Michael Smith Laboratories and Chemical & Biological Engineering, University of British Columbia, 2185 East Mall, Vancouver, British Columbia, Canada V6T 1Z4.
Biotechnol Bioeng. 2013 Oct;110(10):2706-16. doi: 10.1002/bit.24936. Epub 2013 Apr 29.
Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However, optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs, including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20, they remain highly useful. In particular, CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus, 10(8) CA1S cells can be generated within one week from 10(6) cells to seed 10(6) wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm, a finding that was confirmed with H1 hESCs, although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines, these cells should ease the development of optimized hESC growth and differentiation protocols. In particular, they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses.
人胚胎干细胞（hESCs）可分化为多种具有巨大治疗潜力的细胞类型。然而，为了获得足够的分化细胞产量，通常需要数周的时间来优化培养，这在一定程度上受到即使是平行的 hESC 分化培养也存在高度可变性的限制。我们描述了 CA1 hESC（CA1S）亚系的分离和特征，该亚系显示出非常高的 25%克隆效率，同时保留了亲本 hESC 的许多特性，包括染色体核型正常和生成包含三个胚层的畸胎瘤的能力。尽管更详细的分析表明 CA1S 细胞在 20 号染色体上有一个 3.8Mb 的基因组重复，但它们仍然非常有用。特别是，CA1S 细胞在培养中可以以高产量轻易扩增，即使以 100 个/孔的密度播种，其孔间差异也大大减少。因此，从 10^6 个细胞中可以在一周内生成 10^8 个 CA1S 细胞，以播种 10^6 个孔。我们确定 CA1S 细胞有能力遵循已建立的体外分化方案，生成胰腺祖细胞和随后的激素阳性细胞类型，并使用 CA1S 细胞在高通量筛选中探索明确的内胚层诱导（Z 因子=0.97）。该系统表明，CA1S 细胞不需要 WNT3A 来有效地形成明确的内胚层，这一发现得到了 H1 hESC 的证实，尽管 H1 细胞确实显示出高剂量 WNT3A 的适度益处。CA1S 细胞的增殖指数测量结果表明，在高通量系统中可以快速反映其分化状态。尽管使用 CA1S 细胞获得的结果需要使用常规 hESC 系进行验证，但这些细胞应该简化优化的 hESC 生长和分化方案的开发。特别是，它们应该将使用 hESC 的更艰巨的二次筛选限制在较小数量的变量和剂量。
