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Leukemia inhibitory factor upregulates cytokine expression by a murine
stromal cell line enabling the maintenance of highly enriched competitive
repopulating stem cells
SJ Szilvassy, KP Weller, W Lin, AK Sharma, AS Ho, A Tsukamoto, R Hoffman, KR Leiby and DP Gearing
Department of Cell Biology, SyStemix, Inc, Palo Alto, CA 94304, USA.
Attempts to maintain or expand primitive hematopoietic stem cells in vitro
without the concomitant loss of their differentiative and proliferative
potential in vivo have largely been unsuccessful. To investigate this
problem, we compared the ability of three cloned bone marrow (BM) stromal
cell lines to support the growth of primitive Thy- 1lo Sca-1+H-2Khi cells
isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice
treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly
enriched in cobblestone area-forming cells (CAFC), but their frequency was
dependent on the stromal cell lines used in this assay (1 per 45 cells on
SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia
inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8
cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high
clonogenicity of this primitive stem cell population. More primitive stem
cells with competitive repopulating potential were measured by injecting
the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5)
radioprotective Ly-5.1 BM cells whose long-term repopulating ability has
been "compromised" by two previous cycles of marrow transplantation and
regeneration. Donor-derived lymphocytes and granulocytes were detected in
66% of animals injected with 50 sorted cells. To quantitate the maintenance
of competitive repopulating units (CRU) by stromal cells, sorted cells were
transplanted at limiting dilution before and after being cultured for 2
weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per
55 freshly sorted cells. CRU could be recovered from cocultures supported
by all three stromal cell lines, but their numbers were
approximately-sevenfold less than on day 0. In contrast, the addition of
LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6
cells (approximately two-fold to threefold decline), and enabled their
maintenance on SyS-1. LIF appeared to act indirectly, because alone it did
not support the proliferation of Thy- 1lo Sca-1+H-2Khi cells in stroma-free
cultures. Polymerase chain reaction (RT-PCR) analysis revealed that
Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating
factor, granulocyte macrophage-colony stimulating factor, transforming
growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1
within 1 to 6 hours of LIF-stimulation. To determine if increased
expression of SLF by LIF-stimulated SyS-1 cells could account for their
capacity to support stem cells, sorted calls were cocultured on simian CV-E
cells that were transfected with an expression vector encoding
membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF
synergized with IL-6 produced endogenously by CV-E cells enabling CAFC
growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-
stimulated SyS-1 could also be completely abrogated by an anti-SLF
antibody. These data provide evidence for a role of LIF in the support of
long-term repopulating stem cells by indirectly promoting cytokine
expression by BM stroma. Furthermore, we have used quantitative assays to
show a maintenance of CRU numbers, with retention of in vivo function
following ex vivo culture.
Volume 87,
Issue 11,
pp. 4618-4628,
06/01/1996
Copyright © 1996 by The American Society of Hematology
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