Investigation of the cardiogenic differentiation of human pluripotent stem cells in static cultures and stirred-suspension bioreactors
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Heart disease is a major cause of morbidity and mortality worldwide. For the last few decades, the heart transplantation is the only feasible method to save people's lives. However, it is severely hindered by the limited availability of donor organs. To this end, transplantation of embryonic stem cell (ESC)-derived cardiomyocytes may provide an attractive alternative to current treatments of heart failure. But this application is limited by an effective large-scale cell production and high yield of differentiated cells. So the goal of this research was to produce a scalable bioprocess for production and selection of cardiomyocytes from human embryonic stem cells (hESCs). Experiments on hESCs differentiation toward cardiac lineage on tissue culture dishes, in alginate-PLL (PLL) microcapsules and on microcarriers in stirred suspension bioreactor were studied. After encapsulation, cells proliferated faster in alginate-PLL microcapsules than in alginate micropaticles, which is due to the more free liquid culture microenvironment in microcapsules than in porous solid alginate matrix in microparticles. However, after two weeks expansion and differentiation, the hESCs-derived cells meet the requirement of cardiomyocytes, including expression of genes, proteins and exhibition of functional assays. About 40-50% of mixed population was positive for NKX2.5, GATA4 probed by flow cytometry. Next I developed a method for directing the commitment of hESCs from monolayers to cardiac muscle cells with developmentally relevant factors avoiding fetal bovine serum (FBS). Human ESCs were guided through the mesendoderm, mesoderm, early cardiac and cardiac stages in ∼15-20 days. The cells expressed stage-specific markers during their transition. The hESC-derived cardiomyocyte-like cells were also characterized by quantitative PCR, immunocytochemistry and flow cytometry for expression of heart muscle-specific genes and proteins. Under appropriate conditions, the cells formed clusters exhibiting contractile activity. The differentiation strategy was also successfully applied in stirred suspension bioreactor with microcarriers. The purification of fully differentiated cells from heterogeneous population was achieved by Fluorescence Activated Cell Sorting (FACS) from a genetically modified hESC cell line carrying a GFP protein driven by cardiac specific Atrial Natriuretic Factor (ANF) promoter. Generated human embryonic stem cell line remains pluoripotency to differentiation to three germ layers and allows sufficient selection of cardiac muscle cells. Combination of large quantities of cells from bioreactor and selection of pure cardiac population facilitates the transplantation of cardiomyocytes derived from embryonic stem cells for heart disease in the future. It is first report the expression of Reg gene family in both human and mouse ESCs. More importantly, this expression was modulated by the activation of Wnt/β-Catenin signaling pathway with Wnt activators. The function of Reg1 protein in mouse model is believed to be involved in the differentiation toward definitive endoderm.