Expansion and differentiation of human iPS cells in a three-dimensional culture using hollow fibers and separation of the specific population by magnetic-activated cell sorting
Expansion and differentiation of human iPS cells in a three-dimensional culture using hollow fibers and separation of the specific population by magnetic-activated cell sorting
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DOI：
10.1016/j.jbiosc.2019.03.014
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发表时间：
2019-10-01
影响因子：
2.8
通讯作者：
Mizumoto, Hiroshi
中科院分区：
文献类型：
--
作者：
Matsushita, Sakiko;Kajiwara, Toshihisa;Mizumoto, Hiroshi
为了将多能干细胞用于再生医学领域，需要建立用于细胞分化的大规模培养系统。我们已经开发了一种新的三维方法培养人诱导多能干细胞（iPS），使用中空纤维（HF）。固定在HF内的细胞可以增殖并形成多细胞聚集体，能够实现高细胞密度并促进进一步的自发细胞分化。我们首先在维持其未分化状态的条件下培养人iPS细胞7天，然后转换培养条件以允许自发细胞分化。在7天未分化培养中，实现了约为初始接种密度10倍的高细胞密度。在分化细胞的培养物中观察到用于分化的基因标记物如CXCR 4或SOX 17的上调。在分化培养的第5天，谱系特异性细胞表面标志物CXCR4的表达约为30%，这是传统单层培养中的2倍。HF培养后，我们获得了CXCR4阳性细胞群，并进行单层培养以进一步分化肝谱系。在CXCR4阳性细胞群中，一些肝脏特异性基因标记物的表达水平趋于增加。然而，分离组和非分离组之间没有显著差异，这表明在未来的研究中需要改进细胞分离过程和细胞成熟过程。总之，HF培养方法具有实现人iPS细胞的大规模培养和自发分化的潜力。(C)2019年，日本生物技术学会。All rights reserved.
In order to employ pluripotent stem cells in the field of regenerative medicine, it is necessary to establish a large-scale culture system for cell differentiation. We have developed a novel three-dimensional method for culturing human induced pluripotent stem (iPS) cells, using hollow fibers (HFs). The cells immobilized inside HFs can proliferate and form multicellular aggregates, capable of achieving a high cell density and promoting further spontaneous cell differentiation. We first cultured human iPS cells for 7 days under conditions that maintained their undifferentiated state and then switched the culture conditions to allow spontaneous cell differentiation. In the 7-day undifferentiated culture, a high cell density of approximately 10-fold that of the initial seeding density was achieved. The upregulation of gene markers for differentiation such as CXCR4 or SOX17 was observed in the culture of differentiated cells. Expression of the lineage-specific cell-surface marker CXCR4 was about 30% at day 5 in the differentiation culture, which was 2-fold higher than that in the traditional monolayer culture. After HF culture, we obtained the CXCR4-positive cell population and performed monolayer culture for further differentiation of the hepatic lineage. In the CXCR4-positive cell population, the expression levels of a few liver-specific gene markers tended to increase. However, there were no significant differences between the separation and non-separation groups, which indicates the need for refinement of the cell separation process and cell maturation procedure in future studies. In conclusion, the HF culture method has potential for achieving the large-scale culturing and spontaneous differentiation of human iPS cells. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.