Bone Marrow Mesenchymal Stem Cells Carrying FANCD2 Mutation Differ from the Other Fanconi Anemia Complementation Groups in Terms of TGF-β1 Production

Abstract

Transforming growth factor beta (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. BM mesenchymal stem cells (BM-MSCs), a component of the BM niche, may produce abnormal levels of TGF-β in Fanconi anemia (FA) and may play a role in bone marrow failure. Here, we molecularly and cellularly characterized FA BM-MSCs by addressing their immunophenotype, proliferation- and differentiation- capacity, reactive oxygen species (ROS) production, senescence activity as well as expression and secretion levels of TGF-β isoforms. In ten FA patients, mutations were detected in FANCA (n = 7), FANCG (n = 1) and FANCD2 (n = 2) genes. The immunophenotype, with the exception of CD29, and differentiation capacity of FA BM-MSCs were similar to healthy donors. FA BM-MSCs showed decreased proliferation, increased ROS level and an arrest in G2 following DEB treatment. β-galactosidase staining indicated elevated senescence of FANCD2-deficient cells. FA BM-MSCs displayed TGF-β1 mRNA levels similar to donor BM-MSCs, and was not affected by DEB treatment. However, secretion of TGF-β was absent in FA-D2 BM-MSCs. Absence of TGF-β secretion may be related to early onset of senescence of the FANCD2-deficient BM-MSCs. The proliferative response of FA-D2 BM-MSCs to rTGF-β1 was not different from FANCA-deficient and donor cells and raises the possibility that rTGF-β1 may reverse the senescence of the FANCD2-deficient BM-MSCs which needs to be investigated further.

PKNOX2 expression and regulation in the bone marrow mesenchymal stem cells of Fanconi anemia patients and healthy donors

“…Indeed, a whole genome RNA interference (RNAi) study showed that PKNOX2 silencing increased cellular sensitivity to ionizing radiation [45]. However, our study showed that DEB treatment of BM-MSCs did not change the expression of PKNOX2 in either donor or FA patients, except one that possessed a novel deletion of exon 1-2 in FANCA gene, reported in our previous study [25]. PKNOX2 expression is lost by DEB treatment in that patient's BM-MSCs.…”

“…From many (n = 1639) transcription factors found in humans [46], we focused on HOX and TALE transcription factors that are strictly under epigenetic control during adult life. TGF-β signaling interacts with HOX genes [20][21][22][23], and we previously showed fluctuation of TGF-β1 secretion from FA BM-MSCs [25]. Deregulated TGF-β signaling may disturb PKNOX2 expression in FA BM-MSCs and trigger disease progression, as seen in FA HSCs [19].…”

“…Passage 3 BM-MSCs were used in the following experiments. Characterization of BM-MSCs was published previously [24,25]. Informed consent was obtained from FA patients and donors enrolled in this study.…”

“…Once treated with DEB, patient cells acquire chromosome breaks and undergo cell cycle arrest as well as genomic instability [18]. BM-MSCs derived from FA patients (n = 6) and donors (n = 3) were treated with 0.1 µg/ mL DEB (Sigma-Aldrich) in DMF10 medium, as outlined previously [25]. Untreated cells cultured in DMF10 medium were used as control.…”

Pathogenesis of Acquired Aplastic Anemia and the Role of the Bone Marrow Microenvironment

“…Indeed a comprehensive comparison of MSCs derived from 18 patients with Fanconi anemia ( n = 18) and age-matched healthy controls ( n = 15) revealed increased spontaneous chromosomal fragility leading to precocious senescence and significantly reduced survival when compared to MSCs derived from age-matched healthy donors (59). These data were further confirmed in a subsequent study from Cagnan et al which documented decreased proliferation, increased ROS levels and an arrest in G2 following DEB (Diepoxybutane) treatment in MSCs from 10 Fanconi anemia patients, with especially absent transforming growth factor (TGF)- β secretion and elevated senescence levels in the FANCD2 mutated cases (60). Analysis of MSCs from Shwachman-Diamond syndrome (SDS) patients, another major subgroup of congenital BMF, also revealed profound functional defects and failed to recreate a BM niche in an in vivo heterotopic ossicle model (61).…”