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.
BACKGROUND
Cord 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.


RESULTS
CB 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.


CONCLUSIONS
Our 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.
V M
Sangeetha
S
Sangeetha
V M
VM
Kale
Vaijayanti P
VP
Limaye
Lalita S
LS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology*; D005615:Freezing; D006410:Hematopoiesis / Q000187:drug effects; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D022423:Myeloid Cells / Q000166:cytology; D009842:Oligopeptides / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D018952:Antigens, CD34; D009842:Oligopeptides; D011480:Protease Inhibitors; C544876:benzyloxycarbonyl-leucyl-glucyl-tyrosine fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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