Previous studies have demonstrated that Bcl-xL offers protection from apoptosis induced by growth factor withdrawal, allowing cells to arrest in Gl/GO until grojwth factors are returned. In addition, studies have shown that a stem cell population transdi ced with Bcl-2 or Bcl-xL increased engraftment. Based on these findings we hypothes zed that Bcl-xL may be critical for maintaining a stem cell phenotype, and that loss of Be -xL expression may be a step leading to loss of engraftment potential. B6D2 Fl mouse hone marrow cells can be separated by elutriation into long-term reconstituting stem cjells (FR25Lin-) a.nd short term reconstituting early and late progenitors(R/O).The FR25Lincells are quiescent and the R/O are in cycle. We now show by Northern analysis that the expression of Bcl-xL in the R/O population is very low compared to the Fr25Lin- cells. We utilized the retroviral vector MGIN2, which contains a murine stem cell virus (MSCV) promoter as well as the genes for Enhanced Green Fluorescent Protein(EGFP) and Neomycin resistance. R/OLin- cells were transduced with supematants containing either MGIN2 or MGIN2/Bcl-xL retroviruses to increase the probability of infecting progenitor cells that were actively cycling. Cells were then selected on the basis of EGFP expression and viable cells were used in the CFU-C, CFU-S and transplant assays. The CFU-C assay resulted in fewer colonies from cells transduced with MGIN2/Bcl-xL (1±.48) as compared to the control vector (19.3+4.7) per 2×103 cells plated. This is consistent with the cells exhibiting a stem cell phenotype. CFUS-8 and CPUS-12 also showed that cells transduced with MGIN2/Bcl-xL had fewer colonies than the control group. These results fit our hypothesis that Bcl-xL may contribute to hematopoietic cell quiescence. We also examined the effect of Bcl-xL on short and long term reconstitution capabilities. Male B6D2F1 R/ OLin- cells (5×103) transduced (and positively selected for EGFP) with either MGIN2/ Bcl-xL or the control vector, along with 2×104 female non-transduced R/O cells, to provide a first wave of engraftment, were injected into lethally irradiated B6D2F1 female mice. A control group received only 2×104 female R/O cells. These short and long term engraftment studies are in progress but preliminarily the group of mice receiving the Bcl-xL transduced male R/OLin- cells show increased donor engraftment. Currently, we can conclude that rendering a progenitor population quiescent may result in the potential for re-acquiring of stem cell characteristics.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology