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Investigation of the cardiogenic differentiation of human pluripotent stem cells in static cultures and stirred-suspension bioreactors.

机译：在静态培养和搅拌悬浮生物反应器中研究人类多能干细胞的心源性分化。

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摘要

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/beta-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.
机译：心脏病是全世界发病率和死亡率的主要原因。在过去的几十年中，心脏移植是挽救生命的唯一可行方法。但是，由于供体器官的供应有限，严重地阻碍了这一进程。为此，胚胎干细胞（ESC）衍生的心肌细胞的移植可能为当前心力衰竭治疗提供有吸引力的替代方法。但是，该应用受到有效的大规模细胞生产和分化细胞高产量的限制。因此，本研究的目的是为从人类胚胎干细胞（hESCs）生产和选择心肌细胞提供可扩展的生物过程。在组织培养皿，藻酸盐-PLL（PLL）微胶囊和搅拌悬浮生物反应器中的微载体上研究了hESCs向心脏谱系分化的实验；;封装后，藻酸盐-PLL微胶囊中的细胞增殖速度快于藻酸盐微片中的细胞增殖，这是由于与微颗粒中的多孔固体藻酸盐基质相比，微囊中的液体培养微环境更自由。然而，经过两周的扩增和分化后，hESCs衍生的细胞可以满足心肌细胞的需求，包括基因，蛋白质的表达和功能测定的展示。约40-50％的混合人群通过流式细胞仪检测到NKX2.5，GATA4阳性。接下来，我开发了一种方法，用于指导hESC从单层到心肌细胞的定向，并具有与发育相关的因素，可避免胎牛血清（FBS）。 。在约15至20天的时间内，将人类ESC引导通过中胚层，中胚层，心脏早期和心脏阶段。细胞在过渡过程中表达阶段特异性标记。还通过定量PCR，免疫细胞化学和流式细胞术表征了hESC衍生的心肌样细胞，以表达心肌特异性基因和蛋白质。在适当的条件下，细胞形成展示收缩活性的簇。该分化策略也成功地应用于带有微载体的搅拌悬浮生物反应器中；通过从带有心脏特异性心房驱动的GFP蛋白的转基因hESC细胞系中进行荧光激活细胞分选（FACS），从异质群体中纯化出完全分化的细胞。利钠因子（ANF）启动子。生成的人类胚胎干细胞系仍然具有分化为三个胚层的多能性，并可以选择足够的心肌细胞。生物反应器中大量细胞的结合以及纯心脏群体的选择，为将来用于心脏病的胚胎干细胞衍生的心肌细胞的移植提供了便利。；这是首次报道Reg基因家族在人和小鼠ESC中的表达。更重要的是，此表达是通过Wnt激活剂激活Wnt /β-Catenin信号传导途径来调节的。据信Reg1蛋白在小鼠模型中的功能与向定形内胚层的分化有关。

著录项

  • 作者

    Jing, Donghui.;

  • 作者单位

    State University of New York at Buffalo.;

  • 授予单位 State University of New York at Buffalo.;
  • 学科 Engineering Biomedical.;Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 187 p.
  • 总页数 187
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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