title: 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: 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.
authors.forename: Sangeetha
authors.initials: S
authors.lastname: V M
authors.forename: V M
authors.initials: VM
authors.lastname: Sangeetha
authors.forename: Vaijayanti P
authors.initials: VP
authors.lastname: Kale
authors.forename: Lalita S
authors.initials: LS
authors.lastname: Limaye
mesh_terms: 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
chemical_list: 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
references.citation: Bone Marrow Transplant. 1999 Aug;24(4):397-403
references.pmid: 10467329
references.citation: Exp Hematol. 2007 Apr;35(4 Suppl 1):105-16
references.pmid: 17379095
references.citation: Blood. 2005 May 15;105(10):3786-92
references.pmid: 15677563
references.citation: J Exp Med. 2000 Nov 6;192(9):1365-72
references.pmid: 11067884
references.citation: Transfus Clin Biol. 2001 Jun;8(3):146-54
references.pmid: 11499955
references.citation: Exp Hematol. 2009 Nov;37(11):1364-1377.e4
references.pmid: 19744539
references.citation: Yale J Biol Med. 2009 Mar;82(1):7-18
references.pmid: 19325941
references.citation: Cell Stem Cell. 2008 Jun 5;2(6):595-601
references.pmid: 18522852
references.citation: Bone Marrow Transplant. 2010 May;45(5):955-6
references.pmid: 19784074
references.citation: Exp Hematol. 2008 Feb;36(2):244-52
references.pmid: 18023520
references.citation: Exp Hematol. 2003 Sep;31(9):824-32
references.pmid: 12962729
references.citation: Exp Hematol. 2001 Dec;29(12):1465-73
references.pmid: 11750106
references.citation: PLoS Biol. 2008 Apr 8;6(4):e84
references.pmid: 18399720
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 1;102(9):3312-7
references.pmid: 15728354
references.citation: J Exp Med. 2000 Jan 17;191(2):253-64
references.pmid: 10637270
references.citation: Nat Rev Cancer. 2003 Jul;3(7):526-32
references.pmid: 12835672
references.citation: Transplantation. 2005 Nov 15;80(9):1251-60
references.pmid: 16314793
references.citation: FASEB J. 2002 Oct;16(12):1651-3
references.pmid: 12207004
references.citation: Eur J Haematol. 2009 Feb;82(2):124-32
references.pmid: 19067743
references.citation: J Clin Invest. 2000 Apr;105(7):1013-21
references.pmid: 10749580
references.citation: Bone Marrow Transplant. 2009 Mar;43(6):447-54
references.pmid: 18955980
references.citation: Blood. 1997 Apr 15;89(8):2644-53
references.pmid: 9108381
references.citation: Nat Med. 2010 Feb;16(2):232-6
references.pmid: 20081862
references.citation: Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):14943-8
references.pmid: 10611317
references.citation: Curr Opin Immunol. 2006 Oct;18(5):571-5
references.pmid: 16895752
references.citation: Brain Res. 2005 Dec 14;1065(1-2):8-19
references.pmid: 16309635
references.citation: J Exp Med. 2001 Oct 1;194(7):941-52
references.pmid: 11581316
references.citation: Stem Cells. 2006 Nov;24(11):2592-602
references.pmid: 17071861
references.citation: Bone Marrow Transplant. 2009 Nov;44(10):627-33
references.pmid: 19802026
references.citation: Bone Marrow Transplant. 2009 Nov;44(10):673-81
references.pmid: 19802023
references.citation: Bone Marrow Transplant. 2008 May;41(9):771-8
references.pmid: 18209724
references.citation: Cell Stem Cell. 2008 Jun 5;2(6):584-94
references.pmid: 18522851
references.citation: Bone Marrow Transplant. 2007 Jan;39(1):11-23
references.pmid: 17164824
references.citation: J Hematother Stem Cell Res. 2003 Oct;12(5):553-64
references.pmid: 14594512
references.citation: Stem Cells Dev. 2010 Apr;19(4):461-8
references.pmid: 19686047
references.citation: Blood. 1999 Jun 1;93(11):3736-49
references.pmid: 10339480
references.citation: Blood. 2003 Jun 15;101(12):5061-7
references.pmid: 12595310
references.citation: J Hematother Stem Cell Res. 2001 Oct;10(5):709-18
references.pmid: 11672518
references.citation: J Biol Chem. 2003 Apr 18;278(16):14162-7
references.pmid: 12576481
references.citation: Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):413-8
references.pmid: 11782553
references.citation: Stem Cells. 2006 Nov;24(11):2437-47
references.pmid: 16888285