The ROCK Inhibitor Y-27632 Negatively Affects the Expansion/Survival of Both Fresh and Cryopreserved Cord Blood-Derived CD34+Hematopoietic Progenitor Cells
The ROCK Inhibitor Y-27632 Negatively Affects the Expansion/Survival of Both Fresh and Cryopreserved Cord Blood-Derived CD34+Hematopoietic Progenitor Cells
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DOI：
10.1007/s12015-010-9118-5
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发表时间：
2010-06-01
影响因子：
4.8
通讯作者：
Menendez, Pablo
中科院分区：
文献类型：
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作者：
Bueno, Clara;Montes, Rosa;Menendez, Pablo
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来源的CD 34 + HSPC在儿童中的使用是成功的，并且通常在移植后导致快速造血恢复。然而，目前用于HSPC的离体扩增的方法仍然导致多谱系分化潜力的丧失，并且目前的冻融方案导致显著的细胞死亡和CD 34 + HSPC的丧失。低温冷冻后细胞活力丧失的主要原因是冷冻损伤直接诱导细胞凋亡。最近的报道已经证明，Y-27632，一种选择性的和稳健的ROCK抑制剂，是一种有效的细胞凋亡抑制剂，并且有效地增强不同人类干细胞（包括人类胚胎、hESC、诱导多能干细胞和间充质干细胞）的解冻后存活和恢复。在这里，我们分析了这种抑制剂在CB衍生的CD 34 + HSPC中的作用。将CB衍生的CD 34 + HSPC进行MACS分离，并用或不用10 μ M Y-27632处理。在新鲜和冷冻保存的CB来源的CD 34 + HSPC中测定Y-27632对培养物稳态的影响。我们的结果表明，Y-27632不仅显著抑制新鲜和冷冻保存的CD 34 + HSPC的细胞扩增，而且在解冻时损害CD 34 + HSPC的存活/恢复，无论Y-27632是否添加到冷冻保存和扩增培养基两者中和/或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来源的CD 34 + HSPC的扩增和存活的不利影响，表明Y-27632可能对不同的谱系/组织特异性干细胞产生不同的影响。我们的数据表明Y-27632对悬浮生长的人干细胞与附着在经处理的组织培养塑料或细胞外基质上生长的人干细胞具有不同的功能。我们不鼓励Y-27632在潜在技术开发中的任何临床应用，这些技术开发旨在改善CB衍生细胞的冷冻保存程序和/或HSPC的体外扩增而不进行自发分化。
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 mu M 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.