Fanconi anemia (FA) is a devastating blood disease associated with bone marrow (BM) failure. Currently the effective treatment for FA is BM transplantation. We and others showed that FA murine hematopoietic stem and progenitor cells (HSC/Ps) have defective hematopoietic reconstitution after BM transplantation. In addition, several clinical gene therapy trials in FA patients have failed to show sustained engraftment of FA HSC/Ps complemented with a functional FA gene. The mechanism underlying this engraftment defect remains unknown. To determine whether the poor engraftment of FA HSC/Ps observed in mouse knockout models and clinical gene therapy trials in FA patients results from an intrinsic defect of the FA HSC/Ps or an impaired microenvironment in the FA bone marrow, we have assessed the self-renewal ability of HSC/Ps and hematopoietic supportive capacity of BM stromal cells from FA-A patients. First, we performed quantitative cobblestone area-forming cell (CAFC) assays using MS-5 stromal layer. CAFCs were enumerated at 2 and 5 weeks of cocultures between BM mononuclear cells from a normal donor and three FA-A patients. Early-appearing CAFCs (week 2) represent transient repopulating cells equivalent to progenitor colony-forming units (CFUs), whereas late-appearing CAFCs (week 5) are representative of long-term repopulating stem cells. The frequency of FA-A CAFCs was 15–20 folds decreased compared with that of the normal donor at both week 2 and week 5. Second, we carried out long-term culture-initiating cell (LTC-IC) assays using BM mononuclear cells and BM-derived stromals from normal donors or FA-A patients. Compared to coculture with normal stromal cells, the recovery of colony forming cells (CFCs) that grew on FA-A stromal layer after 5–6 weeks incubation was significantly decreased for either normal or FA-A HSC/Ps (41.4% ± 3.1 CFC in normal HSC/Ps - normal stroma compared to 14.3% ± 1.6 CFC in normal HSC/Ps – FA-A stroma and 2.4 ± 0.6 CFC in FA-A HSC/Ps- FA-A stroma). Finally, we conducted CFC and cell-cell adhesion assays using BM-derived stromal cells from normal donors and FA-A patients. We observed that FA-A BM stroma failed to support HSC/P cells from either normal donors or FA-A patients. Specifically, severe adhesion defect was found in adhesion of normal and FA-A HSC/Ps with FA-A stromal layer (5.5% ± 0.6 CFC adhesion in normal HSC/Ps - normal stroma compared to 2.4% ± 0.5 CFC adhesion in normal HSC/Ps – FA-A stroma and 1.0 ± 0.7 CFC adhesion in FA-A HSC/Ps- FA-A stroma). These results suggest that both a cell-autonomous defect of FA HSC/Ps and an impaired FA BM microenvironment may contribute to the abnormal hematopoiesis in FA.

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