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Mesenchymal stromal cells (MSCs) have been employed in vitroto support hematopoietic stem and progenitor cell (HSPC) expansion and in vivoto promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro. The use of BM-MSCs resulted in superior human engraftment and increased clonal output of GE-HSPCs contributing to the early phase of hematological reconstitution in the peripheral blood of transplanted mice. In conclusion, our work poses the biological bases for a novel clinical use of BM-MSCs to promote engraftment of GE-HSPCs and improve their transplantation outcome.

Marktel: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding.

Bone marrow (BM) contains a population of mesenchymal stromal cells (MSC) that, together with endothelial cells and osteoblasts, provide a specific microenvironment to support hematopoietic stem cell homeostasis. Beta-thalassemia (BT) is an hereditary blood disorder characterized by reduced or absent synthesis of hemoglobin beta-chains. Data on the mesenchymal compartment in BT patients are scarce. We isolated and characterized MSCs from BT and healthy donor (HD) BM samples. BT-MSCs showed a reduced clonogenic capacity, delay in colony formation, lower numbers of CFU-Fs and longer population doubling time. Similarly, we observed an altered differentiation capacity into adipocytes and osteoblasts. Both HD- and BT-MSCs express the canonical mesenchymal markers. On the contrary, the expression of CD146 and CD271 was extremely reduced in BT-MSCs, indicating a pauperization of the most primitive stem cell pool. We analyzed the expression of genes involved in the crosstalk between MSCs and HSCs and found a reduced expression of Cxcl12, SCFand Angp1in BT-MSCs. Several genes relevant for MSC functionality were also downregulated in BT-MSCs. We demonstrated that MSCs are able to uptake and store iron. ROS levels were increased in BT-MSCs possibly due to altered anti-oxidant response and iron overload associated with blood transfusions. Importantly, higher level of ROS and reduced expression of primitive markers were observed in HD-MSCs cultured in presence of iron, underlying the importance of iron level for normal MSC function. In conclusion, we showed an impairment in the mesenchymal niche of BT-BM possibly associated with prolonged iron exposure.