Bone marrow stromal cells from β-thalassemia patients have impaired hematopoietic supportive capacity

BACKGROUND. The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS. MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS. We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche–associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION. Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING. This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.

Osteogenic differentiation was evaluated after 21 days by Alizarin Red S staining (Sigma-Aldrich, A5533) and RT-qPCR. For Alizarin Red S staining, cells were fixed with 4% PFA, washed with water, stained with Alizarin Red solution and fixed with FlourSave Reagent for microscopic evaluation of osteogenic differentiation.

PBMC proliferation assay
Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood of healthy donors by conventional Ficoll -Hypaque gradient for 20 min at 600 x g, after obtaining written informed consent. The proliferation of PBMCs in RPMI 1640 medium (ThermoFisher, 11875-093) supplemented with 10% FBS, in response to PHA-P (Sigma-Aldrich, L8754), in presence or absence of MSCs, was evaluated in triplicate in flat-bottom 96-well tissue culture plates. Briefly, MSCs were seeded overnight at MSCs:PBMCs ratio of 1:2, 1:20, and 1:200. The day after, 1x10 5 PBMCs per well were added with or without PHA-P (4 µg/ml) and pulsed with 3 H-thymidine (1µCi/well, specific activity 6.7 µCi/mmole, Perkin Elmer, Waltham, MA) after 48 hours of culture.  The presence of apoptotic cells after iron treatment was evaluated in MSCs from healthy donors exposed to 40µM iron for 5 days. 5 x 10 5 MSCs were plated in basal medium. The day after 40µM iron (FAC) was added to the culture. 5 day later the caspase 3/7 activity was measured using the Caspase-Glo® 3/7 Assay (Promega, G8090) according to the manufacturer's instructions.
Experiments were performed in triplicates for each HD samples. Untreated cells cultured for 5 days were used as negative control. Results were expressed as relative to Caspase 3/7 activity in untreated controls.

Iron and deferoxamine (DFO) treatment experiments
In specific experiments BT-MSCs were exposed in vitro to increasing doses of Ferric Ammonium Citrate (FAC; 5-10-20-40 µM) for 5 to 21 days. In some experiments BT-MSCs were treated in vitro with an iron chelating agent (DFO 100µM for 24 h) (1) in the presence or in the absence of FAC.

Luminex Assay
R&D system Luminex Kit was used to analyse cytokine's secretion in supernatant derived from HD-and BT-MSC. Supernatant were collected, filtered with a 0.22µm PVDF filter (Millipore) and stored at -80° or used freshly. Custumized Luminex plates were obtained to screen for: IL1a, IL1b, IL15, MIP-1a. Assays were run as per manufacturers' instructions with standards and samples in duplicate. Data were acquired on a calibrated Bio-Plex MAGPIX multiple reader system (Bio-Rad) and visualized with Bio-Plex manager Software.

Implantation of humanized ossicles into NSG mice.
Humanized ossicles were established according to published protocols (89) (88). In particular, 1 x 10 5 MSCs from 3 different HDs and BT patients were pre-seeded on gelatin scaffold (Gelfoam) in combination with 1 x 10 5 endothelial cells (HUVECs), purified from different cord donors after obtaining written informed consent, and 1 x 10 5 CD34 + cells. Scaffolds were surgically implanted subcutaneouslly in the flank of non-irradiated NSG mice, under anesthetized with 0,5% isofluorane.
All procedures were performed according to protocols approved by the Committee for Animal Care and Use of San Raffaele Scientific Institute. 5 or 12 weeks post implantation humanized scaffolds were explanted from the skin and processed for histological (hematoxylin-eosin staining), flow cytometry (for huCD45 expression) and immunofluorescence (huCD45, hu-vimentin) analysis.