Protein Methyltransferase Inhibition Decreases Endocrine Specification Through the Upregulation of Aldh1b1 Expression

Abstract

Understanding the mechanisms that promote the specification of pancreas progenitors and regulate their self-renewal and differentiation will help to maintain and expand pancreas progenitor cells derived from human pluripotent stem (hPS) cells. This will improve the efficiency of current differentiation protocols of hPS cells into β-cells and bring such cells closer to clinical applications for the therapy of diabetes. Aldehyde dehydrogenase 1b1 (Aldh1b1) is a mitochondrial enzyme expressed specifically in progenitor cells during mouse pancreas development, and we have shown that its functional inactivation leads to accelerated differentiation and deficient β-cells. In this report, we aimed to identify small molecule inducers of Aldh1b1 expression taking advantage of a mouse embryonic stem (mES) cell Aldh1b1 lacZ reporter line and a pancreas differentiation protocol directing mES cells into pancreatic progenitors. We identified AMI-5, a protein methyltransferase inhibitor, as an Aldh1b1 inducer and showed that it can maintain Aldh1b1 expression in embryonic pancreas explants. This led to a selective reduction in endocrine specification. This effect was due to a downregulation of Ngn3, and it was mediated through Aldh1b1 since the effect was abolished in Aldh1b1 null pancreata. The findings implicated methyltransferase activity in the regulation of endocrine differentiation and showed that methyltransferases can act through specific regulators during pancreas differentiation. Stem Cells 2019;37:640-651.

Keywords: Aldehyde dehydrogenase 1b1; Embryonic stem cell pancreas differentiation; Endocrine specification; Pancreas development; Pancreatic progenitors; β-Cells.

Figure 1

Differentiation of mouse ES cells toward pancreatic‐like progenitors to monitor Aldh1b1 expression analysis. (A): Schematic diagram of the differentiation procedure for the ES^{Aldh1b1lacZ/+} and ES^TgCAGlacZ lines. (B): Immunofluorescence analysis of ES^{Aldh1b1lacZ/+} derived EEBs for expression of Sox17 and PPs for expression of Pdx1, Nkx6‐1, and Aldh1b1. (C): Chromogenic detection of β‐galactosidase activity in EB and PP clusters. (D): Fluorescent detection of β‐galactosidase activity in EB and PP clusters. (E): Diagram showing the z‐score mean intensity readings for eight candidate compounds that may upregulate Aldh1b1 expression. The circles represent the z‐score of log mean intensity of different screening replicates (n = 5) and the lines the median z‐score intensity for each compound. Abbreviations: ES, embryonic stem; LIF, leukemia inhibitory factor; SR, serum replacement; ActA, Activin a; RA, retinoic acid; cycl, cyclopamine; PETG, 2‐Phenylethyl β‐d‐thiogalactoside; PP, pancreatic‐like progenitors. Scale bars: 50 μm.

Figure 2

Validation of AMI‐5 as an inducer of Aldh1b1 expression in mES derived pancreatic progenitors. (A): qPCR analysis of Aldh1b1 expression in PP clusters following a 16 hour treatment with different concentrations of AMI‐5 or DMSO relative to Aldh1b1 expression in EB clusters (n = 5). (B): Median fluorescence intensity of PP clusters assessed by flow cytometry following a 16 hour treatment with different concentrations of AMI‐5 relative to fluorescence intensity of PP clusters treated during 24 hours with DMSO (n = 3). (C): Western blots illustrating expression levels of the AMI‐5 target proteins Prmt1, Dot1l, and Setd7. Abbreviation: mES, mouse embryonic stem; MFI, median fluorescence intensity; PP, pancreatic‐like progenitors. *, p < .05, error bars show SEM.

Figure 3

AMI‐5 prolongs Aldh1b1 expression and delays endocrine differentiation in mouse embryo pancreatic explants. (A): Immunofluorescence analysis of pancreata at 14.5 dpc and after 2 days in ALI cultures (14.5 dpc + 2 days) shows that Aldh1b1 expression is reduced after 2 days in ALI culture of control pancreata whereas it remains higher in AMI‐5 treated pancreata. (B): Quantitation of relative Aldh1b1 fluorescence signal in 14.5 dpc pancreata and 14.5 dpc pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 4). (C): qPCR analysis of Aldh1b1 expression in 14.5 dpc pancreata and 14.5 dpc pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 6). (D): Immunofluorescence analysis of Aldh1b1 expression of 14.5 dpc pancreata after 2 days in ALI cultures in the presence of the Dot1l inhibitor EPZ004777 (10 μM) or the Prmt1 inhibitor C21 (10 μM) or both (10 μM each). (E): Relative quantification of the Aldh1b1 immunofluorescence signal shows significant upregulation in the presence of both inhibitors. (F): Immunofluorescence analysis of 14.5 dpc pancreata after 2 days in ALI cultures shows a reduction in the C‐pep and Gcg signal in pancreata treated with AMI‐5. (G): Relative quantitation of the C‐pep and Gcg fluorescence signal in 14.5 dpc pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 4). (H): Immunofluorescence analysis of 14.5 dpc pancreata after 2 days in ALI cultures shows an increase of Pdx1^LOW cells in pancreata treated with AMI‐5 and indicates the expression of ins in Pdx1^HIGH cells. (I): Quantitation of the Pdx1^LOW cells in relation to all Pdx1⁺ cells in 14.5 dpc pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 3). (J): Fold regulation of endocrine markers at 14.5 + 2 days in ALI culture in the presence of 10 μM AMI‐5 in relation to untreated controls. Only significantly regulated genes are shown (padj ≤ 0.05). (K): Immunofluorescence analysis of 14.5 dpc pancreata after 2 days in ALI cultures shows a reduction in the number of Ngn3⁺ cells in pancreata treated with AMI‐5. (L): Relative quantitation of the Ngn3⁺ cells in 14.5 dpc pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 4). (M): qPCR analysis of Ngn3 expression in 14.5 dpc pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 3). *, p < .05; **, p < .01; ***, p < .001, error bars show SEM. Scale bar: 50 μm. Abbreviation: ALI, air to liquid interface.

Figure 4

The effects of the protein methyltransferase inhibitor AMI‐5 are mediated through Aldh1b1 expression. (A): Immunofluorescence analysis of 14.5 dpc Aldh1b1 null pancreata after 2 days in ALI cultures in the presence of 10 μM AMI‐5 showed that the number of C‐pep⁺ or Gcg⁺ cells was not affected. (B): Relative quantitation of the C‐pep and Gcg fluorescence signal in 14.5 dpc Aldh1b1 null pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 3). (C): Immunofluorescence analysis of 14.5 dpc Aldh1b1 null pancreata after 2 days in ALI cultures in the presence of 10 μM AMI‐5 showed that the number of Amy⁺ or CK19⁺ cells was not affected. (D): Relative quantitation of the amylase and CK19 fluorescence signal in 14.5 dpc Aldh1b1 null pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 5). (E): Immunofluorescence analysis of 14.5 dpc Aldh1b1 null pancreata after 2 days in ALI cultures shows no effect in the number of Ngn3⁺ cells in pancreata in the presence of 10 μM AMI‐5. (F): Relative quantitation of the Ngn3⁺ cells in 14.5 dpc Aldh1b1 null pancreata cultured in ALI for 2 days in the absence or presence of 10 μM AMI‐5 (n = 3). Error bars show SEM; Abbreviations: ALI, air to liquid interface; ns, not significant. Scale bar: 50 μm.