2012年12月07日 讯 /生物谷BIOON/ --诱导性多能干细胞(induced pluripotent stem cells, iPSCs)有潜力分化为体内任何一种细胞，从而提供一种用于药物开发和再生医学的强大工具。但是在实验室中大规模地诱导这些细胞可靠地选择一种特定的命运一直都是一个挑战。如今，来自新加坡A*STAR的一个干细胞研究人员小组开发出一种细胞分化方法：在三维生物反应器中增殖和扩大iPSCs从而高效地产生神经祖细胞(neural progenitor cell)。
论文通信作者Steve Oh和他的同事们利用他们之前为在三维悬浮系统中的小的固体颗粒表面上培养人胚胎干细胞开发出的一种所谓的微载体平台开始研究。他们为人iPSCs优化了这种技术，从而证实在每天更换两次培养基的被称作搅拌式反应器的搅拌容器中，包被蛋白的圆柱状微载体能够导致重编程的干细胞增殖20倍。这种产量比任何其他报道的成批培养这些细胞的系统高得多。
在正常条件下，科学家们不得不在平直的培养皿中费时费力地操纵iPSCs以便形成更加特化的细胞。但是，在这种新的三维平台下，只需简单地更换培养基，研究人员就可以高达85%的效率诱导iPSCs变成神经祖细胞。利用这种将细胞增殖和细胞分化整合在一起的过程，每个接种的iPSC能够产生333个神经祖细胞。作为比较，大多数科学家利用经典的二维组织培养方法只能让每个初始的干细胞产生53个神经祖细胞。
Oh说，“这种二维方法费时费力，只能获得10%的产量，而且在不同实验室之间也是不同的。基于微载体的培养方法为细胞生长提供更加大的表面面积，因而更加多的细胞能够被加入到这种系统系统来增加集料尺寸和产量。”
Oh和他的同事们也诱导这些神经祖细胞进一步分化为很多不同类型的脑细胞，包括三种主要的神经细胞系：神经元、少突胶质细胞和星形胶质细胞。Oh注意到，在未来，这样的神经元可能能够被用来治疗帕金森病之类的疾病，而移植少突胶质细胞可能能够治疗脊髓损伤。(生物谷Bioon.com)
doi: 10.1089/ten.tec.2012.0146
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Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells
Jo'an Bardy, Allen K. Chen, Yu Ming Lim, Selena Wu, Shunhui Wei, Han Weiping, Ken Chan, Shaul Reuveny, and Steve K.W. Oh
Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×106 cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1–60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×106 cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%–85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin+ neurons, GFAP+ astrocytes, and O4+ oligodendrocytes, showing that cells maintained their multilineage differentiation potential.
doi: 10.1016/j.scr.2011.04.007
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Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells
Allen Kuan-Liang Chena, , , Xiaoli Chena, , Andre Boon Hwa Chooa, , Shaul Reuvenyb, , Steve Kah Weng Oh
A variety of microcarriers may be used for the expansion of human embryonic stem cells (hESC) for cell therapy applications. This study investigated the effects of 10 types of microcarriers on hESC attachment efficiency, growth and pluripotency. High attachment efficiency was observed on uncoated microcarriers, however poor cell growth and/or gradual loss of pluripotency occurred during continuous passaging. Coating of the microcarriers with Matrigel resulted in higher cell yields and stable pluripotent states for at least three passages. Positively charged cylindrical cellulose microcarriers (DE52, DE53 and QA52) and large (190 μm) positively charged spherical microcarriers (Cytodex 1) exhibited high cell expansion potential and levels of pluripotency. Lower cell yields were obtained using smaller diameter spherical (65 μm and 10 μm) or macroporous beads. Instead of Matrigel, laminin coated microcarriers (DE53 and Cytodex 1) are capable of supporting the long term propagation and pluripotency of HES-2 and HES-3 cell lines. HES-2 cell line which was shown earlier to be shear resistant achieved similar cell growth and expression of pluripotent markers when cultured on both Matrigel (84% Tra-1-60, 1.43 × 106 cells/ml) and laminin (74% Tra-1-60, 1.37 × 106 cells/ml) coated microcarriers in spinner flasks. In contrast, HES-3 exhibited a decrease in cell yield, viability and pluripotent markers on laminin as compared with Matrigel coated microcarriers possibly due to shear sensitivity. Conventional microcarriers intended for propagation of mammalian cells are not suitable for long term propagation of hESC. Matrigel or laminin coating is essential for stable long term propagation of hESC on a variety of microcarriers
