Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture.
Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture.
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控制可扩展悬浮培养物中人多能干细胞的扩张和心肌生成分化。
DOI：
10.1016/j.stemcr.2014.09.017
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发表时间：
2014-12-09
影响因子：
5.9
通讯作者：
Zweigerdt R
中科院分区：
文献类型：
--
作者：
Kempf H;Olmer R;Kropp C;Rückert M;Jara-Avaca M;Robles-Diaz D;Franke A;Elliott DA;Wojciechowski D;Fischer M;Roa Lara A;Kensah G;Gruh I;Haverich A;Martin U;Zweigerdt R
为了利用人类多能干细胞（hPSC）的潜力，需要大量供应其后代。在此，使用化学Wnt途径调节剂将悬浮培养物中作为基质非依赖性聚集体的hPSC扩增与心肌样分化相结合。将多孔筛按比例放大到搅拌的锥形瓶，随后放大到罐式生物反应器，应用受控的进料策略（分批和循环灌注）。心肌发生对GSK 3抑制剂CHIR 99021浓度敏感，而聚集体大小在整个培养平台中不是主导因素。然而，在生物反应器中，在扩张阶段的聚集体形成的模式主导了随后的分化。全球分析显示，BMP激动剂/拮抗剂的表达依赖于培养，这表明它们在细胞命运决定中发挥着决定性作用。此外，金属硫蛋白被发现是hPSC中潜在的应激相关标志物。在100 ml生物反应器中，能够生产4000万个主要是心室样心肌细胞（纯度高达85%），这些细胞可直接用于生物人工心脏组织形成。通过化学Wnt调节剂的悬浮液中的有效心脏分化方案分化是CHIR浓度依赖性的，但聚集体大小不依赖于生物反应器控制的hPSC扩增决定了随后的谱系分化金属硫蛋白是hPSC培养物的潜在应激诱导标志物在这篇文章中，Zweigerdt及其同事将hPSC扩展显示为矩阵-悬浮培养中的独立聚集体与搅拌罐生物反应器中的有效和可扩展的心脏分化相结合。该策略使得每次运行能够产生4000万个主要为心室样表型的心肌细胞（纯度高达85%），其直接适用于生物人工心脏组织形成。
To harness the potential of human pluripotent stem cells (hPSCs), an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation. Efficient cardiac differentiation protocol in suspension by chemical Wnt modulators Differentiation is CHIR concentration dependent, but aggregate size independent Bioreactor-controlled hPSC expansion dictates subsequent lineage differentiation Metallothionein is a potentially stress-induced marker of hPSC culture In this article, Zweigerdt and colleagues show hPSC expansion as matrix-independent aggregates in suspension culture combined with efficient and scalable cardiac differentiation in stirred tank bioreactors. The strategy enables the generation of 40 million cardiomyocytes (up to 85% purity) of predominantly ventricular-like phenotype per run that were directly applicable to bioartificial cardiac tissue formation.
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