The ROCK Inhibitor Y-27632 negatively affects the expansion/survival of both fresh and cryopreserved cord blood-derived CD34+ hematopoietic progenitor cells

Abstract

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 freezethawing 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 MACSisolated and treated with or without 10 μM 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.