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A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Published on: May 12, 2017
Glycogen synthase kinase 3 (GSK3) inhibitor, SB-216763, promotes pluripotency in mouse embryonic stem cells
Leslie A Kirby1, Jason T Schott, Brenda L Noble
1Department of Biological Science, California State University-Fullerton, Fullerton, California, United States of America.
Abstract:
Canonical Wnt/β-catenin signaling has been suggested to promote self-renewal of pluripotent mouse and human embryonic stem cells. Here, we show that SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, can maintain mouse embryonic stem cells (mESCs) in a pluripotent state in the absence of exogenous leukemia inhibitory factor (LIF) when cultured on mouse embryonic fibroblasts (MEFs). MESCs maintained with SB-216763 for one month were morphologically indistinguishable from LIF-treated mESCs and expressed pluripotent-specific genes Oct4, Sox2, and Nanog. Furthermore, Nanog immunostaining was more homogenous in SB-216763-treated colonies compared to LIF. Embryoid bodies (EBs) prepared from these mESCs expressed early-stage markers for all three germ layers, and could efficiently differentiate into cardiac-like cells and MAP2-immunoreactive neurons. To our knowledge, SB-216763 is the first GSK3 inhibitor that can promote self-renewal of mESC co-cultured with MEFs for more than two months.
Insights
SB-216763, a GSK3 inhibitor, effectively maintains mouse embryonic stem cells (mESCs) pluripotency without LIF. This compound supports self-renewal and differentiation potential, offering a novel approach for stem cell research.
Area of Science:
- Stem Cell Biology
- Developmental Biology
- Molecular Signaling
Background:
- Canonical Wnt/β-catenin signaling is implicated in maintaining pluripotency of embryonic stem cells.
- Leukemia inhibitory factor (LIF) is commonly used to sustain mouse embryonic stem cell (mESC) self-renewal.
- Alternative methods for maintaining mESC pluripotency are sought.
Purpose of the Study:
- To investigate the potential of SB-216763, a glycogen synthase kinase-3 (GSK3) inhibitor, in maintaining mESC pluripotency.
- To assess the self-renewal and differentiation capacity of mESCs cultured with SB-216763 in the absence of LIF.
Main Methods:
- Mouse embryonic stem cells (mESCs) were cultured with SB-216763 on mouse embryonic fibroblasts (MEFs) without LIF.
- Morphological and gene expression analyses (Oct4, Sox2, Nanog) were performed.
- Nanog immunostaining assessed colony homogeneity.
- Embryoid bodies (EBs) were generated for differentiation studies.
Main Results:
- SB-216763 successfully maintained mESC pluripotency for over a month, comparable to LIF treatment.
- mESCs exhibited pluripotent marker expression (Oct4, Sox2, Nanog) and homogenous Nanog staining.
- EBs differentiated into derivatives of all three germ layers, including cardiac-like cells and neurons.
Conclusions:
- SB-216763 is the first GSK3 inhibitor demonstrated to promote mESC self-renewal on MEFs for extended periods (over two months) without LIF.
- This finding provides a novel chemical approach for maintaining mESC pluripotency and their developmental potential.
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