Dataset Information

CRISPR-Cpf1 Activation of Endogenous BMP4 Gene for Osteogenic Differentiation of Umbilical-Cord-Derived Mesenchymal Stem Cells.

ABSTRACT: The CRISPR systems provide powerful genome-editing tools for wide applications in biological and medical research fields. However, the safety issue due to off-target effects of CRISPR has been one of the major hindrances of its application to regenerative medicine. The conventional CRISPR system has the intrinsic danger of inducing unpredictable mutations at non-targeted genomic loci via erroneous double-strand DNA breaks (DSBs). In this study, we demonstrate a safety-enhanced application of a recently discovered CRISPR-Cpf1 for targeted gene activation, without DNA double-strand break, to facilitate osteogenic differentiation of human umbilical-cord-derived mesenchymal stem cells (UC-MSCs). To this end, we developed a catalytically inactive AsCpf1 fused to tripartite transcription activator domain (dAsCpf1-VPR) that can induce upregulation of targeted gene expression in mammalian cells. We observed that the CRISPR-dAsCpf1-VPR activator can be applied to enhance the osteogenic differentiation of human UC-MSCs, via increasing the expression level of endogenous BMP4 gene. The results suggested that the CRISPR-Cpf1 activator provides versatile methods applicable for bone regeneration and regenerative medicine.

SUBMITTER: Choi J

PROVIDER: S-EPMC6994413 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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CRISPR-Cpf1 Activation of Endogenous <i>BMP4</i> Gene for Osteogenic Differentiation of Umbilical-Cord-Derived Mesenchymal Stem Cells.

Choi Jaehoon J Bae Taegeun T Byambasuren Ninj N Park Seong-Ho SH Jo Chris H CH Kim Dokyoung D Hur Junho K JK Hwang Nathaniel S NS

Molecular therapy. Methods & clinical development 20200109

The CRISPR systems provide powerful genome-editing tools for wide applications in biological and medical research fields. However, the safety issue due to off-target effects of CRISPR has been one of the major hindrances of its application to regenerative medicine. The conventional CRISPR system has the intrinsic danger of inducing unpredictable mutations at non-targeted genomic loci via erroneous double-strand DNA breaks (DSBs). In this study, we demonstrate a safety-enhanced application of a r ...[more]

PMID: 32021879

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Project description:Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a novel and promising strategy for tissue engineering because of their ability to differentiate into many cell types. We characterized the differentiation of WJ-MSCs into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells and assessed the effect of 17?-estradiol and 8-Br-cAMP on the differentiation system.WJ-MSCs were treated in two ways to differentiate into EEC-like and ESC-like cells respectively: cocultured with ESCs in control/differentiation medium (17?-estradiol, growth factors); and cultured in control/differentiation medium (8-Br-cAMP alone or 8-Br-cAMP plus 17?-estrogen and growth factors). Three signaling pathway inhibitors (SB203580, PD98059, H89) were used to investigate the mechanism of WJ-MSC differentiation into ESC-like cells. Immunofluorescence, western blot and flow cytometry analyses were used to analyze expression of epithelial markers and stromal cell markers. Enzyme-linked immunosorbent assays were used to test the production of secretory proteins associated with the differentiation of ESC-like cells.17?-estradiol at 1 ?M downregulated vimentin and CD13 and upregulated cytokeratin and CD9 proteins, promoting the differentiation of WJ-MSCs into EEC-like cells in the coculture system. 8-Br-cAMP at 0.5 mM upregulated vimentin and CD13 and downregulated CK and CD9, promoting the differentiation of WJ-MSCs into ESC-like cells. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were upregulated and the protein kinase A (PKA) signaling pathway was activated, whereas extracellular signal-regulated (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were not affected.17?-estradiol at 1 ?M is a good inducer for facilitating the differentiation of WJ-MSCs into EEC-like cells. 8-Br-cAMP plus estrogen and growth factors can induce the differentiation of WJ-MSCs into ESC-like cells. During the differentiation of WJ-MSCs into ESC-like cells, PRL and IGFBP1 were upregulated by the treatment and the PKA signaling pathway was activated, whereas ERK1/2 and p38 MAPK were not affected. These findings suggest a promising approach to the treatment of endometrial damage and other endometrial diseases and suggest new applications for WJ-MSCs in clinical practice.

Dataset Information

CRISPR-Cpf1 Activation of Endogenous BMP4 Gene for Osteogenic Differentiation of Umbilical-Cord-Derived Mesenchymal Stem Cells.

Publications

CRISPR-Cpf1 Activation of Endogenous <i>BMP4</i> Gene for Osteogenic Differentiation of Umbilical-Cord-Derived Mesenchymal Stem Cells.

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