Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum.
Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum.
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DOI：
10.1186/1471-213x-10-60
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发表时间：
2010-06-02
影响因子：
--
通讯作者：
Duncan SA
中科院分区：
文献类型：
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作者：
Nagaoka M;Si-Tayeb K;Akaike T;Duncan SA
为了在无饲养层培养中维持人胚胎干（huES）细胞的多能性，有必要提供基质胶基质，其是来源于小鼠Engelbreth-Holm-Swarm肉瘤细胞的不明确的细胞外基质和生长因子的复合物。在不明确的条件下培养干细胞会抑制将细胞维持在多能状态的有效性，并降低分化方案的再现性。此外，最近几批基质胶被发现被单链RNA病毒乳酸脱氢酶升高病毒（LDEV）污染，引起了对在治疗环境中使用在基质胶上培养的干细胞的安全性的担忧。为了避免这些问题，我们试图确定一种重组基质，可以作为替代基质胶的人类多能干细胞的培养。使用mTeSR 1培养基，使huES和人诱导多能干细胞（hiPS）在包被有由E-钙粘蛋白和IgG Fc结构域组成的融合蛋白的平板上生长。在这些条件下生长的细胞保持与在Matrigel上生长的细胞相似的形态和生长速率，并保留所有多能干细胞特征，包括在畸胎瘤测定中分化成多个细胞谱系的能力。因此，我们提出了一种在完全确定的条件下维持huES和hiPS细胞多能性的培养系统。我们建议，该系统应促进干细胞的生长，使用良好的生产规范（GMP），这将是必要的多能干细胞及其衍生物的临床使用。
To maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum, which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Culture of stem cells under ill-defined conditions can inhibit the effectiveness of maintaining cells in a pluripotent state and reduce reproducibility of differentiation protocols. Moreover recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus, Lactate Dehydrogenase Elevating Virus (LDEV), raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns, we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. huES and human induced pluripotent stem (hiPS) cells were grown on plates coated with a fusion protein consisting of E-cadherin and the IgG Fc domain using mTeSR1 medium. Cells grown under these conditions maintained similar morphology and growth rate to those grown on Matrigel and retained all pluripotent stem cell features, including an ability to differentiate into multiple cell lineages in teratoma assays. We, therefore, present a culture system that maintains the pluripotency of huES and hiPS cells under completely defined conditions. We propose that this system should facilitate growth of stem cells using good manufacturing practices (GMP), which will be necessary for the clinical use of pluripotent stem cells and their derivatives.