We previously demonstrated that fractions of ex vivo expanded murine Sca-1+ in- cells possess different bone marrow (BM) engraftment potentials depending on their proliferation history. Mitotically active Sca-1+ cells which have undergone extensive cellular proliferation displayed diminished repopulatlng potential, while cells which maintained mifotic quiescence, by virtue of being cytokine nonresponsive (CNR), retained an enhanced BM repopulatmg ability. We extended our studies to investigate whether re-entry of proliferating hematopoietic progenitor cells (HPC) into mitotic quiescence following in vitro cell division would impart on these cells an enhanced marrow repopulating potential. Ex vivo proliferation of Sca-1+ lin- cells from B6.Hbbd congenic mice was tracked by the membrane dye PKH2, and Sca-1+ cells having undergone one or more cellular divisions were isolated and subsequently fractionated into those belonging to GO or S/G2+M phases of cell cycle using the DNA dye Hoechst 33342 and the RNA dye Pyronin Y. Isolated fractions were transplanted into lethally irradiated C57/BI6 recipients and engraftment was monitored by hemoglobin typing and isoenzyme analysis of CD3+, B220+ and GR-1+ cells. In agreement with our earlier studies, CNR cells displayed enhanced long-term marrow repopulating potentials compared to Sca-1+ cells that had divided during culture (Sca-l+ PKH2Dim). When Sca-1+ PKH2Dim cells were further fractionated on the basis of cell cycle position, we found that early engraftment with donor cells as well as the rate of chimerism were higher among mice receiving GO cells than among those transplanted with S/G2+M cells. However, at 6 mo. posttransplantation, both groups of cells mediated similar degrees of chimerism as demonstrated by hemoglobin typing. Similarly, at 8 mo. post-transplantation, the levels of Trilineage chimerism realized in recipients of GO cells were very similar to those observed in mice transplanted with S/G2+M cells, suggesting that GO cells isolated from ex vivo expanded Sca-l+ PKH2Dim cells were not superior in longterm engraftment potential compared to their counterparts in S/G2+M. These data demonstrate that re-entry of HPC into mitotic quiescence following in vitro proliferation may not be sufficient for the restoration of primitive hematopoietic activity.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
- Cancer Research