Andalusian Stem Cell Bank, Centro de Investigación Biomédica, Consejería de Salud-Universidad de Granada, Granada, Spain.
Stem Cell Rev Rep. 2010 Jun;6(2):215-23. doi: 10.1007/s12015-010-9118-5.
Cord blood (CB) is an unlimited source of hematopoietic stem and progenitor cells (HSPC). The use of cryopreserved CB-derived CD34+ HSPCs is successful in children and usually leads to rapid hematopoietic recovery upon transplantation. However, current methods for ex vivo expansion of HSPCs still result in a loss of multilineage differentiation potential and current freeze-thawing protocols result in significant cell death and loss of CD34+ HSPCs. The major cause for the loss of viability after slow freezing is apoptosis induced directly by cryoinjury. Very recent reports have demonstrated that Y-27632, a selective and robust ROCK inhibitor is a potent inhibitor of the apoptosis and is efficient in enhancing the post-thaw survival and recovery of different human stem cells including human embryos, hESCs, induced pluripotent stem cells and mesenchymal stem cells. Here, we analyzed the effect of such an inhibitor in CB-derived CD34+ HSPCs. CB-derived CD34+ HSPCs were MACS-isolated and treated with or without 10 microM of Y-27632. The effect of Y-27632 on culture homeostasis was determined in both fresh and cryopreserved CB-derived CD34+ HSPCs. Our results indicate that the Y-27632 not only dramatically inhibits cell expansion of both fresh and cryopreserved CD34+ HSPCs but also impairs survival/recovery of CD34+ HSPCs upon thawing regardless whether Y-27632 is added to both the cryopreservation and the expansion media and or just to the expansion culture medium with or without hematopoietic cytokines. This study identifies for the first time a detrimental effect of Y-27632 on the expansion and survival of both fresh and cryopreserved CB-derived CD34+ HSPCs, suggesting that Y-27632 may have a differential impact on distinct lineage/tissue-specific stem cells. Our data suggest different functions of Y-27632 on human stem cells growing in suspension versus those growing attached to either treated tissue culture plastic or extracellular matrix. We discourage any clinical application of Y-27632 in potential technical developments aimed at improving cryopreservation procedures of CB-derived cells and/or in vitro expansion of HSPCs without spontaneous differentiation.
脐带血（CB）是造血干细胞和祖细胞（HSPC）的无限来源。冷冻保存的 CB 衍生 CD34+ HSPC 的使用在儿童中是成功的，通常在移植后导致快速造血恢复。然而，目前用于 HSPC 体外扩增的方法仍然导致多谱系分化潜力的丧失，并且当前的冻融方案导致显著的细胞死亡和 CD34+ HSPC 的丧失。在缓慢冷冻后活力丧失的主要原因是由冷冻损伤直接诱导的细胞凋亡。最近的报道表明，Y-27632，一种选择性和强大的 ROCK 抑制剂，是细胞凋亡的有效抑制剂，并且有效地增强了不同人类干细胞（包括人类胚胎、hESCs、诱导多能干细胞和间充质干细胞）的冻后存活和恢复。在这里，我们分析了这种抑制剂在 CB 衍生 CD34+ HSPC 中的作用。CB 衍生的 CD34+ HSPC 用 MACS 分离并用或不用 10 μM 的 Y-27632 处理。在新鲜和冷冻保存的 CB 衍生 CD34+ HSPC 中确定 Y-27632 对培养平衡的影响。我们的结果表明，Y-27632 不仅显著抑制新鲜和冷冻保存的 CD34+ HSPC 的细胞扩增，而且还损害 CD34+ HSPC 的存活/恢复，无论 Y-27632 是添加到冷冻保存和扩增培养基中，还是仅添加到具有或不具有造血细胞因子的扩增培养基中。这项研究首次确定了 Y-27632 对新鲜和冷冻保存的 CB 衍生 CD34+ HSPC 的扩增和存活的有害影响，这表明 Y-27632 可能对不同的谱系/组织特异性干细胞有不同的影响。我们的数据表明，Y-27632 对悬浮生长的人类干细胞和附着于处理过的组织培养塑料或细胞外基质的人类干细胞具有不同的功能。我们不鼓励在旨在改善 CB 衍生细胞的冷冻保存程序和/或 HSPC 的体外扩增而不自发分化的潜在技术发展中应用 Y-27632。
Stem Cells Transl Med. 2016-6
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