Single-cell Lineage Analysis Reveals Extensive Multimodal Transcriptional Control During Directed Beta-cell Differentiation

Overview

Journal Nat Metab

Publisher Springer Nature

Specialty Endocrinology

Date 2020 Dec 1

PMID 33257854

Citations 29

Authors

Chen Weng

Jiajia Xi

Haiyan Li

Jian Cui

Anniya Gu

Sisi Lai

Konstantin Leskov

Luxin Ke

Fulai Jin

Yan Li

Affiliations

Soon will be listed here.

Abstract

The in vitro differentiation of insulin-producing beta-like cells can model aspects of human pancreatic development. Here, we generate 95,308 single-cell transcriptomes and reconstruct a lineage tree of the entire differentiation process from human embryonic stem cells to beta-like cells to study temporally regulated genes during differentiation. We identify so-called 'switch genes' at the branch point of endocrine/non-endocrine cell fate choice, revealing insights into the mechanisms of differentiation-promoting reagents, such as NOTCH and ROCKII inhibitors, and providing improved differentiation protocols. Over 20% of all detectable genes are activated multiple times during differentiation, even though their enhancer activation is usually unimodal, indicating extensive gene reuse driven by different enhancers. We also identify a stage-specific enhancer at the TCF7L2 locus for diabetes, uncovered by genome-wide association studies, that drives a transient wave of gene expression in pancreatic progenitors. Finally, we develop a web app to visualize gene expression on the lineage tree, providing a comprehensive single-cell data resource for researchers studying islet biology and diabetes.

Citing Articles

ETVs dictate hPSC differentiation by tuning biophysical properties.

Ziojla N, Socha M, Guerra M, Kizewska D, Blaszczyk K, Urbaniak E Nat Commun. 2025; 16(1):1999.

PMID: 40011454 PMC: 11865489. DOI: 10.1038/s41467-025-56591-6.

Extracellular matrix proteins refine microenvironments for pancreatic organogenesis from induced pluripotent stem cell differentiation.

Hu M, Liu T, Huang H, Ogi D, Tan Y, Ye K Theranostics. 2025; 15(6):2229-2249.

PMID: 39990212 PMC: 11840725. DOI: 10.7150/thno.104883.

Mechanistic elucidation of human pancreatic acinar development using single-cell transcriptome analysis on a human iPSC differentiation model.

Mima A, Kimura A, Ito R, Hatano Y, Tsujimoto H, Mae S Sci Rep. 2025; 15(1):4668.

PMID: 39920294 PMC: 11806057. DOI: 10.1038/s41598-025-88690-1.

Controlling human stem cell-derived islet composition using magnetic sorting.

Kelley A, Shunkarova M, Maestas M, Gale S, Hogrebe N, Millman J bioRxiv. 2024; .

PMID: 39605713 PMC: 11601561. DOI: 10.1101/2024.11.19.624394.

Depolymerizing F-actin accelerates the exit from pluripotency to enhance stem cell-derived islet differentiation.

Hogrebe N, Schmidt M, Augsornworawat P, Gale S, Shunkarova M, Millman J bioRxiv. 2024; .

PMID: 39484596 PMC: 11526947. DOI: 10.1101/2024.10.21.618465.

References

Lu L, Liu X, Huang W, Giusti-Rodriguez P, Cui J, Zhang S . Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases. Mol Cell. 2020; 79(3):521-534.e15. PMC: 7415676. DOI: 10.1016/j.molcel.2020.06.007. View

Kelly O, Chan M, Martinson L, Kadoya K, Ostertag T, Ross K . Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat Biotechnol. 2011; 29(8):750-6. DOI: 10.1038/nbt.1931. View

Rezania A, Bruin J, Riedel M, Mojibian M, Asadi A, Xu J . Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes. 2012; 61(8):2016-29. PMC: 3402300. DOI: 10.2337/db11-1711. View

Rezania A, Bruin J, Arora P, Rubin A, Batushansky I, Asadi A . Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. 2014; 32(11):1121-33. DOI: 10.1038/nbt.3033. View

Lyssenko V, Lupi R, Marchetti P, Del Guerra S, Orho-Melander M, Almgren P . Mechanisms by which common variants in the TCF7L2 gene increase risk of type 2 diabetes. J Clin Invest. 2007; 117(8):2155-63. PMC: 1934596. DOI: 10.1172/JCI30706. View

Perez-Alcantara M, Honore C, Wesolowska-Andersen A, Gloyn A, McCarthy M, Hansson M . Patterns of differential gene expression in a cellular model of human islet development, and relationship to type 2 diabetes predisposition. Diabetologia. 2018; 61(7):1614-1622. PMC: 6354904. DOI: 10.1007/s00125-018-4612-4. View

Lun A, Bach K, Marioni J . Pooling across cells to normalize single-cell RNA sequencing data with many zero counts. Genome Biol. 2016; 17:75. PMC: 4848819. DOI: 10.1186/s13059-016-0947-7. View

Hogrebe N, Augsornworawat P, Maxwell K, Velazco-Cruz L, Millman J . Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells. Nat Biotechnol. 2020; 38(4):460-470. PMC: 7274216. DOI: 10.1038/s41587-020-0430-6. View

Bruin J, Erener S, Vela J, Hu X, Johnson J, Kurata H . Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells. Stem Cell Res. 2013; 12(1):194-208. DOI: 10.1016/j.scr.2013.10.003. View

10.

Bailey T, Boden M, Buske F, Frith M, Grant C, Clementi L . MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009; 37(Web Server issue):W202-8. PMC: 2703892. DOI: 10.1093/nar/gkp335. View

11.

Scavuzzo M, Hill M, Chmielowiec J, Yang D, Teaw J, Sheng K . Endocrine lineage biases arise in temporally distinct endocrine progenitors during pancreatic morphogenesis. Nat Commun. 2018; 9(1):3356. PMC: 6105717. DOI: 10.1038/s41467-018-05740-1. View

12.

Gerst J . Pimp My Ribosome: Ribosomal Protein Paralogs Specify Translational Control. Trends Genet. 2018; 34(11):832-845. DOI: 10.1016/j.tig.2018.08.004. View

13.

DAmour K, Bang A, Eliazer S, Kelly O, Agulnick A, Smart N . Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. 2006; 24(11):1392-401. DOI: 10.1038/nbt1259. View

14.

Ghazizadeh Z, Kao D, Amin S, Cook B, Rao S, Zhou T . ROCKII inhibition promotes the maturation of human pancreatic beta-like cells. Nat Commun. 2017; 8(1):298. PMC: 5563509. DOI: 10.1038/s41467-017-00129-y. View

15.

Riedel M, Asadi A, Wang R, Ao Z, Warnock G, Kieffer T . Immunohistochemical characterisation of cells co-producing insulin and glucagon in the developing human pancreas. Diabetologia. 2011; 55(2):372-81. DOI: 10.1007/s00125-011-2344-9. View

16.

Chiou J, Zeng C, Cheng Z, Han J, Schlichting M, Miller M . Single-cell chromatin accessibility identifies pancreatic islet cell type- and state-specific regulatory programs of diabetes risk. Nat Genet. 2021; 53(4):455-466. PMC: 9037575. DOI: 10.1038/s41588-021-00823-0. View

17.

Sharon N, Vanderhooft J, Straubhaar J, Mueller J, Chawla R, Zhou Q . Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development. Cell Rep. 2019; 27(8):2281-2291.e5. PMC: 6933053. DOI: 10.1016/j.celrep.2019.04.083. View

18.

Macosko E, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M . Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015; 161(5):1202-1214. PMC: 4481139. DOI: 10.1016/j.cell.2015.05.002. View

19.

Chen Q, Hu G . Post-transcriptional regulation of the pluripotent state. Curr Opin Genet Dev. 2017; 46:15-23. PMC: 6476425. DOI: 10.1016/j.gde.2017.06.010. View

20.

Murtaugh L, Stanger B, Kwan K, Melton D . Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A. 2003; 100(25):14920-5. PMC: 299853. DOI: 10.1073/pnas.2436557100. View