Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cincinnati, OH 45867, USA.
Cytotherapy. 2011 Oct;13(9):1057-65. doi: 10.3109/14653249.2011.597380. Epub 2011 Aug 17.
GATA-4 is a cardiac transcription factor and plays an important role in cell lineage differentiation during development. We investigated whether overexpression of GATA-4 increases adult mesenchymal stromal cell (MSC) transdifferentiation into a cardiac phenotype in vitro.
MSC were harvested from rat bone marrow (BM) and transduced with GATA-4 (MSC(GATA-4)) using a murine stem cell virus (pMSCV) retroviral expression system. Gene expression in MSC(GATA-4) was analyzed using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Native cardiomyocytes (CM) were isolated from ventricles of neonatal rats. Myocardial transdifferentiation of MSC was determined by immunostaining and electrophysiologic recording. The transdifferentiation rate was calculated directly from flow cytometery.
The expression of cardiac genes, including brain natriuretic peptide (BNP), Islet-1 and α-sarcomeric actinin (α-SA), was up-regulated in MSC(GATA-4) compared with control cells that were transfected with Green Fluorescent Protein (GFP) only (MSC(Null)). At the same time, insulin-like growth factor-binding protein (IGFBP)-4 was significantly up-regulated in MSC(GATA-4). A synchronous beating of MSC with native CM was detected and an action potential was recorded. Some GFP (+) cells were positive for α-SA staining after MSC were co-cultured with native CM for 7 days. The transdifferentiation rate was significantly higher in MSC(GATA-4). Functional studies indicated that the differentiation potential of MSC(GATA-4) was decreased by knockdown of IGFBP-4.
Overexpression of GATA-4 significantly increases MSC differentiation into a myocardial phenotype, which might be associated with the up-regulation of IGFBP-4.
GATA-4 是一种心脏转录因子，在发育过程中的细胞谱系分化中发挥重要作用。我们研究了过表达 GATA-4 是否会增加成年间充质基质细胞（MSC）在体外向心肌表型的转分化。
从大鼠骨髓（BM）中收获 MSC，并使用鼠干细胞病毒（pMSCV）逆转录病毒表达系统将 GATA-4 转导至 MSC（MSC(GATA-4)）。使用定量逆转录聚合酶链反应（RT-PCR）和 Western 印迹分析 MSC(GATA-4)中的基因表达。从新生大鼠心室中分离天然心肌细胞（CM）。通过免疫染色和电生理记录来确定 MSC 的心肌转分化。直接从流式细胞仪计算转分化率。
与仅转染绿色荧光蛋白（GFP）的对照细胞（MSC(Null)）相比，MSC(GATA-4)中心脏基因的表达，包括脑钠肽（BNP）、胰岛 1 基因（Islet-1）和α-横纹肌肌动蛋白（α-SA），上调。同时，胰岛素样生长因子结合蛋白（IGFBP）-4 在 MSC(GATA-4)中显著上调。检测到 MSC 与天然 CM 同步跳动并记录动作电位。在 MSC 与天然 CM 共培养 7 天后，一些 GFP（+）细胞对 α-SA 染色呈阳性。MSC(GATA-4)的转分化率明显更高。功能研究表明，IGFBP-4 的敲低降低了 MSC(GATA-4)的分化潜能。
过表达 GATA-4 可显著增加 MSC 向心肌表型的分化，这可能与 IGFBP-4 的上调有关。
