Abstract
Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry–based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1+PAX6+ neuroectoderm and FOXG1+ cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage.
Highlights
Human embryonic stem cells are a type of pluripotent stem cells that are used to investigate their differentiation into diverse mature cell types for molecular studies
Human insulin receptor (IR) signaling regulates stem cell differentiation extracellular matrix (ECM) genes. When these shIR-human pluripotent stem cell (hPSC) were differentiated toward the neural lineage, we observed aberrant neuroectoderm and cerebral organoid development. These results demonstrate the importance of IRmediated signaling in the maintenance of pluripotency, the ECM which contributes to the hPSC niche [11], and longlasting effects on tissue and neural differentiation
The rationale for adding >100× physiological concentration of recombinant insulin/ insulin-like growth factor (IGF) to hPSC media is unclear, one implication is that insulin/ IGF signaling is important for the maintenance of hPSCs
Summary
Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. Our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage. The use of KnockOut Serum Replacement (KOSR) and defined media for culturing hPSCs such as StemPro, mTeSR1, and Essential 8 (E8) has shed light on the extracellular components necessary for maintaining human pluripotency and metabolic homeostasis These hPSC media that contain supraphysiological levels of insulin and insulin-like growth factors (IGFs) suggest a requirement for insulin and/or IGF-I receptors in regulating hPSCs [4]. Human IR signaling regulates stem cell differentiation extracellular matrix (ECM) genes When these shIR-hPSCs were differentiated toward the neural lineage, we observed aberrant neuroectoderm and cerebral organoid development. These results demonstrate the importance of IRmediated signaling in the maintenance of pluripotency, the ECM which contributes to the hPSC niche [11], and longlasting effects on tissue and neural differentiation
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