Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells
Pluripotent stem cells
Generation of pancreatic β cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived β (SC-β) cells with improved in vitro and in vivo function. SC-β cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.
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Fig. 1: The state of the cytoskeleton controls expression of the transcription factors NEUROG3 and NKX6-1 in pancreatic progenitors.
Fig. 2: Single-cell RNA sequencing demonstrates that cytoskeletal state directs pancreatic progenitor fate.
Fig. 3: Latrunculin A treatment during stage 5 increases the efficiency of SC-β cell specification for plated pancreatic progenitors.
Fig. 4: SC-β cells differentiated with the new planar protocol express β cell markers and function in vitro.
Fig. 5: SC-β cells generated with the new planar protocol can rapidly cure preexisting diabetes in mice.
Fig. 6: The state of the cytoskeleton influences endodermal cell fate.
All single-cell and bulk RNA sequencing data have been deposited in the Gene Expression Omnibus under accession number GSE137659 . Any other data and protocol information used in the manuscript are available from the corresponding author upon reasonable request.
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This work was supported by the JDRF Career Development Award (5-CDA-2017-391-A-N), the National Institutes of Health (NIH) (5R01DK114233) and startup funds from the Washington University School of Medicine Department of Medicine. N.J.H. was supported by the NIH (T32DK007120). K.G.M. was supported by the NIH (T32DK108742). L.V.C. was supported by the NIH (R25GM103757). Microscopy was performed through the Washington University Center for Cellular Imaging, which is supported by the Washington University School of Medicine, the Childrenʼs Discovery Institute (CDI-CORE-2015-505) and the Foundation for Barnes-Jewish Hospital (3770). Microscopy was supported by the Washington University Diabetes Research Center (P30DK020579). Sequencing work was performed by the Washington University Genome Technology Access Center in the Department of Genetics (NIH P30CA91842 and UL1TR000448) and supported by the Washington University Institute of Clinical and Translational Sciences (NIH UL1TR002345). We thank M. Kim for technical assistance.
Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, MO, USA
Nathaniel J. Hogrebe
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA
N.J.H. and J.R.M. conceived of the experimental design. N.J.H., P.A., K.G.M., L.V.C. and J.R.M. contributed to in vitro experiments. P.A., K.G.M. and J.R.M. performed all in vivo experiments. N.J.H. and J.R.M. wrote the manuscript. All authors edited and reviewed the manuscript.
N.J.H., L.V.C. and J.R.M. are inventors on patents and patent applications related to SC-β cell differentiation approaches described in this manuscript.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 Plating pancreatic progenitors on ECM-coated tissue culture polystyrene prevents NEUROG3 expression.
(a) Images of pancreatic progenitors plated at the beginning of stage 4 onto ECM-coated tissue culture polystyrene as per Fig. 1a. Scale bar = 200 µm. (b) A colorimetric antibody-based integrin adhesion assay at the beginning and end of stage 4 confirmed high expression of integrin subunits that bind to collagens I and IV (α1, α2, β1), fibronectin (αV, β1, α5β1), vitronectin (αV, β1, αVβ5) and some but not all laminin isoforms (α3, β1) (n = 3). Data is normalized to an isotype control. (c) Immunostaining confirmed that cells at the beginning of stage 4 lack expression of both NKX6-1 and NEUROG3. Scale bar = 25 µm. (d) qRT-PCR of plated cells at the end of stage 4 (n = 4). All data was generated with HUES8. All data is represented as the mean, and all error bars represent SEM. Individual data points are shown for all bar graphs.
Supplementary Figure 2 Latrunculin treatment induces NEUROG3 expression in plated pancreatic progenitors.
(a) qRT-PCR of pancreatic gene expression at the end of stage 4 in response to different concentrations of latrunculin A added during stage 4 to plated pancreatic progenitors from the 1013-4FA and 1016SeVA iPSC lines (n = 4). (b) qRT-PCR of pancreatic gene expression at the end of stage 4 in response to latrunculin B dosing during stage 4 on plated HUES8 (ANOVA, n = 4). (c) qRT-PCR at the end of stage 4 of untreated HUES8 plated cells, untreated reaggregated clusters, and reaggregated clusters treated with the actin polymerizer jasplakinolide (unpaired two-sided t-tests, n = 4). (d) Western blots of the G/F actin ratio within cells under different culture formats and treated with latrunculin A are shown with the corresponding protein ladder (n = 3). All data was generated by plating down suspension clusters at the beginning of stage 4 and culturing the cells on collagen 1 coated plates throughout stage 4. All data is represented as the mean, and all error bars represent SEM. Individual data points are shown for all bar graphs. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Supplementary Figure 3 Single-cell RNA sequencing demonstrates that the state of the cytoskeleton influences gene expression in pancreatic progenitors.
(a) Immunostaining of F-actin in plated stage 4 PDX1-expressing pancreatic progenitors with and without a 5 µM nocodazole treatment for 24 hours. (b) tSNE plots generated from single-cell RNA sequencing data of plated HUES8 pancreatic progenitors showing expression of pancreatic and off-target genes. Red indicates increased gene expression levels (n = 1,062 total cells). All data was generated by plating down HUES8 suspension clusters at the beginning of stage 4 and culturing the cells on collagen 1 coated plates throughout stage 4.
Supplementary Figure 4 Latrunculin A treatment enables a planar protocol for making SC-β cells.
(a)qRT-PCR of HUES8 cells differentiated with the new planar protocol to the end of stage 4, untreated or treated throughout stage 4 with 0.5 µM latrunculin A (unpaired two-sided t-tests, n = 4). (b-d) qRT-PCR of HUES8 cells differentiated with the planar protocol to stage 6 with or without a 24 hour 1 µM latrunculin A treatment at the beginning of stage 5, (b, c) showing expression of islet and β cell genes and (d) non-endocrine genes (unpaired two-sided t-tests, n = 4). (e, f) Immunostaining of aggregates generated from the planar protocol with (e) 1013-4FA and (f) 1016SeVA iPSC lines. (g) MAFB immunostaining of stage 6 cells generated with the planar protocol from HUES8. MAFB is shown in both planar cells and in histological sections of clusters aggregated from planar cells. All data is represented as the mean, and all error bars represent SEM. Individual data points are shown for all bar graphs. Scale bars = 50 µm.
Supplementary Figure 5 SC-β cells generated with the planar protocol demonstrate high levels of functionality.
(a, b) Static GSIS of SC-β cells generated with the planar protocol from HUES8 in response to (a) sequential glucose challenges (paired two-sided t-test, n = 4) and (b) various secretagogues (paired two-sided t-test, n = 4). (c) Electron microscopy image of SC-β cells generated with the planar protocol from HUES8 demonstrating the presence of insulin granules. (d) Calcium flux of SC-β cells generated with the planar protocol from HUES8 and human islets in response to high glucose and KCL (n = 10 clusters). (e) Quantified cumulative population doublings of stem cells cultured in suspension bioreactors comparing the BJFF.6 iPSC line with HUES8. (f) Stage 6 clusters generated from the BJFF.6 iPSC line with the planar differentiation methodology. Scale bar = 250 µm. (g) Dynamic GSIS of stage 6 clusters generated from the BJFF.6 iPSC line with the planar differentiation methodology. (h) Dynamic (n = 2) and (i) static (paired two-sided t-test, n = 6) GSIS of SC-β cells generated from HUES8 with the planar protocol in a T-75 flask. (j) Quantification of mouse c-peptide with ELISA of serum from mice (untreated control, n = 5; STZ no transplant, n = 3; STZ planar transplant, n = 12). (k) Quantification of human insulin in the serum of mice without a transplant (untreated control, n = 5; STZ no transplant, n = 3) (l) A heatmap of genes associated with insulin processing and secretion was generated from the bulk RNA sequencing data in Figures 6a-d, comparing stage 6 cells produced with the suspension protocol to those of plated cells receiving the optimal stage 5 latrunculin A treatment. All data is represented as the mean, and all error bars represent SEM. Individual data points are shown for all bar graphs. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Supplementary Figure 6 SC-β cells generated from the planar and suspension protocols with the HUES8 line have similar gene expression and functionality.
(a) qRT-PCR of islet and disallowed (LDHA, SLC16A1) genes for stage 6 cells generated with the planar protocol compared to those generated with the suspension protocol and to human islets (Dunnett’s multiple comparisons test; planar, n = 4; suspension, n = 6; human islets, n = 3). (b) Insulin content of planar stage 6 cells compared to those generated with the suspension protocol (planar, n = 5; suspension, n = 4). (c) Proinsulin/insulin content ratio for planar stage 6 cells compared to those generated with the suspension protocol (planar, n = 5; suspension, n = 4). (d) Static GSIS for planar stage 6 cells compared to those generated with the suspension protocol (paired two-sided t-tests; planar, n = 11; suspension, n = 8). All data was generated with HUES8. All data is represented as the mean, and all error bars represent SEM. Individual data points are shown for all bar graphs. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.