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Long-term generation and expansion of human primitive hematopoietic
progenitor cells in vitro
EF Srour, JE Brandt, RA Briddell, S Grigsby, T Leemhuis and R Hoffman
Department of Medicine, Hematology/Oncology, Indiana University School of
Medicine, Indianapolis 46202-5121.
Although sustained production of committed human hematopoietic progenitor
cells in long-term bone marrow cultures (LTBMC) is well documented,
evidence for the generation and expansion of human primitive hematopoietic
progenitor cells (PHPC) in such cultures is lacking. For that purpose, we
attempted to determine if the human high proliferative potential
colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor
cell, is capable of generation and expansion in vitro. To that effect,
stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine
123dull bone marrow cells and were maintained with repeated addition of
c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage
colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater
than twofold increase in the number of assayable HPP-CFC was detected.
Furthermore, the production of HPP-CFC in LTBMC continued for up to 4
weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly
phenotypic analyses of cells harvested from LTBMC showed that the number of
CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells
increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further
the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies
were serially cloned. Secondary cloning of individual, day 28 primary
HPP-CFC indicated that 46% of these colonies formed an average of nine
secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived
colonies, whereas 43% of primary HPP-CFC gave rise to between one and six
secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+
HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone
marrow highly enriched for HPP-CFC and that based on their regeneration and
proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these
studies indicate that in the presence of appropriate cytokine stimulation,
it is possible to expand the number of PHPC in vitro.
Volume 81,
Issue 3,
pp. 661-669,
02/01/1993
Copyright © 1993 by The American Society of Hematology
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