Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Abstract

BackgroundCord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34+ cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion.Methodology/Principal FindingsCB CD34+ cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34+ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone.Conclusion/SignificanceOur data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34+ cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.

Differential ability of MSCs isolated from placenta and cord as feeders for supporting ex vivo expansion of umbilical cord blood derived CD34+ cells

“…We have earlier reported that the addition of anti-apoptotic agents resulted in increased expansion efficiency of suspension cultures of UCB CD34 + cells [ 17 ]. Since higher levels of apoptosis were detected in the C-MSCs:CD34 + co-cultures, we checked if this system could be made equivalent to the P-MSCs co-cultures by the addition of anti-apoptotic molecules.…”

Pharmacological Inhibition of Caspase and Calpain Proteases: A Novel Strategy to Enhance the Homing Responses of Cord Blood HSPCs during Expansion

“…Previously, we identified a distinctive aspect of apoptosis (negative regulation) that influenced the expansion of CD34 + cells. Our study revealed that the cell-permeable inhibitors of caspase and calpain proteases, augmented the expansion and long-term engraftment of cord blood CD34 + cells [13]. We speculated that the mechanism behind the sustained engraftment might lie in the ability of the inhibitors to influence the migratory and adhesive interactions of these cells in vitro.…”

Cryopreservation of Hematopoietic and Non-Hematopoietic Stem Cells – A Review for the Clinician

“…This led to the study of caspase inhibitors as cryopreservatives. The initial encouraging results on cell cultures (Heng, Clement, & T. Cao, 2007;Stroh et al, 2002), were recently supported by in vivo experiments (Sangeetha, Kale, & Limaye, 2010). Other cryo-preservative additives, such as α tocopherol (Neunert et al, 2009;E J Woods et al, 2000), are under investigation and may be of future interest.…”

“…Whilst platelet recovery and the median amount of blood products transfused did not differ in between the two groups, the patients obtaining cell concentrates, cryopreserved with the DMSO/HES combination obtained neutrophil recovery in average one day earlier and needed one day less antibiotic administration(p=0.04) (Rowley et al, 2003). Unfortunately hematopoietic and other stem cells, particularly human embryonic stem cells, undergo apoptotic transformation during the cryopreservation process (Sangeetha, Kale, & Limaye, 2010;Stroh et al, 2002). This led to the study of caspase inhibitors as cryopreservatives.…”