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• W2999894002 abstract "•Single-cell profiling identifies genes induced in proliferating β cells•Activation of the LIF pathway induces proliferation of human β cells•LIFR+ β cells represent a subpopulation of transcriptionally distinct β cells•STAT3 and CEBPD are targets of LIF and regulators of β-cell proliferation The beta (β)-cell mass formed during embryogenesis is amplified by cell replication during fetal and early postnatal development. Thereafter, β cells become functionally mature, and their mass is maintained by a low rate of replication. For those few β cells that replicate in adult life, it is not known how replication is initiated nor whether this occurs in a specialized subset of β cells. We capitalized on a YAP overexpression system to induce replication of stem-cell-derived β cells and, by single-cell RNA sequencing, identified an upregulation of the leukemia inhibitory factor (LIF) pathway. Activation of the LIF pathway induces replication of human β cells in vitro and in vivo. The expression of the LIF receptor is restricted to a subset of transcriptionally distinct human β cells with increased proliferative capacity. This study delineates novel genetic networks that control the replication of LIF-responsive, replication-competent human β cells. The beta (β)-cell mass formed during embryogenesis is amplified by cell replication during fetal and early postnatal development. Thereafter, β cells become functionally mature, and their mass is maintained by a low rate of replication. For those few β cells that replicate in adult life, it is not known how replication is initiated nor whether this occurs in a specialized subset of β cells. We capitalized on a YAP overexpression system to induce replication of stem-cell-derived β cells and, by single-cell RNA sequencing, identified an upregulation of the leukemia inhibitory factor (LIF) pathway. Activation of the LIF pathway induces replication of human β cells in vitro and in vivo. The expression of the LIF receptor is restricted to a subset of transcriptionally distinct human β cells with increased proliferative capacity. This study delineates novel genetic networks that control the replication of LIF-responsive, replication-competent human β cells. Approaches that induce the proliferation of insulin-producing pancreatic β cells may hold promising therapeutic potential to treat insulin-dependent diabetes. However, factors that regulate this process in humans, essential to control proper β-cell mass, remain elusive. Here, Rosado-Olivieri et al. developed a method to identify genes and pathways regulating this process based on single-cell RNA-sequencing analysis of proliferating human β cells. They identified a subpopulation of such cells that can be expanded in vitro and in vivo upon activation of the molecular pathway downstream of leukemia inhibitory factor (LIF), a cytokine known to regulate cell growth but previously little studied in β-cell biology. This signaling pathway represents a potential therapeutic target for the regeneration of β cells in diabetic patients. Pancreatic β cells are essential for the control of glucose homeostasis, and deficits in β-cell mass contribute to the development of type 1 and type 2 diabetes (Gepts, 1965Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus.Diabetes. 1965; 14: 619-633Crossref PubMed Scopus (982) Google Scholar, Pipeleers and Ling, 1992Pipeleers D. Ling Z. Pancreatic beta cells in insulin-dependent diabetes.Diabetes Metab. Rev. 1992; 8: 209-227Crossref PubMed Scopus (128) Google Scholar). As self-duplication of pre-existing β cells is the main mechanism controlling β-cell mass in adults (Dor et al., 2004Dor Y. Brown J. Martinez O.I. Melton D.A. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.Nature. 2004; 429: 41-46Crossref PubMed Scopus (1906) Google Scholar, Georgia and Bhushan, 2004Georgia S. Bhushan A. Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass.J. Clin. Invest. 2004; 114: 963-968Crossref PubMed Scopus (359) Google Scholar, Meier et al., 2008Meier J.J. Butler A.E. Saisho Y. Monchamp T. Galasso R. Bhushan A. Rizza R.A. Butler P.C. β-cell replication is the primary mechanism subserving the postnatal expansion of β-cell mass in humans.Diabetes. 2008; 57: 1584-1594Crossref PubMed Scopus (524) Google Scholar), approaches to regenerate β-cell mass in diabetics may offer an alternative therapeutic strategy. In humans, the highest β-cell replication rates occur during the postnatal expansion of β-cell mass (Meier et al., 2008Meier J.J. Butler A.E. Saisho Y. Monchamp T. Galasso R. Bhushan A. Rizza R.A. Butler P.C. β-cell replication is the primary mechanism subserving the postnatal expansion of β-cell mass in humans.Diabetes. 2008; 57: 1584-1594Crossref PubMed Scopus (524) Google Scholar), and this rate declines with postnatal functional maturation and age (Blum et al., 2012Blum B. Hrvatin S. Schuetz C. Bonal C. Rezania A. Melton D.A. Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3.Nat. Biotechnol. 2012; 30: 261-264Crossref PubMed Scopus (249) Google Scholar, Helman et al., 2016Helman A. Klochendler A. Azazmeh N. Gabai Y. Horwitz E. Anzi S. Swisa A. Condiotti R. Granit R.Z. Nevo Y. et al.p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion.Nat. Med. 2016; 22: 412-420Crossref PubMed Scopus (184) Google Scholar, Jermendy et al., 2011Jermendy A. Toschi E. Aye T. Koh A. Aguayo-Mazzucato C. Sharma A. Weir G.C. Sgroi D. Bonner-Weir S. Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells.Diabetologia. 2011; 54: 594-604Crossref PubMed Scopus (94) Google Scholar; Martens et al., 2013Martens G.A. Motté E. Kramer G. Stangé G. Gaarn L.W. Hellemans K. Nielsen J.H. Aerts J.M. Ling Z. Pipeleers D. Functional characteristics of neonatal rat β cells with distinct markers.J. Mol. Endocrinol. 2013; 52: 11-28Crossref PubMed Scopus (31) Google Scholar, Qiu et al., 2017Qiu W.L. Zhang Y.W. Feng Y. Li L.C. Yang L. Xu C.R. Deciphering pancreatic islet b cell and a cell maturation pathways and characteristic features at the single-cell level.Cell Metab. 2017; 25: 1194-1205.e4Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, Zeng et al., 2017Zeng C. Mulas F. Sui Y. Guan T. Miller N. Tan Y. Liu F. Jin W. Carrano A.C. Huising M.O. et al.Pseudotemporal ordering of single cells reveals metabolic control of postnatal β cell proliferation.Cell Metab. 2017; 25: 1160-1175.e11Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Induction of replication in adult mouse and human β cells by genetically manipulating cell-cycle effectors reverts them to an immature state and represses genes involved in glucose sensing, insulin secretion, and maturation (Kim et al., 2010Kim H. Toyofuku Y. Lynn F.C. Chak E. Uchida T. Mizukami H. Fujitani Y. Kawamori R. Miyatsuka T. Kosaka Y. et al.Serotonin regulates pancreatic beta cell mass during pregnancy.Nat. Med. 2010; 16: 804-808Crossref PubMed Scopus (424) Google Scholar, Klochendler et al., 2016Klochendler A. Caspi I. Corem N. Moran M. Friedlich O. Elgavish S. Nevo Y. Helman A. Glaser B. Eden A. et al.The genetic program of pancreatic β-cell replication in vivo.Diabetes. 2016; 65: 2081-2093Crossref PubMed Scopus (54) Google Scholar, Puri et al., 2018Puri S. Roy N. Russ H.A. Leonhardt L. French E.K. Roy R. Bengtsson H. Scott D.K. Stewart A.F. Hebrok M. Replication confers β cell immaturity.Nat. Commun. 2018; 9: 485Crossref PubMed Scopus (83) Google Scholar). Nonetheless, it is not understood whether or how β cells decouple physiological function to support cell cycle re-entry and regeneration in humans. Recent efforts have identified subpopulations of β cells that differ in functionality and gene expression. In mice, β-cell subpopulations have been defined based on gene markers that subdivide replicating and mature beta cells (Bader et al., 2016Bader E. Migliorini A. Gegg M. Moruzzi N. Gerdes J. Roscioni S.S. Bakhti M. Brandl E. Irmler M. Beckers J. et al.Identification of proliferative and mature β-cells in the islets of Langerhans.Nature. 2016; 535: 430-434Crossref PubMed Scopus (215) Google Scholar), insulin mRNA expression (Farack et al., 2019Farack L. Golan M. Egozi A. Dezorella N. Bahar Halpern K. Ben-Moshe S. Garzilli I. Tóth B. Roitman L. Krizhanovsky V. Itzkovitz S. Transcriptional heterogeneity of beta cells in the intact pancreas.Dev. Cell. 2019; 48: 115-125.e4Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and electrochemical properties (Johnston et al., 2016Johnston N.R. Mitchell R.K. Haythorne E. Pessoa M.P. Semplici F. Ferrer J. Piemonti L. Marchetti P. Bugliani M. Bosco D. et al.Beta cell hubs dictate pancreatic islet responses to glucose.Cell Metab. 2016; 24: 389-401Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), yet whether such heterogeneity exists in the human context has not been addressed (Liu and Hebrok, 2017Liu J.S. Hebrok M. All mixed up: defining roles for β-cell subtypes in mature islets.Genes Dev. 2017; 31: 228-240Crossref PubMed Scopus (50) Google Scholar). Multiple studies have defined subpopulations of human β cells based on single-cell transcriptomic analysis and cell surface markers (Segerstolpe et al., 2016Segerstolpe Å. Palasantza A. Eliasson P. Andersson E.M. Andréasson A.C. Sun X. Picelli S. Sabirsh A. Clausen M. Bjursell M.K. et al.Single-cell transcriptome profiling of human pancreatic islets in health and type 2 diabetes.Cell Metab. 2016; 24: 593-607Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar, Muraro et al., 2016Muraro M.J. Dharmadhikari G. Grün D. Groen N. Dielen T. Jansen E. van Gurp L. Engelse M.A. Carlotti F. de Koning E.J. van Oudenaarden A. A single-cell transcriptome atlas of the human pancreas.Cell Syst. 2016; 3: 385-394.e3Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, Baron et al., 2016Baron M. Veres A. Wolock S.L. Faust A.L. Gaujoux R. Vetere A. Ryu J.H. Wagner B.K. Shen-Orr S.S. Klein A.M. et al.A single-cell transcriptomic map of the human and mouse pancreas reveals inter- and intra-cell population structure.Cell Syst. 2016; 3: 346-360.e4Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar, Wang et al., 2016Wang Y.J. Schug J. Won K.J. Liu C. Naji A. Avrahami D. Golson M.L. Kaestner K.H. Single-cell transcriptomics of the human endocrine pancreas.Diabetes. 2016; 65: 3028-3038Crossref PubMed Scopus (220) Google Scholar, Dorrell et al., 2016Dorrell C. Schug J. Canaday P.S. Russ H.A. Tarlow B.D. Grompe M.T. Horton T. Hebrok M. Streeter P.R. Kaestner K.H. Grompe M. Human islets contain four distinct subtypes of β cells.Nat. Commun. 2016; 7: 11756Crossref PubMed Scopus (224) Google Scholar). An analysis of how this heterogeneity reflects replication competence and responsiveness to proliferative cues in subpopulations of human β cells is incomplete. While several growth factors, small molecules, hormones, and nutrients can induce rodent β cell replication, they fail to induce replication in human β cells (Stewart et al., 2015Stewart A.F. Hussain M.A. García-Ocaña A. Vasavada R.C. Bhushan A. Bernal-Mizrachi E. Kulkarni R.N. Human β-cell proliferation and intracellular signaling: part 3.Diabetes. 2015; 64: 1872-1885Crossref PubMed Scopus (101) Google Scholar). The replication and expansion of adult human β cells can be induced by inhibition of DYRK1A and GSK3β signaling (Aamodt et al., 2016Aamodt K.I. Aramandla R. Brown J.J. Fiaschi-Taesch N. Wang P. Stewart A.F. Brissova M. Powers A.C. Development of a reliable automated screening system to identify small molecules and biologics that promote human β-cell regeneration.Am. J. Physiol. Endocrinol. Metab. 2016; 311: E859-E868Crossref PubMed Scopus (24) Google Scholar, Abdolazimi et al., 2018Abdolazimi Y. Zhao Z. Lee S. Xu H. Allegretti P. Horton T.M. Yeh B. Moeller H.P. Nichols R.J. McCutcheon D. et al.CC-401 promotes b-cell replication via pleiotropic consequences of DYRK1A/B inhibition.Endocrinology. 2018; 159: 3143-3157Crossref PubMed Scopus (37) Google Scholar, Dirice et al., 2016Dirice E. Walpita D. Vetere A. Meier B.C. Kahraman S. Hu J. Dančík V. Burns S.M. Gilbert T.J. Olson D.E. et al.Inhibition of DYRK1A stimulates human b-cell proliferation.Diabetes. 2016; 65: 1660-1671Crossref PubMed Scopus (117) Google Scholar, Shen et al., 2015Shen W. Taylor B. Jin Q. Nguyen-Tran V. Meeusen S. Zhang Y.Q. Kamireddy A. Swafford A. Powers A.F. Walker J. et al.Inhibition of DYRK1A and GSK3B induces human β-cell proliferation.Nat. Commun. 2015; 6: 8372Crossref PubMed Scopus (133) Google Scholar, Wang et al., 2015Wang P. Alvarez-Perez J.C. Felsenfeld D.P. Liu H. Sivendran S. Bender A. Kumar A. Sanchez R. Scott D.K. Garcia-Ocaña A. Stewart A.F. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.Nat. Med. 2015; 21: 383-388Crossref PubMed Scopus (246) Google Scholar, Wang et al., 2019Wang P. Karakose E. Liu H. Swartz E. Ackeifi C. Zlatanic V. Wilson J. González B.J. Bender A. Takane K.K. et al.Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells.Cell Metab. 2019; 29: 638-652.e5Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, dissecting the genetic determinants of cell-cycle re-entry in human β cells has been limited due to a lack of model systems relevant to the human context (Stewart et al., 2015Stewart A.F. Hussain M.A. García-Ocaña A. Vasavada R.C. Bhushan A. Bernal-Mizrachi E. Kulkarni R.N. Human β-cell proliferation and intracellular signaling: part 3.Diabetes. 2015; 64: 1872-1885Crossref PubMed Scopus (101) Google Scholar). Finding factors that control this process in human β cells may provide therapeutic targets and inform the development of therapies for the regeneration of β-cell mass in diabetics. We developed a method to induce human β-cell proliferation by the overexpression of a constitutively active, non-phosphorylatable form of the Hippo pathway effector YAP (YAPS6A; Rosado-Olivieri et al., 2019Rosado-Olivieri E.A. Anderson K. Kenty J.H. Melton D.A. YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells.Nat. Commun. 2019; 10: 1464Crossref PubMed Scopus (73) Google Scholar) in stem-cell-derived β (SC-β) cells (Figure 1A). As YAP promotes cell-cycle re-entry in this cell type (Rosado-Olivieri et al., 2019Rosado-Olivieri E.A. Anderson K. Kenty J.H. Melton D.A. YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells.Nat. Commun. 2019; 10: 1464Crossref PubMed Scopus (73) Google Scholar) and native adult counterparts (George et al., 2015George N.M. Boerner B.P. Mir S.U.R. Guinn Z. Sarvetnick N.E. Exploiting expression of hippo effector, Yap, for expansion of functional islet mass.Mol. Endocrinol. 2015; 29: 1594-1607Crossref PubMed Scopus (25) Google Scholar, Yuan et al., 2016Yuan T. Rafizadeh S. Azizi Z. Lupse B. Gorrepati K.D.D. Awal S. Oberholzer J. Maedler K. Ardestani A. Proproliferative and antiapoptotic action of exogenously introduced YAP in pancreatic β cells.JCI Insight. 2016; 1: e86326Crossref PubMed Scopus (20) Google Scholar), this allows us to induce cell-cycle re-entry, enrich for replicating cells, and identify early effectors of the replication process. Sustained activation of YAPS6A leads to a significant increase in cell-cycle re-entry in SC-β and non-β cells, as measured by EdU incorporation in cells (Figures 1B and 1C). We performed droplet-based single-cell RNA sequencing 4 days after the induction of YAP or LacZ expression and sequenced 11,517 stage-matched cells comprising 3 biological replicates per condition (Figures S1A–S1F). We used canonical correlation analysis to align datasets (Butler et al., 2018Butler A. Hoffman P. Smibert P. Papalexi E. Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species.Nat. Biotechnol. 2018; 36: 411-420Crossref PubMed Scopus (4156) Google Scholar) and identified major cell subpopulations based on lineage marker expression (Veres et al., 2019Veres A. Faust A.L. Bushnell H.L. Engquist E.N. Kenty J.H.R. Harb G. Poh Y.C. Sintov E. Gürtler M. Pagliuca F.W. et al.Charting cellular identity during human in vitro β-cell differentiation.Nature. 2019; 569: 368-373Crossref PubMed Scopus (227) Google Scholar), including β (insulin+, NKX6.1+), alpha (glucagon+, ARX+), and other endocrine and non-endocrine subpopulations of cells (Figures 1D–1H and S1G). YAP target genes, including CTGF, were expressed in most cell subpopulations (Figure 1G) and were enriched in YAPS6A-overexpressing cells (Figure S1F). The proportion of cell subpopulations was similar between LacZ-control and YAPS6A-overexpressing cell preparations (Figure 1I). Within β cells (Figures 1J and S2A–S2F), we identified major expression differences between control and YAPS6A-overexpressing cells (Figure 1K). Genes downregulated in YAPS6A-expressing β cells are involved in insulin secretion and β-cell function (Figure 1L), in agreement with recent reports suggesting a reversion of replicating β cells into an immature state (Klochendler et al., 2016Klochendler A. Caspi I. Corem N. Moran M. Friedlich O. Elgavish S. Nevo Y. Helman A. Glaser B. Eden A. et al.The genetic program of pancreatic β-cell replication in vivo.Diabetes. 2016; 65: 2081-2093Crossref PubMed Scopus (54) Google Scholar, Puri et al., 2018Puri S. Roy N. Russ H.A. Leonhardt L. French E.K. Roy R. Bengtsson H. Scott D.K. Stewart A.F. Hebrok M. Replication confers β cell immaturity.Nat. Commun. 2018; 9: 485Crossref PubMed Scopus (83) Google Scholar, Rosado-Olivieri et al., 2019Rosado-Olivieri E.A. Anderson K. Kenty J.H. Melton D.A. YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells.Nat. Commun. 2019; 10: 1464Crossref PubMed Scopus (73) Google Scholar). Genes induced in YAPS6A-overexpressing β cells are enriched for Hippo signaling, the leukemia inhibitory factor (LIF) pathway, and serine biosynthesis (Figure 1M), a biosynthetic pathway that has been recently implicated in rodent fetal β-cell proliferation (Zeng et al., 2017Zeng C. Mulas F. Sui Y. Guan T. Miller N. Tan Y. Liu F. Jin W. Carrano A.C. Huising M.O. et al.Pseudotemporal ordering of single cells reveals metabolic control of postnatal β cell proliferation.Cell Metab. 2017; 25: 1160-1175.e11Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We further resolved a subpopulation of YAPS6A-overexpressing human β cells with an enrichment of gene ontology terms related to cell-cycle progression including translation initiation and cell-cycle G1/S phase transition (Figures S2G–S2K). Non-dividing β cells display an enrichment of genes involved in hormone transport, extracellular signaling, and immune regulation (Figures S2G–S2K). To gain insights into transcriptional dynamics associated with cell-cycle re-entry, we performed pseudotime ordering of β cells (Trapnell et al., 2014Trapnell C. Cacchiarelli D. Grimsby J. Pokharel P. Li S. Morse M. Lennon N.J. Livak K.J. Mikkelsen T.S. Rinn J.L. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.Nat. Biotechnol. 2014; 32: 381-386Crossref PubMed Scopus (2437) Google Scholar) and identified a continuous transition of YAPS6A-overexpressing cells along a single trajectory (Figures S2L–S2N). Genes expressed in functional β cells including insulin (INS), amylin (IAPP), ERO1B, PCSK1, and others are gradually downregulated along the pseudotime trajectory (Figures 1N and S2N). Conversely, cell cycle markers (CCND1, PCNA, MKI67, and MYC), YAP targets (CYR61 and CTGF) and effectors of the LIF pathway (LIF, LIFR, and IL6ST) are upregulated along the trajectory (Figures 1N and S2N). Thus, our analysis suggests that cell-cycle re-entry in β cells correlates with repression of genes involved in β-cell function and an induction of components of the LIF pathway. The LIF pathway has pleiotropic effects in multiple cell types and regulates cell differentiation, proliferation, and survival (Nicola and Babon, 2015Nicola N.A. Babon J.J. Leukemia inhibitory factor (LIF).Cytokine Growth Factor Rev. 2015; 26: 533-544Crossref PubMed Scopus (249) Google Scholar). While gene variants in the LIF gene are associated with type 2 diabetes (Mahajan et al., 2018Mahajan A. Taliun D. Thurner M. Robertson N.R. Torres J.M. Rayner N.W. Payne A.J. Steinthorsdottir V. Scott R.A. Grarup N. et al.Fine-mapping type 2 diabetes loci to single-variant resolution using high-density imputation and islet-specific epigenome maps.Nat. Genet. 2018; 50: 1505-1513Crossref PubMed Scopus (745) Google Scholar; http://www.type2diabetesgenetics.org), whether it controls β-cell proliferation is unknown. Several genes in the pathway, including LIF, LIFR (also known as CD118), IL6ST (also known as gp130/CD130), and STAT3 are all upregulated in YAPS6A-overexpressing replicating β cells (Figures 2A and 2B ). Cardiotrophin-1 (CTF1), a related cytokine that can also activate LIFR/IL6ST receptor complex (Nicola and Babon, 2015Nicola N.A. Babon J.J. Leukemia inhibitory factor (LIF).Cytokine Growth Factor Rev. 2015; 26: 533-544Crossref PubMed Scopus (249) Google Scholar), is also induced in replicating β cells (Figure 2A). Applying the ligands LIF and CTF1 induces replication of monohormonal β cells (Figures 2C, S3A, and S3B). Increased replication following LIF pathway activation was also observed in polyhormonal (INS+, glucagon+) cells (Figure S4A), which are fated to become alpha cells (Sharon et al., 2019Sharon N. Chawla R. Mueller J. Vanderhooft J. Whitehorn L.J. Rosenthal B. Gürtler M. Estanboulieh R.R. Shvartsman D. Gifford D.K. et al.A peninsular structure coordinates asynchronous differentiation with morphogenesis to generate pancreatic islets.Cell. 2019; 176: 790-804.e13Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, Veres et al., 2019Veres A. Faust A.L. Bushnell H.L. Engquist E.N. Kenty J.H.R. Harb G. Poh Y.C. Sintov E. Gürtler M. Pagliuca F.W. et al.Charting cellular identity during human in vitro β-cell differentiation.Nature. 2019; 569: 368-373Crossref PubMed Scopus (227) Google Scholar). Notably, LIF does not stimulate replication of Chromogranin A(CHGA)-negative non-endocrine cells (Figures S3C, S4B, and S4C). To test whether LIF pathway activation increases endocrine mass, we treated differentiated SC-β-cell clusters with LIF for 14 days (Figure 2D) in suspension cultures and observed a significant increase in cluster size (Figures 2E and 2F). Using an insulin reporter stem cell line, we detected an increase in the total number of SC-β cells by flow cytometry upon stimulation with LIF for 7 days (Figure S4D). In addition, LIF pathway activation significantly increases cell number but does not affect the proportion of monohormonal SC-β cells (Figures S4E and S4F), which reflects a net increase in β-cell number upon LIF pathway activation. Importantly, sustained LIF pathway activation in SC-β cells does not impair function as determined by an in vitro glucose-stimulated insulin secretion (GSIS) assay (Figures 2G and 2H). LIF pathway stimulation in SC-β cells induces the expression of multiple cell cycle regulators including cyclins CCNA2, CCNB1, CCNB2, and CCNE2, cyclin-dependent kinases CDK2 and CDK4 as well as suppressors CDKN2A/C/D, as measured by qPCR (Figure 2I). As some of these cell-cycle effectors are also regulated by the combined inhibition of DYRK1A and TGFβ signaling in human β cells (Wang et al., 2019Wang P. Karakose E. Liu H. Swartz E. Ackeifi C. Zlatanic V. Wilson J. González B.J. Bender A. Takane K.K. et al.Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells.Cell Metab. 2019; 29: 638-652.e5Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we sought to determine whether the LIF pathway would synergize with these signaling axes to promote β-cell proliferation. Interestingly, combined modulation of the LIF pathway with the DYRK1A inhibitor, harmine, and the TGFβ inhibitor, LY364947 significantly increases the proportion of EdU+-replicating SC-β cells compared to combined treatment with harmine and LY364947 (Figure 2J). Thus, the LIF pathway synergizes with DYRK1A and TGFβ signaling to support cell-cycle re-entry in human β cells. JAK/STAT3 signaling is a downstream target of the LIF pathway in multiple tissues and cell types (Nicola and Babon, 2015Nicola N.A. Babon J.J. Leukemia inhibitory factor (LIF).Cytokine Growth Factor Rev. 2015; 26: 533-544Crossref PubMed Scopus (249) Google Scholar). Interestingly, LIF treatment robustly induced STAT3 activity in β and non- β cells as assayed by the expression of phosphorylated active STAT3 (pTyr705) in clusters of SC-β cells (Figures 3A and 3B ). To further delineate a dependency of the LIF pathway on STAT3 signaling for the induction of β-cell replication, we treated SC-β cells with the STAT3 inhibitor, stattic, or overexpressed a dominant-negative phospho-deficient form of STAT3 (STAT3-DN), which both prevent STAT3 dimerization and subsequent activation (Hillion et al., 2008Hillion J. Dhara S. Sumter T.F. Mukherjee M. Di Cello F. Belton A. Turkson J. Jaganathan S. Cheng L. Ye Z. et al.The high-mobility group A1a/signal transducer and activator of transcription-3 axis: an achilles heel for hematopoietic malignancies?.Cancer Res. 2008; 68: 10121-10127Crossref PubMed Scopus (84) Google Scholar). Both pharmacological and genetic inhibition of STAT3 activity blocked LIF-induced cell-cycle re-entry in SC-β cells (Figures 3C and 3D). In addition, the induction of most cell-cycle effectors upon LIF pathway activation in SC-β cells, identified previously by qPCR, is impaired when STAT3 activity is blocked with stattic (Figure 3E). Thus, LIF induces replication of β cells in a STAT3-dependent manner. LIF pathway activation also increases the proliferation rates of adult human and mouse islet β cells in vitro (Figures 4A–4C ). Similar to SC-β cells, its activation in human islets in vitro induces the expression of many cell-cycle effectors, including CCNB1, CCNB2, CCND1, CCNE2, and CDK2 (Figure 4D). Moreover, daily intraperitoneal injections of recombinant mouse LIF for 7 days increased the replication rates of islet mouse β cells (Figures 4E and 4F). Similarly, injection of human recombinant LIF (hrLIF) significantly increased the replication rates of human β cells transplanted into the kidney capsule of euglycemic NOD-SCID IL2Rgnull (NSG) mice (Figures 4G and 4H). To determine whether LIF pathway agonism can improve diabetes outcomes in vivo, we followed a human islet transplant model in streptozotocin-induced diabetic NSG mice in which treatment with human β cell mitogens improves glycemic control due to increased β-cell mass (Wang et al., 2015Wang P. Alvarez-Perez J.C. Felsenfeld D.P. Liu H. Sivendran S. Bender A. Kumar A. Sanchez R. Scott D.K. Garcia-Ocaña A. Stewart A.F. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.Nat. Med. 2015; 21: 383-388Crossref PubMed Scopus (246) Google Scholar). In streptozocin-treated NSG mice transplanted with human islets, daily injection of hrLIF for 14 days enhances glycemic control as assessed by fed blood glucose levels, a glucose tolerance test, and glucose-induced insulin secretion during in vivo GSIS (Figures 4I–4K). Thus, LIF pathway activation induces replication of human adult islet β cells. LIF pathway activation depends on the cell surface expression of the receptor LIFR and co-receptor subunit GP130 (Nicola and Babon, 2015Nicola N.A. Babon J.J. Leukemia inhibitory factor (LIF).Cytokine Growth Factor Rev. 2015; 26: 533-544Crossref PubMed Scopus (249) Google Scholar). Expression of these receptors was observed in a subset of SC-β cells by single-cell RNA sequencing and immunostaining (Figures 2B, 5A, 5B , and S5E). In both SC-β cells and adult human islet β cells, LIFR is expressed in less than 20% of all insulin-expressing β cells (Figures 5A–5F), which suggests that there are subpopulations of β cells defined by the expression of LIFR. To test that, we employed the SC-β-cell-specific cell surface marker CD49a (Veres et al., 2019Veres A. Faust A.L. Bushnell H.L. Engquist E.N. Kenty J.H.R. Harb G. Poh Y.C. Sintov E. Gürtler M. Pagliuca F.W. et al.Charting cellular identity during human in vitro β-cell differentiation.Nature. 2019; 569: 368-373Crossref PubMed Scopus (227) Google Scholar) to sort LIFR+ and LIFR− β cells and interrogate transcriptional differences by RNA sequencing (Figures 5G–5H and S5A–S5D). We identified a set of 169 genes that are differentially expressed (adjusted p value < 0.05), suggesting that that they may represent subpopulations of β cells with distinct gene expression signatures (Figure 5I). Genes upregulated in LIFR+ β cells are involved in cell junction organization (CLDN6, FN1, AMOT, and ITGB4), proliferation (CDC20B and NABP1), and oxidative phosphorylation (MT-ND1/2/3/5/6 subunits, MT-RNR1) (Figures 5J–5L). These gene expression differences may confer differences in proliferative potential, as LIFR+ SC-β cells display higher proliferation rates than do LIFR− cells under unstimulated conditions (Figures 5M, S5F, and S5G). Genes enriched in LIFR− β cells are involved in hormone secretion such as insulin, CHGA, and carboxypeptidase-E (CPE), suggesting that there may also be differences in functionality (Figures 5J–5L). Interestingly, we did not find differences in expression" @default.
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• W2999894002 date "2020-02-01" @default.
• W2999894002 modified "2023-10-16" @default.
• W2999894002 title "Identification of a LIF-Responsive, Replication-Competent Subpopulation of Human β Cells" @default.
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• W2999894002 doi "https://doi.org/10.1016/j.cmet.2019.12.009" @default.
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