Bone marrow stromal cells from β-thalassemia patients have impaired hematopoietic supportive capacity
Stefania Crippa, … , Giuliana Ferrari, Maria Ester Bernardo
Stefania Crippa, … , Giuliana Ferrari, Maria Ester Bernardo
Published February 25, 2019
Citation Information: J Clin Invest. 2019;129(4):1566-1580. https://doi.org/10.1172/JCI123191.
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Clinical Research and Public Health

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

Abstract

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.

Authors

Stefania Crippa, Valeria Rossella, Annamaria Aprile, Laura Silvestri, Silvia Rivis, Samantha Scaramuzza, Stefania Pirroni, Maria Antonietta Avanzini, Luca Basso-Ricci, Raisa Jofra Hernandez, Marco Zecca, Sarah Marktel, Fabio Ciceri, Alessandro Aiuti, Giuliana Ferrari, Maria Ester Bernardo

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Figure 8

Impaired hematopoietic supportive capacity of BT-MSCs.

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Impaired hematopoietic supportive capacity of BT-MSCs. (A) Transwell mig...
(A) Transwell migration assay of cord blood (CB) CD34+ toward HD-MSCs (n = 5; blue squares), BT-MSCs (n = 5; orange circles), and BT-MSCs (purple squares) treated with 100 μM DFO for 24 hours. Migration capacity is represented as absolute number of CD45+ cells migrated into the bottom chamber. Positive control (pos ctrl): SDF-1 (100 ng/ml). Negative control (neg ctrl): basal medium. Each error bar shows mean ± SEM. (B) Total number of live cord blood (CB) CD34+ cells after 3 days of coculture with HD-MSCs (red) and BT-MSCs (blue) in the presence or absence of proper cytokines. CB CD34+ cells cultured for 3 days in the presence or absence of proper cytokines were used as control (black). Data are mean ± SEM (HD: n = 3; BT: n = 3). (C) Absolute number of primitive HSPCs identified as Lin–, CD34hi, CD90+, CD45RA– on CD45+ cells after 3 days of coculture with HD-MSCs (red) and BT-MSCs (blue) in the presence or absence of proper cytokines. CB CD34+ cells cultured alone in the presence or absence of proper cytokines were used as control (black) (HD: n = 3; BT: n = 3). (D) Percentage (left panel) and absolute number (right panel) of human CD45+ cells detected in the peripheral blood of NSG mice 6 weeks after intra tail vein coinfusion of 2.5 × 105 human CB CD34 with 1 × 106 HD- or BT-MSCs. (E) Percentage (left panel) and absolute number (right panel) of human CD45+ cells detected in the peripheral blood of NSG mice at a later time point (12 weeks). (F) Percentage (left panel) and absolute number (right panel) of human T lymphocytes (CD3+) detected in the peripheral blood of NSG mice 12 weeks after transplantation. Mice transplanted with CB CD34+ cells alone were used as controls. Data are mean ± SEM. In all panels, each dot represents an irradiated mouse transplanted with CB CD34+ + HD-MSCs (light blue), CB CD34+ + BT-MSCs (orange), or CB CD34+ (black). P values were determined by Student’s t test (*P < 0.05; **P < 0.001).