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.

中文翻译：

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糖原合酶激酶 3β 抑制剂 - CHIR 99021 增强间充质干细胞的分化潜能

AIM 间充质干细胞 (MSC) 是胚胎或诱导多能干细胞（ESC 或 iPSC）的免疫调节、非致畸和多能替代品。然而，MSCs 的效力并不等同于 ESCs 或 iPSCs 的多能性。我们使用 CHIR 99021 来改进当前的方案和分化方法，以增强 MSC 的转分化能力。主要方法 我们使用荧光激活细胞分选仪 (FACS) 进行 MSC 免疫表型分析和生化分析，以证明 MSCs 的三系潜力。我们使用实时聚合酶链反应、免疫细胞化学和蛋白质印迹分析来分析谱系特异性标记的表达。主要发现 CHIR 99021 处理 MSCs 导致增强的转分化为神经、具有标准化分化方案的肝源性和心肌细胞谱系。CHIR 99021 处理的 MSC 显示出 β-连环蛋白的核定位增加。这些 MSC 显示活性组蛋白标记（H3K4Me3、H3K36Me3）的沉积显着增加，而在抑制标记（H3K9Me3、H3K27Me3）中没有观察到变化。差异甲基化分析显示转录因子 OCT4 启动子区域的去甲基化，随后的分析显示基因表达和蛋白质含量增加。CHIR 99021 处理的 MSC 及其分化的细胞类型中不存在 HLA-DR 抗原，表明它们的免疫特权状态。核型分析表明，经 CHIR 99021 处理的 MSC 在基因组上是稳定的。对注射了 CHIR 99021 处理的 MSC 的裸鼠进行畸胎瘤分析，结果表明注射部位中胚层来源的细胞类型增加。意义 用 CHIR 99021 预处理的 MSC 可以成为有效、丰富的干细胞替代来源，具有增强的分化能力，非常适合基于细胞的再生治疗。