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Large-scale expansion of human stem and progenitor cells from bone marrow
mononuclear cells in continuous perfusion cultures
MR Koller, SG Emerson and BO Palsson
Aastrom Biosciences, Inc, Ann Arbor, MI 48106.
There is a growing consensus that clinical practice in the areas of bone
marrow (BM) transplantation and gene therapy will rely on the ex vivo
expansion of hematopoietic cells. We report here on the development of
continuously perfused culture systems (bioreactor systems) that expand
human stem and progenitor cells from BM mononuclear cell (MNC) populations
obtained without cell enrichment. In three separate experiments, 10
bioreactors were each inoculated with 3 x 10(7) BM MNC from patients
undergoing marrow harvest for autologous transplantation. At various times
thereafter (between days 6 and 16), duplicate bioreactors were harvested
and cells were analyzed. The bioreactors contained three cell populations
that were analyzed separately: nonadherent cells; cells that were loosely
adherent to the endogenously formed stromal layer; and an adherent cell
layer that required trypsinization for removal. Total cell numbers
increased continuously to give an overall 10-fold (range, 8- to 11-fold)
expansion by day 14. The adherent stromal layer significantly expanded to
more than 2 x 10(7) cells, but remained less than 6% of the total cell
population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers
expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this
expansion was greater than that for total cells, CFU-GM were enriched by as
much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers
peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to
18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were
predominantly nonadherent, BFU-E were more evenly distributed between the
three cell populations. Stem cell activity was measured by the long-term
culture-initiating cell (LTC-IC) limiting dilution assay. The number of
LTC-IC per reactor consistently increased with time in all cultures,
resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary,
more than 3 billion cells, containing 12 million CFU-GM, were reproducibly
generated from the equivalent of a 10 to 15 ml BM aspirate. These data
indicate that small numbers of BM MNC can be readily expanded ex vivo in
continuous perfusion cultures, and that such ex vivo expansion may have
direct applications in clinical and experimental BM transplantation.
Volume 82,
Issue 2,
pp. 378-384,
07/15/1993
Copyright © 1993 by The American Society of Hematology
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