The role of insulin as a key regulator of seeding, proliferation, and mRNA transcription of human pluripotent stem cells.


Journal

Stem cell research & therapy
ISSN: 1757-6512
Titre abrégé: Stem Cell Res Ther
Pays: England
ID NLM: 101527581

Informations de publication

Date de publication:
29 07 2019
Historique:
received: 24 10 2018
accepted: 30 06 2019
revised: 06 05 2019
entrez: 31 7 2019
pubmed: 31 7 2019
medline: 26 6 2020
Statut: epublish

Résumé

Human-induced pluripotent stem cells (hiPSCs) show a great promise as a renewable source of cells with broad biomedical applications. Since insulin has been used in the maintenance of hiPSCs, in this study we explored the role of insulin in culture of these cells. We report conditions for insulin starvation and stimulation of hiPSCs. Crystal violet staining was used to study the adhesion and proliferation of hiPSCs. Apoptosis and cell cycle assays were performed through flow cytometry. Protein arrays were used to confirm phosphorylation targets, and mRNA sequencing was used to evaluate the effect of transcriptome. Insulin improved the seeding and proliferation of hiPSCs. We also observed an altered cell cycle profile and increase in apoptosis in hiPSCs in the absence of insulin. Furthermore, we confirmed phosphorylation of key components of insulin signaling pathway in the presence of insulin and demonstrated the significant effect of insulin on regulation of the mRNA transcriptome of hiPSCs. Insulin is a major regulator of seeding, proliferation, phosphorylation and mRNA transcriptome in hiPSCs. Collectively, our work furthers our understanding of human pluripotency and paves the way for future studies that use hiPSCs for modeling genetic ailments affecting insulin signaling pathways.

Sections du résumé

BACKGROUND
Human-induced pluripotent stem cells (hiPSCs) show a great promise as a renewable source of cells with broad biomedical applications. Since insulin has been used in the maintenance of hiPSCs, in this study we explored the role of insulin in culture of these cells.
METHODS
We report conditions for insulin starvation and stimulation of hiPSCs. Crystal violet staining was used to study the adhesion and proliferation of hiPSCs. Apoptosis and cell cycle assays were performed through flow cytometry. Protein arrays were used to confirm phosphorylation targets, and mRNA sequencing was used to evaluate the effect of transcriptome.
RESULTS
Insulin improved the seeding and proliferation of hiPSCs. We also observed an altered cell cycle profile and increase in apoptosis in hiPSCs in the absence of insulin. Furthermore, we confirmed phosphorylation of key components of insulin signaling pathway in the presence of insulin and demonstrated the significant effect of insulin on regulation of the mRNA transcriptome of hiPSCs.
CONCLUSION
Insulin is a major regulator of seeding, proliferation, phosphorylation and mRNA transcriptome in hiPSCs. Collectively, our work furthers our understanding of human pluripotency and paves the way for future studies that use hiPSCs for modeling genetic ailments affecting insulin signaling pathways.

Identifiants

pubmed: 31358052
doi: 10.1186/s13287-019-1319-5
pii: 10.1186/s13287-019-1319-5
pmc: PMC6664730
doi:

Substances chimiques

IGF1R protein, human 0
Insulin 0
RNA, Messenger 0
Receptor, IGF Type 1 EC 2.7.10.1

Types de publication

Journal Article Research Support, N.I.H., Extramural

Langues

eng

Sous-ensembles de citation

IM

Pagination

228

Subventions

Organisme : Doris Duke Charitable Foundation
ID : 2016097
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK106236
Pays : United States
Organisme : NIH HHS
ID : U01HL107388
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK120565
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK116750
Pays : United States
Organisme : NIDDK NIH HHS
ID : P30 DK116074
Pays : United States

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Auteurs

Mohammad Shahbazi (M)

Stanford Cardiovascular Medicine and Cardiovascular Institute, Stanford School of Medicine, Stanford University, Falk CVRC, Room CV273, MC 5406 300 Pasteur Drive, Stanford, CA, 94305, USA.

Paige Cundiff (P)

Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, Mount Sinai, New York, NY, 10029, USA.

Wenyu Zhou (W)

Stanford Center for Genomics and Personalized Medicine, Stanford University, Stanford, CA, 94305, USA.
Stanford Diabetes Research Center, Stanford University, Stanford, CA, 94305, USA.
Genetics Bioinformatics Service Center, Stanford University, Stanford, CA, 94305, USA.

Philip Lee (P)

Stanford Cardiovascular Medicine and Cardiovascular Institute, Stanford School of Medicine, Stanford University, Falk CVRC, Room CV273, MC 5406 300 Pasteur Drive, Stanford, CA, 94305, USA.

Achchhe Patel (A)

Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, Mount Sinai, New York, NY, 10029, USA.

Sunita L D'Souza (SL)

Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, Mount Sinai, New York, NY, 10029, USA.

Fahim Abbasi (F)

Stanford Cardiovascular Medicine and Cardiovascular Institute, Stanford School of Medicine, Stanford University, Falk CVRC, Room CV273, MC 5406 300 Pasteur Drive, Stanford, CA, 94305, USA.

Thomas Quertermous (T)

Stanford Cardiovascular Medicine and Cardiovascular Institute, Stanford School of Medicine, Stanford University, Falk CVRC, Room CV273, MC 5406 300 Pasteur Drive, Stanford, CA, 94305, USA.

Joshua W Knowles (JW)

Stanford Cardiovascular Medicine and Cardiovascular Institute, Stanford School of Medicine, Stanford University, Falk CVRC, Room CV273, MC 5406 300 Pasteur Drive, Stanford, CA, 94305, USA. knowlej@stanford.edu.
Stanford Diabetes Research Center, Stanford University, Stanford, CA, 94305, USA. knowlej@stanford.edu.

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Classifications MeSH