine differentiation in vitro.

Fig. 1

YAP downregulation in SC-endocrine and insulin-producing β cells. a Diagram of the directed differentiation of hPSCs into insulin-producing β cells. b–e Immunofluorescence micrographs of YAP expression in PDX1 + early pancreatic progenitors, NKX6.1 + late pancreatic progenitors, NGN3 + endocrine progenitors, and C-peptide + β cells. Representative images and cropped blots (bottom panels) are shown. Scale bar: 50 µm. White arrows denote NKX6.1 + /YAP1- (c), NGN3 + /YAP1- (d), and C-peptide + /NKX6.1 + /YAP1-cells (e). f Proportion of cells expressing YAP (green), C-peptide (purple) and CHGA (blue) from stage 3 through stage 6 as estimated by flow cytometry. g–i Proportion of YAP + and YAP- cells of all NKX6.1 + (g), CHGA + (h) and CHGA- (i) cells as quantified by flow cytometry. hPSCs human pluripotent stem cells, DE definitive endoderm, GTE gut tube endoderm, PP1 pancreatic progenitor 1, PP2 pancreatic progenitor 2, EN endocrine precursor, β insulin-producing β cells. Data represent mean values ± SEM, ****p < 0.0001, two-sided student’s t test (n = 3 biologically independent samples per group)

Fig. 2

YAP activity regulates the specification and proliferation of NKX6.1 + progenitors. a Diagram of experimental design for b–g. b–d Flow cytometry of PDX1 and NKX6.1 expression in DMSO (b) or veterporfin-treated (c) MPPs and quantification of the proportion of PDX1/NKX6.1 co-positive pancreatic progenitors (d) from b and c, as assayed at the end of stage 4. e–g Flow cytometry analysis of PDX1 and the proliferation marker Ki67 in DMSO (e) and veterporfin-treated pancreatic progenitors (f), and quantification of coexpression of PDX1 and Ki67 in MPPs from e and f (g) as assayed at the end of stage 4. h Experimental design for i–n. i, j Effects of YAP shRNA expression during pancreatic differentiation on the expression of YAP and target genes assayed by qPCR for RNA (i) and western blot for protein (j), as assayed at the end of stage 4. qPCR values normalized to the average expression value of shControl samples. k–n Effects of the expression of non-targeting control (k) and YAP shRNAs (l) during pancreatic progenitor differentiation on NKX6.1 and CHGA expression and quantification of the proportion of NKX6.1 + (m) and NKX6.1-/CHGA + (n) cells from i and j, as assayed at the end of stage 4. Data represent mean ± SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided student’s t test (n = 3 biologically independent samples per group)

Fig. 3

YAP inhibition enhances the generation of endocrine progenitors and SC-β cells. a Experimental design for b–g. b–d Flow cytometry analysis of NGN3 expression at stage 5, day 3 of DMSO and verteporfin-treated pancreatic progenitors and proportion of NGN3 + cells from b and c. e–g Flow cytometry analysis of NKX6.1 and C-peptide expression upon completion of the differentiation protocol of DMSO and verteporfin-treated pancreatic progenitors and quantification of the proportion of C-peptide + /NKX6.1 + SC-β cells from e and f. h Effects of non-targeting control and YAP shRNAs during endocrine differentiation on lineage marker mRNAs assayed by Nanostring assayed at the end of stage 5. Differentially expressed genes (adjusted p-value < 0.05) are displayed with genes relevant to endocrine induction highlighted in red. Data presented as z-scores. i–k Flow cytometry of C-peptide and NKX6.1 expression in non-targeting control and YAP shRNA-expressing cells and proportion of cells co-expressing both markers from i-j at the end of stage 6 differentiation (stage 6 day 14). l Proportion of cells expressing YAP in cultures of cells expressing non-targeting control and YAP shRNAs assayed at the end of stage 6 differentiation (stage 6 day 14) by flow cytometry. Data represent mean ± SEM, *p < 0.05, ***p < 0.001, two-sided student’s t test (n = 3 biologically independent samples per group)

Fig. 4

YAPS6A overexpression impairs differentiation of β cells and promotes proliferation. a Experimental workflow for the lentiviral overexpression of a stabilized form of YAP (YAPS6A) during β-cell differentiation. b, c Flow cytometry of YAP expression and quantification of YAP + cells in LacZ control and YAP-overexpressing stem cell-derived cells during β-cell differentiation (assayed at stage 6, day 14). d–f Flow cytometry analysis and quantification of C-peptide and NKX6.1 coexpression in LacZ and YAPS6A-overexpressing cells at the end of stage 6 differentiation (stage 6, day 14). g–j Flow cytometry analysis and quantification of C-peptide and Ki67 expression in LacZ and YAPS6A-overexpressing cells. k–n Quantification of EdU staining (k, l) and cleaved Caspase-3 staining (m-n) in monohormonal C-peptide + and C-peptide- cells by flow cytometry at the end of stage 6 differentiation (stage 6, day 14). Data represent mean (center line) ± min to max (bounds of box), **p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided student’s t test (n = 3 biologically independent samples per group)

Fig. 5

Functionality of SC-β cells upon YAP inhibition and overexpression. a, b Insulin secretion levels for DMSO or verteporfin-treated stem cell-derived β cells during sequential rounds of glucose and KCl challenge and insulin secretion stimulation indexes. Insulin secretion levels were normalized to total insulin content for each sample. Stimulation indexes were calculated as a ratio of insulin secretion at high glucose (20 mM) relative to the basal secretion (2.8 mM glucose). c, d Insulin secretion levels of LacZ and YAPS6A-overexpressing stem cell-derived β cells during sequential rounds of glucose and KCl stimulation and insulin secretion stimulation indexes. Data represent mean ± SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. non-significant, two-sided student’s t test, VTPF verterporfin (n = 3 biologically independent samples per group)

Fig. 6

YAP inhibition reduces Sox9 + progenitors in vitro. a Experimental design for b–e. b–d Flow cytometry of SOX9 expression in the control and verteporfin-treated differentiating progenitors and quantification of the proportion of SOX9 + cells from b and c at the end of stage 6 differentiation (stage 6, day 14). mCFP is an unstained control. Data represent mean ± SEM, **p < 0.01 (n = 3 biologically independent samples per group). e Proportion of Ki67 + in control or verteporfin-treated differentiating progenitors as assayed by flow cytometry at the end of stage 6 differentiation. Data represent mean ± SEM, ***p < 0.001 (n = 3 biologically independent samples per group). f–h Flow cytometry analysis of SOX9 in non-targeting control and YAP shRNA-expressing pancreatic progenitors and quantification of SOX9 + cell proportion (h) at the end of stage 6 differentiation. Data represent mean ± SEM, ***p < 0.001 (n = 3 biologically independent samples per group). i Experimental design for j–n. j, k in vivo GSIS of control and verteporfin-derived SC-β cell grafts as assayed by serum human insulin before and 30 min after a glucose injection 8 weeks post transplantation. l, m Immunofluorescence micrographs of grafts 12 weeks post transplantation of control and verteporfin-derived SC-β cell grafts stained for C-peptide and SOX9. Representative images and cropped blots (bottom panels) are shown. Asterisk (*) denotes nonspecific staining. Scale bar: 50 µm. n Quantification of SOX9 + of DAPI + cells within grafts 12 weeks after transplantation. Data represent mean (center line) ± min to max (whiskers) and lower and upper quartiles (bounds of box), ***p < 0.001, two-sided student’s t test (n = 4 animals per group, values correspond to the average proportion of SOX9 + cells from four histological sections per animal)