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Titolo:
ORDERLY PROCESS OF SEQUENTIAL CYTOKINE STIMULATION IS REQUIRED FOR ACTIVATION AND MAXIMAL PROLIFERATION OF PRIMITIVE HUMAN BONE-MARROW CD34(+) HEMATOPOIETIC PROGENITOR CELLS RESIDING IN G(0)
Autore:
LADD AC; PYATT R; GOTHOT A; RICE S; MCMAHEL J; TRAYCOFF CM; SROUR EF;
Indirizzi:
INDIANA UNIV,SCH MED,DIV HEMATOL ONCOL,975 W WALNUT ST,IB 442 INDIANAPOLIS IN 46202 INDIANA UNIV,SCH MED,DIV HEMATOL ONCOL INDIANAPOLIS IN 46202 INDIANA UNIV,SCH MED,INDIANA ELKS CANC RES CTR,DEPT MED INDIANAPOLIS IN 46202 INDIANA UNIV,SCH MED,HERMAN B WELLS CTR PEDIAT RES,DEPT PEDIAT INDIANAPOLIS IN 46202
Titolo Testata:
Blood
fascicolo: 2, volume: 90, anno: 1997,
pagine: 658 - 668
SICI:
0006-4971(1997)90:2<658:OPOSCS>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
LONG-TERM CULTURE; STEM-CELLS; GROWTH-FACTORS; CORD-BLOOD; INVITRO; RHODAMINE-123; FLUORESCENCE; ENRICHMENT; SEPARATION; EXPANSION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
40
Recensione:
Indirizzi per estratti:
Citazione:
A.C. Ladd et al., "ORDERLY PROCESS OF SEQUENTIAL CYTOKINE STIMULATION IS REQUIRED FOR ACTIVATION AND MAXIMAL PROLIFERATION OF PRIMITIVE HUMAN BONE-MARROW CD34(+) HEMATOPOIETIC PROGENITOR CELLS RESIDING IN G(0)", Blood, 90(2), 1997, pp. 658-668

Abstract

Bone marrow (BM) CD34(+) cells residing in the G(0) phase of cell cycle may be the most suited candidates for the examination of cell cycleactivation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labelling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34(+) cells residing in G(0)(G(0)CD34(+)). Using long-term BM cultures and limiting dilution analysis, G(0)CD34(+) cells were found to be enriched for primitive HPCs. In vitro proliferation of G(0)CD34(+) cells in response to sequential cytokine stimulation wasexamined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 far 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G(0)CD34(+) cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained inphase I. Cells cultured in SCF or FL for the entire 14-day period didnot progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G(o)CD34(+) cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34(+), detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G(0)/G(1), unable toprogress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggestthat a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34(+) cells residing in G(0) may result in disruption of cell-cycle regulation. (C) 1997 by The American Society of Hematology.

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Documento generato il 05/07/20 alle ore 07:15:35