Title: Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation

Abstract

Objectives: Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells, however, its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. The main objective of this study is to understand the role of IR signaling in pluripotency and lineage development. Methods: To explore the role of IR signaling, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors (Oct4, Sox2, Klf4, cMyc). We performed pluripotency characterization and directed differentiation of control and IR-KO iPSCs into neural progenitors (ectoderm), adipocyte progenitors (mesoderm) and pancreatic beta-like cells (endoderm). We mechanistically confirmed these findings via phosphoproteomics analyses of control and IR-KO iPSCs. Results: Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3 and cMyc were upregulated while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold respectively in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, p-mTor and p-Erk, and an increase in the total mTor and Erk proteins in IRKO iPSCs at basal level. Stimulation with leukemia inhibitory factor (LIF) showed a ~33% decrease of phospho-ERK in IRKO iPSCs. Onmore » the contrary, Erk phosphorylation was increased during in vitro spontaneous differentiation of iPSCs lacking IRs. Lineage-specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Ascl1 and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Ppar?, Fabp4, C/ebpa and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1 and Sox9). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network and several key proteins involved in diverse aspects of growth and embryonic development. Conclusion: We report, for the first time to our knowledge, the phosphoproteome of insulin, IGF1 and LIF stimulation in mouse iPSCs and reveal the importance of insulin receptor signaling for the maintenance of pluripotency and lineage determination.« less

Authors:

Gupta, Manoj K.; De Jesus, Dario F. ;

• Search DOE PAGES for author "De Jesus, Dario F."
• Search DOE PAGES for ORCID "0000-0001-8826-9095"

Kahraman, Sevim; Valdez, Ivan A.; Shamsi, Farnaz; Yi, Lian; Swensen, Adam C. ;

• Search DOE PAGES for author "Swensen, Adam C."
• Search DOE PAGES for ORCID "0000-0001-8135-1766"

Tseng, Yu-Hua; Qian, Wei-Jun ;

• Search DOE PAGES for author "Qian, Wei-Jun"
• Search DOE PAGES for ORCID "0000-0002-5393-2827"

Kulkarni, Rohit N.

Publication Date:

Sat Dec 01 00:00:00 EST 2018

Research Org.:

Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Org.:

USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Fuel Cell Technologies Office; USDOE

OSTI Identifier:

1772093

Alternate Identifier(s):

OSTI ID: 1480130; OSTI ID: 1507758

Report Number(s):

PNNL-SA-136923
Journal ID: ISSN 2212-8778; S2212877818306999; PII: S2212877818306999

Grant/Contract Number:  

AC05-76RL0 1830; SFRH/BD/51699/2011; AC05-76RL01830; 3-APF-2017-393-A-N; R01 DK67536; R01 DK103215; UC4 DK104167; DP3 DK110844; R01DK077097; R01DK102898; #1-18-PDF-169

Resource Type:

Published Article

Journal Name:

Molecular Metabolism

Additional Journal Information:

Journal Name: Molecular Metabolism Journal Volume: 18 Journal Issue: C; Journal ID: ISSN 2212-8778

Publisher:

Elsevier

Country of Publication:

Germany

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Insulin receptor signaling; Pluripotency; Lineage differentiation; Adipocyte; Beta cells; Neurons; Stem cells; Phosphoproteomics; Reprogramming