Inhibition of ROCK-Myosin II Signaling Pathway Enables Culturing of Human Pluripotent Stem Cells on Microcarriers Without Extracellular Matrix Coating
Inhibition of ROCK-Myosin II Signaling Pathway Enables Culturing of Human Pluripotent Stem Cells on Microcarriers Without Extracellular Matrix Coating
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DOI：
10.1089/ten.tec.2013.0191
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发表时间：
2014-03-01
影响因子：
3
通讯作者：
Oh, Steve Kah Weng
中科院分区：
文献类型：
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作者：
Chen, Allen Kuan-Liang;Chen, Xiaoli;Oh, Steve Kah Weng
治疗应用所需的大量人类多能干细胞(HPSCs)可以在可扩展的悬浮微载体培养中培养。这些微载体包裹有动物或人类细胞外基质(ECM)蛋白，以促进细胞生长并保持多能性。然而，这种涂层对于大规模培养来说是昂贵的，而且存在安全风险。这项研究表明hPSCs可以在含有ROCK抑制剂(Y27632)或肌球蛋白抑制剂Blebbistatin的无血清介质中在非包被的带正电的纤维素微载体上增殖。在这两种抑制剂的存在下，肌球蛋白磷酸酶1和肌球蛋白轻链2被去磷酸化，这表明肌球蛋白收缩能力的降低是hPSC在无ECM涂层的微载体上存活和生长的原因。细胞在未包被的微载体上培养12代后，保持了其多能性和核型稳定性。在100毫升的旋转瓶中，细胞浓度达到2.3×10(6)个/毫升，扩张率为11.5倍(HES-3)，证明了可伸缩性。通过体外类胚体和体内畸胎瘤的形成，证实了这些细胞向三个主要谱系分化的能力。定向分化为多唾液酸化神经细胞黏附分子阳性(PSA-NCAM+)神经前体细胞的细胞密度高(9.1+/-1.2×10(6)个/毫升)，细胞产量为412+/-77个神经前体细胞/HES-3，PSA-NCAM表达水平为91+/-1.1%。这种明确的无血清和无涂层的可伸缩微载体培养系统是一种更安全、更便宜的方法，可以产生大量的hPSCs用于细胞治疗。
Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free microcarriers. Cells propagated on the noncoated microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3x10(6) cells/mL with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1 +/- 1.2x10(6) cells/mL) with a cell yield of 412 +/- 77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91 +/- 1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.