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Porcine brain microvascular endothelial cells support the in vitro
expansion of human primitive hematopoietic bone marrow progenitor cells
with a high replating potential: requirement for cell-to-cell interactions
and colony-stimulating factors
TA Davis, DH Robinson, KP Lee and SW Kessler
Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD
20889-5055, USA.
Primary autologous as well as allogeneic and xenogeneic stroma will support
human stem cell proliferation and differentiation for several months. In
the present study, we investigated the capacity of porcine microvascular
endothelial cells (PMVECs) together with combinations of cytokines
(granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor
[SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6)
to support the expansion and development of purified human CD34+ bone
marrow cells. In short-term cultures (7 days), the greatest expansion of
nonadherent hematopoietic cells and clonogenic progenitors was seen with
CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact),
followed by PMVEC noncontact and liquid suspension cultures, respectively.
Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells
(12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 +
SCF + IL-6, with similar increases in the number of granulocyte-macrophage
colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units
(BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells.
Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased
148.1-fold and 8.0-fold, respectively. Replating studies show that cells
from day 7 dispersed blast cell colonies generated on cytokine-treated
PMVEC monolayers have a high replating potential for multilineage
progenitor cells. In long- term PMVEC contact cultures, CD34+ cells seeded
onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total
calculated expansion of over 5,000,000-fold of nonadherent cells over 35
days in culture. Maximal clonogenic cell production was observed at day 28,
with 6,353- fold for total CFC and comparable increases for CFU-GM,
CFU-mix, CFU- blast, BFU-E, and CFU-Mk. The total number of CD34+ cells
increased 2,584-fold at day 28. Furthermore, the extended growth kinetics
of these cultures indicates that these phenotypically primitive progenitor
cells are also functionally expanded on PMVEC monolayers. These results
support the hypothesis that direct contact with a PMVEC monolayer supports
the initial expansion of hematopoietic progenitor cells with a high
replating potential and, possibly, a more primitive phenotype (CD34+,
CD34+/CD38-).
Volume 85,
Issue 7,
pp. 1751-1761,
04/01/1995
Copyright © 1995 by The American Society of Hematology
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