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

Abstract

BackgroundExpansion of hematopoietic stem/progenitor cells (HSPCs) is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB) derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion.Methodology/Principal FindingsCB derived CD34+ cells were expanded using a combination of growth factors with and without Caspase inhibitor -zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homing-related molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors) caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice.Conclusion/SignificanceOur present study reveals another novel aspect of the regulation of caspase and calpain proteases in the biology of HSPCs. The priming of the homing responses of the inhibitor-cultured HSPCs compared to the cytokine-graft suggests that the modulation of these proteases may help in overcoming the major homing defects prevalent in the expansion cultures thereby facilitating the manipulation of cells for transplant procedures.

Rhizoctonia Bataticola Lectin (RBL) Induces Caspase-8-Mediated Apoptosis in Human T-Cell Leukemia Cell Lines but Not in Normal CD3 and CD34 Positive Cells

“…The cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS), 100 µg/ml streptomycin and 100units/ml penicillin. CD34+ve hematopoietic stem and progenitor cells (HSPCs) were isolated from human umbilical cord blood and expanded in vitro according to protocol reported earlier [19] . The cells were maintained in IMDM medium supplemented with 20% FCS and 25000 cells were seeded per well in 96 well tissue culture plates.…”