Expansion and Differentiation of Human Embryonic Stem Cells to Endoderm Progeny in a Microcarrier Stirred-Suspension Culture
Expansion and Differentiation of Human Embryonic Stem Cells to Endoderm Progeny in a Microcarrier Stirred-Suspension Culture
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DOI：
10.1089/ten.tea.2008.0455
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发表时间：
2009-08-01
影响因子：
4.1
通讯作者：
Tzanakakis, Emmanuel S.
中科院分区：
文献类型：
--
作者：
Lock, Lye T.;Tzanakakis, Emmanuel S.
胚胎干细胞（ESC）具有广泛增殖和多向分化的能力，可作为再生医学中细胞材料的可再生来源。然而，在基于hESC的疗法成为现实之前，开发用于大规模产生人ESC（hESC）或其后代的方法将是必要的。我们假设，微载体搅拌悬浮生物反应器的特点是可扩展性，简单的操作，和严格控制的培养环境，可用于人胚胎干细胞培养和定向分化。在适当的条件下，在微载体生物反应器中培养的hESC的浓度在8天内增加了34- 45倍。通过定量PCR、免疫细胞化学和流式细胞术评估，细胞保留了多能性标志物如OCT 3/4A、NANOG和SSEA 4的表达。我们进一步假设，在微载体上的人胚胎干细胞可以诱导定形内胚层（DE）与生理相关因素孵育时。与胚状体培养物相反，微载体上的所有hESC暴露于本体培养基中的可溶性刺激物，从而促进向DE的有效转变。在达到峰值浓度后，将微载体培养物中的hESC在含有激活素A、Wnt 3a和低浓度血清的培养基中孵育。超过80%的分化的hESC共表达FOXA 2和SOX 17，除了其他DE标记，而非DE基因的表达是不存在或最小的。我们还证明了在微载体培养中的hESC至DE诱导是可扩展的。我们的研究结果支持使用微载体生物反应器从hESC产生内胚层后代，包括治疗上有用的量的胰岛和肝细胞。
Embryonic stem cells (ESCs) with their abilities for extensive proliferation and multi-lineage differentiation can serve as a renewable source of cellular material in regenerative medicine. However, the development of processes for large-scale generation of human ESCs (hESCs) or their progeny will be necessary before hESC-based therapies become a reality. We hypothesized that microcarrier stirred-suspension bioreactors characterized by scalability, straightforward operation, and tight control of the culture environment can be used for hESC culture and directed differentiation. Under appropriate conditions, the concentration of hESCs cultured in a microcarrier bioreactor increased 34- to 45-fold over 8 days. The cells retained the expression of pluripotency markers such as OCT3/4A, NANOG, and SSEA4, as assessed by quantitative PCR, immunocytochemistry, and flow cytometry. We further hypothesized that hESCs on microcarriers can be induced to definitive endoderm (DE) when incubated with physiologically relevant factors. In contrast to embryoid body cultures, all hESCs on microcarriers are exposed to soluble stimuli in the bulk medium facilitating efficient transition to DE. After reaching a peak concentration, hESCs in microcarrier cultures were incubated in medium containing activin A, Wnt3a, and low concentration of serum. More than 80% of differentiated hESCs coexpressed FOXA2 and SOX17 in addition to other DE markers, whereas the expression of non-DE genes was either absent or minimal. We also demonstrate that the hESC-to-DE induction in microcarrier cultures is scalable. Our findings support the use of microcarrier bioreactors for the generation of endoderm progeny from hESCs including pancreatic islets and liver cells in therapeutically useful quantities.