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Titolo:
LEUKEMIA INHIBITORY FACTOR UP-REGULATES CYTOKINE EXPRESSION BY A MURINE STROMAL CELL-LINE ENABLING THE MAINTENANCE OF HIGHLY ENRICHED COMPETITIVE REPOPULATING STEM-CELLS
Autore:
SZILVASSY SJ; WELLER KP; LIN WY; SHARMA AK; HO ASY; TSUKAMOTO A; HOFFMAN R; LEIBY KR; GEARING DP;
Indirizzi:
SYSTEMIX INC,DEPT CELL BIOL,3155 PORTER DR PALO ALTO CA 94304 SYSTEMIX INC,DEPT MOLEC BIOL PALO ALTO CA 94304
Titolo Testata:
Blood
fascicolo: 11, volume: 87, anno: 1996,
pagine: 4618 - 4628
SICI:
0006-4971(1996)87:11<4618:LIFUCE>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
LONG-TERM CULTURES; COLONY-FORMING-UNITS; BONE-MARROW CULTURE; LIMITING-DILUTION; HEMATOPOIETIC-CELLS; C-KIT; FUNCTIONAL-CHARACTERIZATION; INVITRO HEMATOPOIESIS; HUMAN INTERLEUKIN; THY-1 ANTIGEN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
57
Recensione:
Indirizzi per estratti:
Citazione:
S.J. Szilvassy et al., "LEUKEMIA INHIBITORY FACTOR UP-REGULATES CYTOKINE EXPRESSION BY A MURINE STROMAL CELL-LINE ENABLING THE MAINTENANCE OF HIGHLY ENRICHED COMPETITIVE REPOPULATING STEM-CELLS", Blood, 87(11), 1996, pp. 4618-4628

Abstract

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-1(lo)Sca-1(+)H-2K(hi) cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with ti-fluorouracil. 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 2weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented1 per 55 freshly sorted cells. CRU could be recovered from coculturessupported by all three stromal cell lines, but their numbers were similar to sevenfold less than on day 0. In contrast, the addition of LIFto stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (similar to 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-1(lo)Sca-1(+)H-2K(hi) cells instroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1 beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factor-beta, 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 cells 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-stimulatedSyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence fora role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, wehave used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture. (C) 1996 by The American Society of Hematology.

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Documento generato il 14/07/20 alle ore 03:42:46