Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-modifying agents.
OBJECTIVE
We investigated whether the addition of two chromatin-modifying agents, 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), to cord blood (CB) CD34(+) cells in culture results in expansion of the numbers of severe combined immunodeficient (SCID) repopulating cells (SRC).


METHODS
Human CB CD34(+) cells were cultured with cytokines in the presence or absence of 5azaD/TSA. After 9 days of culture, the fold expansion of CD34(+) and CD34(+)CD90(+) cell numbers, colony-forming unit (CFU)-mix, cobblestone area-forming cell (CAFC), and SRC numbers were determined.


RESULTS
A 12.5-fold expansion of CD34(+)CD90(+) cells was observed in the 5azaD/TSA-treated cultures in comparison to the input cell numbers. Expansion of CD34(+)CD90(+) cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold increase in CAFC. 5azaD/TSA treatment of the CB CD34(+) cells resulted in a 9.6-fold expansion of the absolute number of SRC following 9 days of culture as determined by limiting dilution analysis. Expansion of cells maintaining CD34(+)CD90(+) phenotype was not due to the retention of a quiescent population of cells because all of the CD34(+)CD90(+) cells in the culture had undergone cellular division. 5azaD/TSA-treated CD34(+)CD90(+) cells, but not CD34(+)CD90(-) cells were responsible for in vivo hematopoietic repopulation potential of nonobese diabetic/SCID mice.


CONCLUSIONS
Ex vivo expansion strategy using chromatin-modifying agents provides a potential avenue by which to expand the number of hematopoietic stem cells (HSC) with a single CB unit for use as an alternative source of HSC grafts for adult recipients.
Araki
Hiroto
H
Section of Hematology/Oncology, Department of Medicine, University of Illinois at Chicago, 60607, USA
Mahmud
Nadim
N
Milhem
Mohammed
M
Nunez
Rafael
R
Xu
Mingjiang
M
Beam
Craig A
CA
Hoffman
Ronald
R
D000818:Animals; D018952:Antigens, CD34 / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured; D002843:Chromatin / Q000187:drug effects; D036101:Cord Blood Stem Cell Transplantation; D016207:Cytokines / Q000494:pharmacology; D000077209:Decitabine; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016511:Severe Combined Immunodeficiency / Q000628:therapy*; D018800:Thy-1 Antigens / Q000187:drug effects; D014183:Transplantation, Heterologous
D018952:Antigens, CD34; D002843:Chromatin; D016207:Cytokines; D006877:Hydroxamic Acids; D018800:Thy-1 Antigens; C012589:trichostatin A; D000077209:Decitabine; D001374:Azacitidine