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 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.

Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease

“…The purity was consistently >95%. [25][26][27] Co-culture of bone marrow mesenchymal stem/stromal cells and cord blood CD34 + cells and in vitro analyses of CD34 + cell homeostasis CD34 + cells were co-cultured on irradiated BM-MSC from normal subjects or patients with AA on serum-free media supplemented with stem cell factor, FLT3 ligand and interleukin-3. In vitro analyses were performed with CD34 + cells without MSC co-culture, as a baseline control for CD34-MSC co-cultures.…”

Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function

“…Mononuclear cells were isolated using Ficoll-Hypaque and after lysing the red cells (Cytognos, Salamanca, Spain), CD34 + cells were purified by magnetic bead separation using the human CD34 MicroBead kit and the AutoMACS Pro separator (Miltenyi Biotec) as instructed by the manufacturer. [17][18][19] The purity of the CD34 + fraction was assessed by flow cytometry using an anti-CD34-PE antibody (Miltenyi Biotec), and only CD34 + fractions showing purity higher than 90% were used. [17][18][19] The CD34 -fraction was irradiated (15 Gy) and used as accessory cells for co-transplantation with CD34 + cells.…”

A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

“…After washing, the cells were suspended in PI buffer as described above. BrdU staining of undifferentiated hESCs and cell cycle distribution of hemogenic precursors was analyzed on a FACS Canto-II cytometer discriminating among apoptotic cells (Sub-G0/G1), quiescent cells (G0/G1), cycling cells (S-phase, BrdU+) and G2/ M cells [46,47]. The apoptotic status of NEO and MLL-AF4 hESC-derived CD45 + hematopoietic cells was assessed using the Annexin-V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions [9].…”

“…CD34 + cells were isolated from hEBs at day 11 of development by magnetic-activated cell sorting (MACS) using the hCD34 MicroBead kit and the AutoMACS Pro separator (Miltenyi Biotech), as per manufacturer's instructions [37,46]. To promote endothelial differentiation, isolated CD34 + cells were seeded on 0.1% gelatincoated plates at 1.2 × 10 4 cells/cm 2 in complete EGM-2 media (Lonza, Walkersville) for 5-7 days.…”