Novel culture strategy for human stem cell proliferation and neuronal differentiation
Novel culture strategy for human stem cell proliferation and neuronal differentiation
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DOI：
10.1002/jnr.21451
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发表时间：
2007-12-01
影响因子：
4.2
通讯作者：
Alves, Paula M.
中科院分区：
文献类型：
--
作者：
Serra, Margarida;Leite, Sofia B.;Alves, Paula M.
胚胎癌（EC）干细胞来源于生殖细胞肿瘤，与胚胎干细胞（ES）非常相似，是研究胚胎发生的重要工具。来源于畸胎瘤的人多能NT2细胞系，经维甲酸处理后可诱导分化为神经元（NT2- n）。为了充分发挥干细胞的潜力，开发干细胞体外增殖和分化的方法是一个关键的挑战。本文提出了一种新的NT2神经元分化培养策略，以扩大NT2- n神经元，减少分化过程所需的时间，并提高NT2- n神经元的最终产量。在维甲酸存在下，使用小型搅拌生物反应器将NT2细胞培养为三维细胞聚集体（“神经球”）；有可能获得均匀的神经球群，这可以转移到进一步的神经元选择涂层表面。与常规静态培养相比，这种培养策略产生的NT2-N神经元数量更高，纯度更高。此外，机械和酶解神经球的方法被评估为恢复神经元的能力，胰蛋白酶消化产生最好的结果。然而，当神经球直接收集到处理表面而没有分离步骤时，获得了最高的回收率。这种新颖的培养策略可以显著提高NT2细胞的神经元分化效率，目前已获得四倍的提高，同时减少了分化过程所需的时间。本文描述的培养方法确保了高效、可复制和可扩展的胚胎干细胞增殖和分化，有助于干细胞生物工程的有用性。(C) 2007 Wiley-Liss, Inc。
Embryonal carcinoma (EC) stem cells derived from germ cell tumors closely resemble embryonic stem (ES) cells and are valuable tools for the study of embryogenesis. Human pluripotent NT2 cell line, derived from a teratocarcinoma, can be induced to differentiate into neurons (NT2-N) after retinoic acid treatment. To realize the full potential of stem cells, developing in vitro methods for stem cell proliferation and differentiation is a key challenge. Herein, a novel culture strategy for NT2 neuronal differentiation was developed to expand NT2-N neurons, reduce the time required for the differentiation process, and increase the final yields of NT2-N neurons. NT2 cells were cultured as 3D cell aggregates ("neurospheres") in the presence of retinoic acid, using small-scale stirred bioreactors; it was possible to obtain a homogeneous neurosphere population, which can be transferred for further neuronal selection onto coated surfaces. This culturing strategy yields higher amounts of NT2-N neurons with increased purity compared with the amounts routinely obtained with static cultures. Moreover, mechanical and enzymatic methods for neurosphere dissociation were evaluated for their ability to recover neurons, trypsin digestion yielding the best results. Nevertheless, the highest recoveries were obtained when neurospheres were collected directly to treated surfaces without dissociation steps. This novel culture strategy allows drastic improvement in the neuronal differentiation efficiency of NT2 cells, insofar as a fourfold increase was obtained, reducing simultaneously the time needed for the differentiation process. The culture method described herein ensures efficient, reproducible, and scaleable ES cell proliferation and differentiation, contributing to the usefulness of stem cell bioengineering. (C) 2007 Wiley-Liss, Inc.