Unraveling the Inconsistencies of Cardiac Differentiation Efficiency Induced by the GSK3β Inhibitor CHIR99021 in Human Pluripotent Stem Cells

Overview

Journal Stem Cell Reports

Publisher Cell Press

Specialty Cell Biology

Date 2018 May 1

PMID 29706502

Citations 48

Authors

Filip Laco

Tsung Liang Woo

Qixing Zhong

Radoslaw Szmyd

Sherwin Ting

Fahima Jaleel Khan

Christina L L Chai

Shaul Reuveny

Allen Chen

Steve Oh

Affiliations

Soon will be listed here.

Abstract

Cardiac differentiation efficiency is hampered by inconsistencies and low reproducibility. We analyzed the differentiation process of multiple human pluripotent stem cell (hPSC) lines in response to dynamic GSK3β inhibition under varying cell culture conditions. hPSCs showed strong differences in cell-cycle profiles with varying culture confluency. hPSCs with a higher percentage of cells in the G1 phase of the cell cycle exhibited cell death and required lower doses of GSK3β inhibitors to induce cardiac differentiation. GSK3β inhibition initiated cell-cycle progression via cyclin D1 and modulated both Wnt signaling and the transcription factor (TCF) levels, resulting in accelerated or delayed mesoderm differentiation. The TCF levels were key regulators during hPSC differentiation with CHIR99021. Our results explain how differences in hPSC lines and culture conditions impact cell death and cardiac differentiation. By analyzing the cell cycle, we were able to select for highly cardiogenic hPSC lines and increase the experimental reproducibility by predicting differentiation outcomes.

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References

Mendjan S, Mascetti V, Ortmann D, Ortiz M, Karjosukarso D, Ng Y . NANOG and CDX2 pattern distinct subtypes of human mesoderm during exit from pluripotency. Cell Stem Cell. 2014; 15(3):310-325. DOI: 10.1016/j.stem.2014.06.006. View

Rao J, Pfeiffer M, Frank S, Adachi K, Piccini I, Quaranta R . Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs. Cell Stem Cell. 2016; 18(3):341-53. DOI: 10.1016/j.stem.2015.11.019. View

Jacobs K, Zambelli F, Mertzanidou A, Smolders I, Geens M, Nguyen H . Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability. Stem Cell Reports. 2016; 6(3):330-41. PMC: 4788786. DOI: 10.1016/j.stemcr.2016.01.015. View

Naujok O, Lentes J, Diekmann U, Davenport C, Lenzen S . Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors. BMC Res Notes. 2014; 7:273. PMC: 4008422. DOI: 10.1186/1756-0500-7-273. View

Laco F, Low J, Seow J, Woo T, Zhong Q, Seayad J . Cardiomyocyte differentiation of pluripotent stem cells with SB203580 analogues correlates with Wnt pathway CK1 inhibition independent of p38 MAPK signaling. J Mol Cell Cardiol. 2014; 80:56-70. DOI: 10.1016/j.yjmcc.2014.12.003. View

Yoshino Y, Ishioka C . Inhibition of glycogen synthase kinase-3 beta induces apoptosis and mitotic catastrophe by disrupting centrosome regulation in cancer cells. Sci Rep. 2015; 5:13249. PMC: 4543981. DOI: 10.1038/srep13249. View

Kapinas K, Grandy R, Ghule P, Medina R, Becker K, Pardee A . The abbreviated pluripotent cell cycle. J Cell Physiol. 2012; 228(1):9-20. PMC: 3667593. DOI: 10.1002/jcp.24104. View

Tan J, Sriram G, Rufaihah A, Neoh K, Cao T . Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embryonic stem cells through GSKi-mediated differentiation. Stem Cells Dev. 2013; 22(13):1893-906. PMC: 3685395. DOI: 10.1089/scd.2012.0590. View

Siller R, Greenhough S, Naumovska E, Sullivan G . Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells. Stem Cell Reports. 2015; 4(5):939-52. PMC: 4437467. DOI: 10.1016/j.stemcr.2015.04.001. View

10.

Gonzalez J, Morgani S, Bone R, Bonderup K, Abelchian S, Brakebusch C . Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency. Stem Cell Reports. 2016; 7(2):177-91. PMC: 4983099. DOI: 10.1016/j.stemcr.2016.07.009. View

11.

Cadigan K . TCFs and Wnt/β-catenin signaling: more than one way to throw the switch. Curr Top Dev Biol. 2012; 98:1-34. DOI: 10.1016/B978-0-12-386499-4.00001-X. View

12.

Huang T, Li L, Moalim-Nour L, Jia D, Bai J, Yao Z . A Regulatory Network Involving β-Catenin, e-Cadherin, PI3k/Akt, and Slug Balances Self-Renewal and Differentiation of Human Pluripotent Stem Cells In Response to Wnt Signaling. Stem Cells. 2014; 33(5):1419-33. PMC: 5297972. DOI: 10.1002/stem.1944. View

13.

Desmarais J, Hoffmann M, Bingham G, Gagou M, Meuth M, Andrews P . Human embryonic stem cells fail to activate CHK1 and commit to apoptosis in response to DNA replication stress. Stem Cells. 2012; 30(7):1385-93. DOI: 10.1002/stem.1117. View

14.

Ding Y, Su S, Tang W, Zhang X, Chen S, Zhu G . Enrichment of the β-catenin-TCF complex at the S and G2 phases ensures cell survival and cell cycle progression. J Cell Sci. 2014; 127(Pt 22):4833-45. DOI: 10.1242/jcs.146977. View

15.

Hodar C, Assar R, Colombres M, Aravena A, Pavez L, Gonzalez M . Genome-wide identification of new Wnt/beta-catenin target genes in the human genome using CART method. BMC Genomics. 2010; 11:348. PMC: 2996972. DOI: 10.1186/1471-2164-11-348. View

16.

Vitale I, Manic G, De Maria R, Kroemer G, Galluzzi L . DNA Damage in Stem Cells. Mol Cell. 2017; 66(3):306-319. DOI: 10.1016/j.molcel.2017.04.006. View

17.

Tighe A, Ray-Sinha A, Staples O, Taylor S . GSK-3 inhibitors induce chromosome instability. BMC Cell Biol. 2007; 8:34. PMC: 1976608. DOI: 10.1186/1471-2121-8-34. View

18.

Yi F, Pereira L, Hoffman J, Shy B, Yuen C, Liu D . Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal. Nat Cell Biol. 2011; 13(7):762-70. PMC: 3129424. DOI: 10.1038/ncb2283. View

19.

Desmarais J, Unger C, Damjanov I, Meuth M, Andrews P . Apoptosis and failure of checkpoint kinase 1 activation in human induced pluripotent stem cells under replication stress. Stem Cell Res Ther. 2016; 7:17. PMC: 4727355. DOI: 10.1186/s13287-016-0279-2. View

20.

Barta T, Vinarsky V, Holubcova Z, Dolezalova D, Verner J, Pospisilova S . Human embryonic stem cells are capable of executing G1/S checkpoint activation. Stem Cells. 2010; 28(7):1143-52. DOI: 10.1002/stem.451. View