Abstract
β cells have a limited capacity for regeneration, which predisposes towards diabetes. Here, we show that, of the MYC family members, Mycl plays a key role in proliferation of pancreatic endocrine cells. Genetic ablation of Mycl causes a reduction in the proliferation of pancreatic endocrine cells in neonatal mice. By contrast, the expression of Mycl in adult mice stimulates the proliferation of β and α cells, and the cells persist after withdrawal of Mycl expression. A subset of the expanded α cells give rise to insulin-producing cells after this withdrawal. Transient Mycl expression in vivo is sufficient to normalize the hyperglycaemia of diabetic mice. In vitro expression of Mycl similarly provokes active replication in islet cells, even in those from aged mice. Finally, we show that MYCL stimulates the division of human adult cadaveric islet cells. Our results demonstrate that the induction of Mycl alone expands the functional β-cell population, which may provide a regenerative strategy for β cells.
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Data availability
The Gene Expression Omnibus accession numbers for the RNA-seq data and the scRNA-seq data reported in this paper are GSE178726 and GSE163947, respectively. The data that support the plots within this paper and other findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank M. Yagi and K. Ito for their critical reading of the manuscript. We are grateful to Y. Yamada, R. Sakamoto, M. Kikuchi and M. Baba for technical assistance. Yasuhiro Y. and T.Y. were supported by AMED-CREST (grant no. 21gm1110004h9905), the Japan Agency for Medical Research and Development (AMED). Yasuhiro Y. was also supported in part by P-CREATE, AMED (grant no. 21cm0106203h0006) and JSPS KAKENHI (grant nos. 18H04026, 20H05384). The Institute for the Advanced Study of Human Biology (ASHBi) is supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan. We thank to Single-CellGenome Information Analysis Core (SignAC) in ASHBi for the RNA sequence analysis.
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M.H. and Yasuhiro Y. designed and conceived the study and wrote the paper. M.H., Y.S., N.S., J.T., Yosuke Y., T.U. and M.O. generated mice, performed experiments, and generated RNA-seq and scRNA-seq libraries. H.S. performed the transmission electron microscopy analysis. S.T., M. Kato and J.N. provided technical instructions for experiments with regard to diabetes. M. Kabata, S.O. and T.Y. analysed RNA-seq and scRNA-seq data in mouse and human.
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Extended data
Extended Data Fig. 1 Expression of MYC family genes during mouse pancreatic development.
a. scRNA-seq of E14.5 pancreatic epithelial cells. scRNA-seq data were obtained from GSE101099. b. Mycl expression in the scRNA-seq analysis of embryonic pancreatic endocrine cells at various time points. Red bars indicate the mean expression levels. scRNA-seq data were obtained from GSE115931. c. Mycl expression in the scRNA-seq analysis of β- and α-cells at various time points. Red bars indicate the mean expression levels. scRNA-seq data were obtained from GSE87375. d. ChIP-seq analysis for H3K27ac in pancreatic endocrine cells. Enrichment of H3K27ac at the Mycl locus was apparent only in E17.5 Ins1+ cells. The Chga locus is shown as a control. ChIP-seq data were obtained from GSE84324 and GSE110648. e. Generation of the Mycl knockout mouse. Genetic aberrations in two independent Mycl knockout mouse lines (nos. 56 and #949) are shown. f. In situ detection of Mycl transcripts. Lymphocytes from Mycl knockout mice (no. 949) displayed no detectable Mycl expression. Scale bars: 100 μm. g. Quantification of Ki67+ cell ratio in hormone-producing cells from P0 Mycl knockout mice (no. 949). Data are presented as means ± s.d. Numbers in parentheses are the numbers of mice analysed. Total numbers of analysed cells are: 963 INS+ and 295 GCG+ cells in +/+ mice; 1,028 INS+ and 418 GCG+ cells in +/- mice; 769 INS+ and 374 GCG+ cells in -/- mice. One-way ANOVA, followed by the Tukey–Kramer post-hoc test. h. IPGTT of Mycl knockout mice (no. 949) at 4 weeks of age. Data are presented as means ± s.d. Numbers in parentheses are the numbers of mice analysed. n.s.: not significant; two-way ANOVA, followed by the Tukey-Kramer post-hoc test.
Extended Data Fig. 2 Systemic induction of MYC family genes in mice.
a. ESCs with inducible alleles for MYC family genes. mCherry signals are detectable in ESCs after Dox treatment. Scale bars: 300 μm. b. qRT-PCR analysis of MYC family genes and mCherry in ESCs. MYC family genes and mCherry were induced by Dox treatment. Data are presented as means ± s.d. of biological triplicates. The mean expression levels in Dox ON cells were set to 1. Unpaired two-tailed t-test. c. Systemic induction of Myc expression in mice. mCherry expression was detectable in most organs after Dox administration in the drinking water for 3 days (2 mg/mL, with sucrose). Scale bars: 1 mm.
Extended Data Fig. 3 Tumour development after systemic induction of MYC family genes in mice.
a. Tumour development in Myc- and Mycn-induced mice. Four-week-old chimeric mice were treated with Dox for 4 weeks. Dashed lines indicate the areas of liver tumours. Arrows indicate intestinal tumours. Mycl-induced mice did not develop tumours in the liver, intestine, stomach, lung, or spleen. Scale bars: 500 μm, except for the intestine (200 μm). b. Incidence of liver tumours and intestinal dysplasia in mice after the induction of MYC family gene expression. Numbers in parentheses are the numbers of mice analysed. Some of Myc-induced mice were sacrificed before the termination of the experiment because of the highly morbid phenotype. c. Ki67 staining for various organs of Mycl ON mice (Fig. 2f). Eight-week-old mice were given Dox for 8 weeks. mCherry expression represents Mycl-expressing cells. Scale bars: 100 μm (Lung, Liver, and Kidney), 200 μm (Intestine). d. Quantification of the Ki67+ cell ratio in each organ of Mycl ON mice. The quantification in each image is shown. Data are presented as means ± s.d. Numbers in parentheses are the numbers of mice analysed. Total numbers of analysed cells are: 4,683 (No Dox) and 3,881 (Mycl ON) in the kidney; 3,182 (No Dox) and 2,228 (Mycl ON) in the liver; 1,966 (No Dox) and 1,390 (Mycl ON) in the lung. e. Expression of mCherry in liver tissues 48 hours after daily intraperitoneal injections of Dox (0.2 ml/day; 1.0 mg/mL) in RNA-seq analysis. f. Gene ontology (GO) term enrichments of indicated clusters in Fig. 2e. GO terms and the p-value (Fisher’s exact test) are shown. DAVID Bioinformatics Resources (v6.8) was used for the analysis. g. Left panel: RNA-seq analysis of liver tissues after induction of either Mycl or Myc for 48 hours. Heatmap showing changes in expression levels of downregulated genes by either Mycl or Myc induction (fold change>2, P<0.05 [FDR-adjusted Wald Test]). Right panel: GO term enrichments of indicated Clusters. GO terms and the p-value (Fisher’s exact test) are shown.
Extended Data Fig. 4 scRNA-seq analysis of murine islet cells after the induction of Mycl expression.
a. Histology of the pancreas in adult control, Mycl ON and Mycl ON-OFF mice. Enlarged islets on histological sections are observed in both Mycl ON and Mycl ON-OFF mice. Scale bars: 1 mm. b. Proportion of hormone-positive area in islets of adult control, Mycl ON and Mycl ON-OFF mice. The quantification in each mouse is shown. Data are presented as means ± s.d. Numbers in parentheses are the numbers of mice analysed. Total numbers of analysed islets are: 52 (control), 60 (Mycl ON), and 41 (Mycl ON-OFF) for INS and SST expression; 48 (control), 58 (Mycl ON), and 55 (Mycl ON-OFF) for GCG and PP expression. c. Expression levels and distribution of representative marker genes for each cell type on a UMAP plot in Fig. 3c. d. Dot plot showing the expression of cell cycle-related genes in islet cells. e. RT-qPCR for representative genes that exhibit the altered expression levels after Mycl induction in scRNA-seq analysis. Isolated islets from control (No Dox) and Mycl ON mice were used in this analysis. Data are presented as means ± s.d. of biological triplicates. Numbers in parentheses are the numbers of mice analysed. The mean expression level of Mycl, Stmn1, and Cdk4 in Mycl ON islets and that of Mafa in control islets was set to 1, respectively. Unpaired two-tailed t test. f. Expression levels and distribution of genes associated with immature islet cells. g. Expression levels and distribution of genes associated with common pancreatic progenitor cells.
Extended Data Fig. 5 scRNA-seq analysis of murine islet cells after the induction of Mycl expression.
a. Expression levels and distribution of representative marker genes for each cell type on a UMAP plot in Fig. 3f. b. UMAP visualization of islet cells from E12.5, E14.5, E17.5, adult control, Mycl ON and Mycl ON-OFF mice. scRNA-seq data of embryonic endocrine cells (GSE101099) were used for the analysis. c. Expression levels and distribution of genes associated with immature islet cells. d. Expression levels and distribution of proliferation-related genes.
Extended Data Fig. 6 scRNA-seq analysis of murine islet cells after the induction of Mycl expression.
Pancreatic islets isolated from Mycl-induced chimeric mice (n=2, male; Dox treatment for 8 weeks; 16 weeks of age) (Fig. 2f) were dissociated into single-cell suspensions in a TripLE solution. ESCs containing Rosa26-M2rtTA; Col1a1::tetO-Mycl-ires-mCherry with a Gcg reporter were utilised to generate the chimeric mice for the scRNA-seq analysis. Islets from the chimeric mice contained both mCherry+ Mycl-induced cells and non-induced cells. a. UMAP visualisation of 7,001 mouse islet cells from Mycl ON chimeric mice. A putative doublet cluster was removed. n=4,307 cells in replicate 1 and 2,694 cells in replicate 2. The UMAP visualisation is coloured for cluster identity, and annotated on the basis of marker gene expression. Expression levels and distribution of mCherry (Mycl) as well as representative marker genes for each cell type are shown. b. Left: Expression levels and distribution of Mki67 and Cdk4. Right: Dot plot showing the expression of cell cycle-related genes in islet cells. c. Expression levels of mature cell-related genes in each cluster. d. Dot plot showing the expression of immature cell-related genes in islet cells.
Extended Data Fig. 7 Lineage tracing analysis of islet cells after the induction of Mycl expression.
a. Schematic representation of the Gcg targeting allele and the generation of Gcg-CreERT2 ESCs. The mTmG reporter-containing ESCs were used for the targeting. b. Schematic illustration of the genomic construct containing the Mycl-inducible allele and reporter alleles for use in mice. c. Seven-week-old mice were injected with Tamoxifen (200 mg/kg) for 3 consecutive days and analysed at 8 weeks of age for reporter expression. Reporter-labelled cells exclusively expressed INS or GCG in Ins1-CreERT2; mTmG mice and Gcg-CreERT2; mTmG mice, respectively. For α-cell tracing, chimeric mice were obtained by injecting Gcg-CreERT2; mTmG ESCs into blastocysts. Scale bars: 20 μm. n=4 mice examined for each reporter. d. Schematic illustration of the experimental protocol for lineage tracing analysis during Mycl-induced expansion of islet cells in adult mice. Chimeric mice were used for α-cell tracing. e. Proportion of the islets containing mGFP+ cells in each mouse for Fig. 4b. Data are presented as means ± s.d. Numbers in parentheses are the numbers of mice analysed. f. Linage tracing analysis of mature β-cells in Mycl ON and Mycl ON-OFF islets for SST and PP expression. Scale bars: Scale bars: 20 μm. g. Linage tracing analysis of mature α-cells in Mycl ON and Mycl ON-OFF islets for SST and PP expression. Chimeric mice were used in all the α-cell tracing experiments in vivo. Scale bars: Scale bars: 20 μm. h. Quantification of SST-positive (left) and PP-positive (right) cells in mGFP+ cells. The quantification in each mouse is shown. Data are presented as means ± s.d. Numbers in parentheses are the numbers of mice analysed. Total numbers of traced mGFP+ cells are: 208 (control), 254 (Mycl ON), and 433 (Mycl ON-OFF) in mice with an Ins1 reporter for SST expression; 205 (control), 248 (Mycl ON), and 429 (Mycl ON-OFF) in mice with an Ins1 reporter for PP expression; 176 (control), 891 (Mycl ON), and 1,105 (Mycl ON-OFF) in mice with a Gcg reporter for SST expression; 203 (control), 855 (Mycl ON), and 1,274 (Mycl ON-OFF) in mice with a Gcg reporter for PP expression.
Extended Data Fig. 8 In vitro expansion of adult islet cells after the induction of Mcyl expression.
a. Lineage tracing analysis of mature α-cells in Mycl ON-OFF islets. A subset of α-cell reporter-labelled cells did not express GCG or SST. Scale bars: 50 μm (large panels), 20 μm (small panels). b. Left: Expression levels and distribution of Arx on a UMAP plot. Right: Expression levels of Arx in α-cells. Black bars in a white box indicate median expression levels. The white bars and lines represent the IQR and the lower/upper adjacent values (±1.5 IQR), respectively. Wilcoxon Rank Sum test. c. Body masses of mice during and after the induction of Mycl expression. Data are presented as means ± s.d. Numbers in parentheses indicate the numbers of mice analysed. n.s.: not significant; one-way ANOVA and Holm–Sidak’s post-hoc test. d. Islet cell clusters after the induction of Myc or Mycl expression. Expansion of mCherry+ clusters at Day 7. No Dox: Mycl-inducible islets without Dox treatment. Scale bars: 50 μm. n=10 mice were examined. e. Ki67+ and cleaved caspase 3+ cells among the INS+ cells 7 days after the induction of either Myc or Mycl commenced. No Dox: Mycl-inducible islets without Dox treatment. Scale bars: 20 μm. Quantification of the Ki67+ and cleaved caspase 3+ cell ratios, relative to the number of INS+ cells, in each mouse is shown. Data are presented as means ± s.d. Numbers in parentheses are the numbers of mice analysed. Total numbers of analysed INS+ cells are: 1,185 (No Dox), 1,001 (Myc-induced), and 1,566 (Mycl-induced) for the Ki67 analysis; 1,176 (No Dox), 1,246 (Myc-induced), and 1,168 (Mycl-induced) for the cleaved caspase 3 analysis. One-way ANOVA, followed by the Tukey-Kramer post-hoc test. f. Left: Schematic representation of the Ins1 targeting allele for the generation of the Ins1-ires-CreERT2 allele. Right: β-cell labelling in Ins1-ires-CreERT2; mTmG chimeric mice. Note that mGFP+ cells exclusively expressed INS. Scale bars: 20 μm. n=4 mice were examined. g. Schematic illustration of the protocol for lineage tracing analysis in vitro. Chimeric mice were used for in vitro lineage tracing analysis. h. Lineage tracing analysis of mature β- and α-cells. mGFP+ cells exclusively expressed the original hormone after induction of Mycl in vitro. Scale bars: 20 μm. n=4 mice examined for each tracing analysis.
Extended Data Fig. 9 In vitro expansion of functional islet cells.
a. Lineage tracing analysis of mature α-cells 7 days after transplantation into the kidney, following the Mycl-induced expansion of islet cell clusters for 7 days. n=4 mice examined. b. Repeated passage of islet cluster cells in vitro. Expanded islet clusters were passaged after dissociation into small clusters and continuously expanded after the second passage, but did not obviously do so after the fourth passage. Scale bars: 50 μm. n=3 mice examined. c. Islet cell clusters after the induction of Mycl expression in RPMI 1640 (D-glucose; 2.0 g/L) with 10% FBS in Matrigel. The expansion of mCherry+ cells is observed even in a low glucose medium. Scale bars: 300 μm. n=4 mice examined. d. Schematic illustration of the genomic construct of the lentivirus containing a Mycl-inducible system. e. Schematic illustration of the experimental protocol for lentivirus-mediated induction of Mycl expression in isolated islet cells from aged mice in vitro. f. Schematic illustration of the experimental protocol for the assessment of insulin secretion. g. Histology of the removed kidney. The transplanted cells included INS+ and SST+ cells. Scale bars: 300 μm. n=3 mice examined.
Extended Data Fig. 10 scRNA-seq analysis of human islet cells after the induction of MYCL expression.
a. Schematic illustration of an experimental protocol for the lentivirus-mediated induction of MYCL in human cadaveric islet cells in vitro. b. mCherry (MYCL)+ islet cells from donors 2 and 3, displaying staining for Ki67. Scale bars: 20 μm. Numbers of analysed cells are shown in Fig. 6e. c. UMAP visualisation of human cadaveric islet cells after the induction of MYCL, Mycl, or zsGreenDR expression. A putative doublet cluster was removed. n=816 MYCL-induced cells, 1,110 Mycl-induced cells and 962 for zsGreenDR-induced cells. d. Expression levels and distribution of mCherry for each transgene. e. Expression levels and distribution of representative marker genes for pancreatic cell types.
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Hirano, M., So, Y., Tsunekawa, S. et al. MYCL-mediated reprogramming expands pancreatic insulin-producing cells. Nat Metab 4, 254–268 (2022). https://doi.org/10.1038/s42255-022-00530-y
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DOI: https://doi.org/10.1038/s42255-022-00530-y
