TGF-β1 Production By Bone Marrow Mesenchymal Stem Cells of FANCD2 Differs from Other Fanconi Anemia Complementation Groups

TGF-β secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. As a component of the BM niche, BM- mesenchymal stem cells (BM-MSCs) may produce abnormal levels of TGF-β in Fanconi anemia (FA) as obtained for HSCs previously and may play a role in bone marrow failure.

Herein, we present the molecular and cellular characterization of FA BM-MSCs by addressing their immunophenotypes, proliferation- and differentiation capacities, as well as expression and secretion levels of transforming growth factor beta (TGF-β) isoforms. Proliferative potency of FA cells was determined between P3 to P7 and compared with healthy donors by calculating cumulative population doubling (cPD) at each passage. Senescence of FA patient BM-MSCs was evaluated at P3 and P6 by SA-β-gal activity and compared with donor BM-MSCs. Diepoxybutane (DEB) effect on TGF-β1 expression of BM-MSCs from FA patients and donors was also assessed.

In ten FA patients, mutations were classified as FANCA (n=7), FANCG (n=1) and FANCD2 (n=2). Of six were novel mutations, five in FANCA and one in FANCD2 . BM-MSCs immunophenotype and differentiation capacity of FA BM-MSCs were similar to healthy donors.The CD200 (OX-2) levels were slightly lower in FA MSCs compared to donors. The percentage of CD106 (VCAM-1) positive cells were slightly high especially in FANCD2 cells. FA BM-MSCs showed arrest in dip G2 following DEB treatment. BM-MSCs derived from FANCA- or FANCG -deficient patients displayed similar ex vivo proliferative capacity as healthy donor cells from P3 to P7. However, FANCD2-deficient cells presented lower proliferative capacity. FANCA BM-MSCs had notably increased β-gal positivity at P6, while cells displaying FANCD2 mutation had higher activity at P3 indicating premature aging of these cells. Relative gene expression of TGF-β1 was similar between donor and FA MSCs and did not change by DEB treatment. All cells did not secrete TGF-β2 and -β3. Since the variation of TGF-β1 secretion by FANCA and donor cells was high, a significant difference between the groups could not be obtained. On the other hand, FANCD2 -deficient cells were lack of TGF-β1 secretion. Our data suggest that absence of TGF-β1 secretion may lead to early onset of senescence of the FA-D2 BM-MSCs and may be of value to understand the Fanconi anaemia bone marrow dysfunction in humans.