Glycogen synthase kinase 3β inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells

Kavitha Govarthanan; Prasanna Vidyasekar; Piyush Kumar Gupta; Nibedita Lenka; Rama Shanker Verma

doi:10.1016/j.jcyt.2019.12.007

Glycogen synthase kinase 3β inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells

Cytotherapy. 2020 Feb;22(2):91-105. doi: 10.1016/j.jcyt.2019.12.007. Epub 2020 Jan 21.

Authors

Kavitha Govarthanan¹, Prasanna Vidyasekar¹, Piyush Kumar Gupta¹, Nibedita Lenka², Rama Shanker Verma³

Affiliations

¹ Stem Cell and Molecular Biology Lab, Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamilnadu, India.
² National Centre for Cell Science, Pune, Maharashtra, India.
³ Stem Cell and Molecular Biology Lab, Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamilnadu, India. Electronic address: vermars@iitm.ac.in.

PMID: 31980369
DOI: 10.1016/j.jcyt.2019.12.007

Abstract

Aim: Mesenchymal stem cells (MSCs) are immunomodulatory, non-teratogenic and multipotent alternatives to embryonic or induced pluripotent stem cells (ESCs or iPSCs). However, the potency of MSCs is not equivalent to the pluripotency of ESCs or iPSCs. We used CHIR 99021 to improve current protocols and methods of differentiation for the enhanced transdifferentiation potency of MSCs.

Main methods: We used Flurescence activated cell sorter (FACS) for MSC immunophenotyping and biochemical assay for demonstrating the trilineage potential of MSCs. We used real-time polymerase chain reaction, immunocytochemistry and Western blotting assay for analyzing the expression of lineage-specific markers.

Key findings: CHIR 99021 treatment of MSCs resulted in enhanced transdifferentiation into neurological, hepatogenic and cardiomyocyte lineages with standardized protocols of differentiation. CHIR 99021-treated MSCs showed increased nuclear localization of β-catenin. These MSCs showed a significantly increased deposition of active histone marks (H3K4Me3, H3K36Me3), whereas no change was observed in repressive marks (H3K9Me3, H3K27Me3). Differential methylation profiling showed demethylation of the transcription factor OCT4 promoter region with subsequent analysis revealing increased gene expression and protein content. The HLA-DR antigen was absent in CHIR 99021-treated MSCs and their differentiated cell types, indicating their immune-privileged status. Karyotyping analysis showed that CHIR 99021-treated MSCs were genomically stable. Teratoma analysis of nude mice injected with CHIR 99021-treated MSCs showed the increased presence of cell types of mesodermal origin at the site of injection.

Significance: MSCs pretreated with CHIR 99021 can be potent, abundant alternative sources of stem cells with enhanced differentiation capabilities that are well suited to cell-based regenerative therapy.

Keywords: CHIR 99021; differentiation; mesenchymal stem cells; multipotency; regenerative therapy.

MeSH terms

Animals
Cell Differentiation
Cell- and Tissue-Based Therapy / methods
Glycogen Synthase Kinase 3 beta / antagonists & inhibitors*
Humans
Immunophenotyping
Induced Pluripotent Stem Cells / cytology*
Liver / cytology
Mesenchymal Stem Cells / cytology*
Mesoderm / cytology
Mice
Mice, Nude
Myocardium / cytology
Myocytes, Cardiac / metabolism
Neurons / cytology
Octamer Transcription Factor-3 / genetics*
Pyridines / pharmacology*
Pyrimidines / pharmacology*
Regeneration
beta Catenin

Substances

CTNNB1 protein, human
Chir 99021
Octamer Transcription Factor-3
POU5F1 protein, human
Pyridines
Pyrimidines
beta Catenin
Glycogen Synthase Kinase 3 beta