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Expansion in vitro of retrovirally marked totipotent hematopoietic stem
cells
CC Fraser, CJ Eaves, SJ Szilvassy and RK Humphries
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
A large number of biologic, technological, and clinical studies await the
development of procedures that will allow totipotent hematopoietic stem
cells to be expanded in vitro. Previous work has suggested that
hematopoiesis can be reconstituted using transplants of cells from long-
term marrow cultures. We have used retrovirus mediated gene transfer to
demonstrate that marked totipotent hematopoietic stem cells are both
maintained and can be amplified in such cultures, and then subsequently
regenerate and sustain lympho-myeloid hematopoiesis in irradiated
recipients. Marrow cells from 5-fluorouracil-treated male mice were
infected with a recombinant virus carrying the neomycin resistence gene and
seeded onto irradiated adherent layers of pre-established, long- term
marrow cultures of female origin. At 4 weeks, cells from individual
cultures were transplanted into single or multiple female recipients.
Southern blot analysis of hematopoietic tissue 45 days posttransplantation
showed retrovirally marked clones common to lymphoid and myeloid tissues in
14 of 23 mice examined. Strikingly, for 3 of 4 long-term cultures, multiple
recipients of cells from a single flask showed marrow and thymus
repopulation with the same unique retrovirally marked clone. These results
establish the feasibility of retroviral-marking techniques to demonstrate
the maintenance of totipotent lympho-myeloid stem cells for at least 4
weeks in the long- term marrow culture system and provide the first
evidence of their proliferation in vitro. Therefore, such cultures may
serve as a starting point for identifying factors that stimulate totipotent
hematopoietic stem cell expansion.
Volume 76,
Issue 6,
pp. 1071-1076,
09/15/1990
Copyright © 1990 by The American Society of Hematology
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