Contributions
Abstract: S845
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 12:15 - 12:30
Location: Room K2
Background
Bone marrow (BM) contains a population of mesenchymal stromal cells (MSCs) that, together with endothelial cells and osteoblasts, provide a specific microenvironment to support hematopoietic stem cell (HSC) homeostasis. Beta-thalassemia (BT) is a hereditary blood disorder characterized by reduced or absent synthesis of hemoglobin beta-chains amenable to allogeneic HSC transplantation and HSC-gene therapy (HSC-GT). Data on the mesenchymal compartment in BT patients are scarce.
Aims
Aim of this work is to characterize the mesenchymal compartment in BT patients before transplant procedures and to test whether these cells are functionally altered in their ability to support HSC.
Methods
We isolated MSCs from the BM of 12 BT patients (9 pediatric and 3 adults) and age-matched healthy donor (HD: 1:1). MSCs were characterized for their classical properties (clonogenic, proliferative and differentiation capacity, immunophenotype analyses) and for their functional properties in terms of BM niche supportive functions and response to iron overload.
Results
BT-MSCs showed a reduced clonogenic capacity (CFU-Fs), delay in colony formation and longer population doubling time. Similarly, we observed an altered differentiation capacity into adipocytes and osteoblasts both by immunohystochemical staining and RT-qPCR. Both HD- and BT-MSCs expressed canonical mesenchymal markers (positive for CD105, CD90, CD73; negative for hematopoietic 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 measured a higher iron content in the BT BM microenvironment; moreover, we demonstrated that MSCs expressed iron transporters (TFR1, ZIP14, ZIP18, DMT1) and were able to uptake iron. Iron overload correlated with increased ROS level in BT-MSCs and robust decrease of primitive MSCs. We showed that ROS levels were increased possibly due to altered anti-oxidant response caused by prolonged iron exposure associated with chronic blood transfusions. Indeed, at basal level BT-MSCs showed a reduced expression of anti-oxidant genes, which were not properly induced in response to iron. Moreover, we found that the expression of genes involved in the crosstalk between MSCs and HSCs (Cxcl12, SCF and Angp1) was reduced in BT-MSCs, leading to an altered capacity to attract CD34+ hematopoietic stem progenitors cells (HSPC) in transwell migration assays and impaired capability to maintain primitive HSPCs in in vitro 2D co-culture models. We demonstrated that iron dependent demethylases were more active in BT-MSCs, leading to an epigenetic remodeling of BT-MSCs associated with an altered MSC functionality.
Conclusion
In conclusion, we showed an impairment in the mesenchymal niche of BT-BM possibly associated with prolonged iron exposure and reduced anti-oxidative response. This underlines the importance of iron level for normal MSC function. Whether the ability of MSC to up-take iron represents a mechanism of protection for the BM niche and how the BT stromal niche impairment influences engraftment and HSC support after allogeneic HSC transplantation and HSC-GT is currently being investigated.
Session topic: 28. Thalassemias
Keyword(s): inflammation, iron overload, Mesenchymal cells, Thalassemia
Abstract: S845
Type: Oral Presentation
Presentation during EHA23: On Saturday, June 16, 2018 from 12:15 - 12:30
Location: Room K2
Background
Bone marrow (BM) contains a population of mesenchymal stromal cells (MSCs) that, together with endothelial cells and osteoblasts, provide a specific microenvironment to support hematopoietic stem cell (HSC) homeostasis. Beta-thalassemia (BT) is a hereditary blood disorder characterized by reduced or absent synthesis of hemoglobin beta-chains amenable to allogeneic HSC transplantation and HSC-gene therapy (HSC-GT). Data on the mesenchymal compartment in BT patients are scarce.
Aims
Aim of this work is to characterize the mesenchymal compartment in BT patients before transplant procedures and to test whether these cells are functionally altered in their ability to support HSC.
Methods
We isolated MSCs from the BM of 12 BT patients (9 pediatric and 3 adults) and age-matched healthy donor (HD: 1:1). MSCs were characterized for their classical properties (clonogenic, proliferative and differentiation capacity, immunophenotype analyses) and for their functional properties in terms of BM niche supportive functions and response to iron overload.
Results
BT-MSCs showed a reduced clonogenic capacity (CFU-Fs), delay in colony formation and longer population doubling time. Similarly, we observed an altered differentiation capacity into adipocytes and osteoblasts both by immunohystochemical staining and RT-qPCR. Both HD- and BT-MSCs expressed canonical mesenchymal markers (positive for CD105, CD90, CD73; negative for hematopoietic 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 measured a higher iron content in the BT BM microenvironment; moreover, we demonstrated that MSCs expressed iron transporters (TFR1, ZIP14, ZIP18, DMT1) and were able to uptake iron. Iron overload correlated with increased ROS level in BT-MSCs and robust decrease of primitive MSCs. We showed that ROS levels were increased possibly due to altered anti-oxidant response caused by prolonged iron exposure associated with chronic blood transfusions. Indeed, at basal level BT-MSCs showed a reduced expression of anti-oxidant genes, which were not properly induced in response to iron. Moreover, we found that the expression of genes involved in the crosstalk between MSCs and HSCs (Cxcl12, SCF and Angp1) was reduced in BT-MSCs, leading to an altered capacity to attract CD34+ hematopoietic stem progenitors cells (HSPC) in transwell migration assays and impaired capability to maintain primitive HSPCs in in vitro 2D co-culture models. We demonstrated that iron dependent demethylases were more active in BT-MSCs, leading to an epigenetic remodeling of BT-MSCs associated with an altered MSC functionality.
Conclusion
In conclusion, we showed an impairment in the mesenchymal niche of BT-BM possibly associated with prolonged iron exposure and reduced anti-oxidative response. This underlines the importance of iron level for normal MSC function. Whether the ability of MSC to up-take iron represents a mechanism of protection for the BM niche and how the BT stromal niche impairment influences engraftment and HSC support after allogeneic HSC transplantation and HSC-GT is currently being investigated.
Session topic: 28. Thalassemias
Keyword(s): inflammation, iron overload, Mesenchymal cells, Thalassemia