zur Nieden Nicole I, Cormier Jaymi T, Rancourt Derrick E, Kallos Michael S
Institute of Maternal & Child Health, University of Calgary, Calgary, Alberta, Canada.
J Biotechnol. 2007 May 1;129(3):421-32. doi: 10.1016/j.jbiotec.2007.01.006. Epub 2007 Jan 21.
Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.
多能胚胎干细胞（ESCs）及其作为各种组织工程应用中主要材料的潜在用途已受到越来越多的关注。该技术的成功临床应用需要一个质量可控的可重复培养系统，用于扩增用于生成功能性组织的细胞。最近，我们表明悬浮生物反应器可用于高度多能的小鼠ESCs的大规模调控扩增。当前研究表明，这些生物反应器方案可适用于长期培养，并且当在悬浮生物反应器中连续传代较长时间时，小鼠ESC培养物仍保持高度未分化状态。在实验开始和结束时进行了流式细胞术分析以及几种多能性标志物的基因表达谱分析，此外还进行了集落和胚状体（EB）形成试验，所有结果均表明ESC培养物在悬浮培养的延长时间内仍保持高度未分化状态。在长期培养结束时进行的体内畸胎瘤形成以及体外分化为神经、心肌细胞、成骨细胞和软骨细胞谱系的实验，进一步支持了扩增群体中存在功能性和未分化的ESCs。总体而言，该系统能够对高度多能的小鼠ESC群体进行可控扩增。
