Contributions
Abstract: S269
Type: Oral Presentation
Session title: Changing the scene on thalassemias
Background
Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating HSCs, with similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from 49f− cell. Recently, in a new study the use of CLEC9A marker highlighted the separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high) within the HSC/MPP compartment.
Aims
Recent evidence showed that the BM microenvironment is perturbed in Bthal. Our aim is to understand how the priming and kinetics of HSC undergoing lineage specification and differentiation are changed under this stress environment.
Methods
Immunophenotype and functional analysis of the lineage commitment allowed to characterize of most primitive Bthal HSC/MPP cells. Gene expression profiling of hematopoietic subpopulations sorted from BM samples of Bthal patients and healthy donors (HDs) delineated the transcriptional networks governing hematopoiesis. We evaluated the expression levels of niche factors involved in the regulation of hematopoiesis/erythropoiesis by ddPCR on RNA purified from Bthal mesenchymal stromal cells. Their protein levels in the BM plasma were analyzed by ELISA. Confirmation experiments are ongoing in vivo in Bthal murine model (Hbbth3/+ strain).
Results
An increased proportion of MPPs was observed in Bthal patients compared to HDs. The Subset1 compartment was actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that resolved My, Ery, and Mk lineage fates, we found a reduction of Ery (CD71+ BAH1-/+) subsets in Bthal samples. By gene expression profiling of Bthal HSC/MPP, we observed down-regulation of TGFß signaling-induced gene expression and enrichment in erythroid signature. The levels of TGFß molecules were altered in Bthal BM micro-environment and treatment of HD Subset 1 cells demonstrates how TGFß/BMP pathway is instrumental for the erythroid cell lineage specification of HSCs. The analysis of molecular pathways driven by TGFß signaling led to the identification of druggable targets with the potential to improve thalassemic hematopoiesis and to normalize erythropoiesis. Ongoing in vivo studies with specific drugs will provide cues to rescue the unbalanced hematopoietic primitive compartment
Conclusion
Overall, these findings show that Bthal HSCs are exiting from the quiescent state towards a preferential erythroid differentiation. The drivers are likely specific niche signals, linked to the TGFß, and chronic BM stimulation or, alternatively, a specific regulation of the ‘erythroid branching’, naturally present in the HSC pool, which is exacerbated by the pathophysiology of the disease.
Keyword(s): Erythroid lineage, Hematopoietic stem cell, TGF-, Thalassemia
Abstract: S269
Type: Oral Presentation
Session title: Changing the scene on thalassemias
Background
Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating HSCs, with similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from 49f− cell. Recently, in a new study the use of CLEC9A marker highlighted the separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high) within the HSC/MPP compartment.
Aims
Recent evidence showed that the BM microenvironment is perturbed in Bthal. Our aim is to understand how the priming and kinetics of HSC undergoing lineage specification and differentiation are changed under this stress environment.
Methods
Immunophenotype and functional analysis of the lineage commitment allowed to characterize of most primitive Bthal HSC/MPP cells. Gene expression profiling of hematopoietic subpopulations sorted from BM samples of Bthal patients and healthy donors (HDs) delineated the transcriptional networks governing hematopoiesis. We evaluated the expression levels of niche factors involved in the regulation of hematopoiesis/erythropoiesis by ddPCR on RNA purified from Bthal mesenchymal stromal cells. Their protein levels in the BM plasma were analyzed by ELISA. Confirmation experiments are ongoing in vivo in Bthal murine model (Hbbth3/+ strain).
Results
An increased proportion of MPPs was observed in Bthal patients compared to HDs. The Subset1 compartment was actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that resolved My, Ery, and Mk lineage fates, we found a reduction of Ery (CD71+ BAH1-/+) subsets in Bthal samples. By gene expression profiling of Bthal HSC/MPP, we observed down-regulation of TGFß signaling-induced gene expression and enrichment in erythroid signature. The levels of TGFß molecules were altered in Bthal BM micro-environment and treatment of HD Subset 1 cells demonstrates how TGFß/BMP pathway is instrumental for the erythroid cell lineage specification of HSCs. The analysis of molecular pathways driven by TGFß signaling led to the identification of druggable targets with the potential to improve thalassemic hematopoiesis and to normalize erythropoiesis. Ongoing in vivo studies with specific drugs will provide cues to rescue the unbalanced hematopoietic primitive compartment
Conclusion
Overall, these findings show that Bthal HSCs are exiting from the quiescent state towards a preferential erythroid differentiation. The drivers are likely specific niche signals, linked to the TGFß, and chronic BM stimulation or, alternatively, a specific regulation of the ‘erythroid branching’, naturally present in the HSC pool, which is exacerbated by the pathophysiology of the disease.
Keyword(s): Erythroid lineage, Hematopoietic stem cell, TGF-, Thalassemia