Fanconi anemia (FA) is an genetic disease characterized by pancytopenia and marrow failure which can only be rescued by allotransplant. Genes responsible for 4 of the 8 complementation groups in FA have been identified. This opens the possibility of genetic correction of FA hematopoietic stem cells. Using a GALV pseudotyped MSCV-eGFP retroviral vector, we transduced marrow CD34+ cells from FA patients, cultured for 2 days with SCF, FU3-L and IL7 in transwells above AFT024 stroma (non-contact=NC) or in the absence of AFT024 (stroma-free=SF), once daily for 2 days. Transduction efficiency was assessed by flow cytometry on CD34+ cells, and colony forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays. (1) FACS analysis showed that recovery of CD34+ cells and CD34+33- cells, and the %GPF+ CD34+ cells was similar in SF or NC cultures. However, (2) more FA CD34+ cells underwent apoptosis when cultured and transduced in SF than in NC cultures as estimated by the expression of Fas and annexin V. (3) Inreased apoptosis in SF culture was not due to depletion of glutathione or generation of free radicals as addition of N-acetylcystein or glutathione did not rescue the apoptosis and no increase in hydrogen peroxide was detected as estimated by oxidation of dihydrorhodamine. (4) PKH26 labeling studies showed that cells cultured in SF conditions proliferated significantly less. (5) Culture in NC conditions did not prevent apoptosis induced by mitomycin C, suggesting that stroma improved cell survival and cell proliferation by mechanisms other than preventing chromosomal damage and reducing oxidative stress. (6) FA CD34+ cells cultured for 4 days in SF, but not in NC cultures, failed to generate CFC and LTC-IC. (7) Consequently, no CFC or LTC-IC transduction was seen for cells from SF cultures whereas 9.6-38.9% of FA CFC and 30% of FA LTC-IC were transduced in NC culture. (7) In contrast to FA CD34+ cells, normal CD34+ cells did not undergo apopotosis, generated CFC and LTC-IC and were transduced following SF culture. We conclude that stroma is critical for supporting FA CD34+ progenitors as FA CFC and LTC-IC apoptose and fail to proliferate in the absence of stroma ex vivo. This underlies the poor transduction efficiency seen in preclinical and clinical studies using murine retroviruses. This also suggests that failure of s(roma in vivo as a result of the FA defect may contribute to bone marrow failure seen in FA.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|