Contributions
Abstract: S249
Type: Oral Presentation
Session title: Stem cell biology and microenvironment
Background
In the last decade many studies unraveled the regulation of the bone marrow (BM) niche and hematopoietic stem cells (HSC) in steady state conditions and malignancies, but HSC-niche interactions are still underexplored in inherited disorders. We have recently provided the first demonstration of impaired HSC function caused by an altered BM stromal niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a congenital severe anemia and it is corrected by HSC transplantation from normal donors or by gene therapy with autologous HSC. In addition to the BM stroma, we found altered levels of multiple local and systemic factors, including reduction of systemic thrombopoietin (TPO).
Aims
Further investigation of the BM alterations is pivotal to develop supportive therapies to ameliorate the BM niche and preserve long-term HSC. To this aim, we defined the role of defective TPO on HSC and BM microenvironment.
Methods
Gene expression profiling of HSC and megakaryocytes (Mk) from Hbbth3/+ (th3) BT mice was assessed by RNA-seq analysis. Flow cytometry characterization, in vitro Mk maturation, histological analysis on the BM of BT mice and patients, in vivo biogenesis, half-life and in vitro phagocytosis assays were performed. In vivo stimulation of TPO was evaluated.
Results
Since TPO is a key regulator of both HSC and Mk, we investigated the dual role of TPO defect in BT. RNA-seq profiling revealed a downregulation of TPO signaling and target stemness genes in th3 HSC, including Cdkn1a, Hoxa9 and Hoxb4, negatively affecting HSC function. The decreased TPO causes a reduced commitment of HSC towards the Mk lineage, with under-expression of Mk-biased genes and lower frequency of CD41+CD9high HSC. In vivo stimulation of TPO axis in th3 mice restored the pool of quiescent HSC, thus demonstrating the contribution of defective TPO signaling in altering BT HSC.
Consistently, histopathological analyses of th3 mice showed dysmegakaryopoiesis and this defect was confirmed in BM sections from BT patients. Evaluation of Mk nuclear content revealed a decreased maturation of Mk, with loss of the mature polyploid profile. These findings correlate with a reduced in vivo Plt biogenesis and impaired in vitro differentiation of th3 Mk. Sorted BT Mk showed the downregulation of niche factors, as Pf4, Cxcl12, TnC, relevant for HSC maintenance. We highlighted a reduced expression of extracellular matrix molecules by BT Mk, whereas overexpression of IFN response genes. Since Mk play a direct role on HSC activity, we are investigating by in vivo and imaging studies their contribution to the impaired HSC-niche crosstalk in BT.
We explored the origin of TPO defect: TPO levels fluctuate in response to platelet (Plt) number and its reduction in th3 mice is associated to the increased count of Plt. A negative correlation between Plt and TPO was confirmed in BT patients. In vivo labeling revealed a higher half-life of BT Plt, which accumulate in the circulation because of a reduced clearance by th3 spleen phagocytes, as assessed by phagocytosis assays. Iron overload and extramedullary erythropoiesis in BT spleen might cause the impaired phagocytic activity.
Conclusion
Our results uncovered the underexplored contribution of TPO alteration to HSC and BM niche defects in BT by unraveling the dual role of TPO on HSC and Mk in a condition of chronically reduced stimulation. This research elucidates the multifactorial alterations in the BM microenvironment, with a potential relevance in improving HSC transplantation approaches for BT.
Keyword(s): Hematopoietic stem cell, Megakaryocyte, Thalassemia, Thrombopoietin (TPO)
Abstract: S249
Type: Oral Presentation
Session title: Stem cell biology and microenvironment
Background
In the last decade many studies unraveled the regulation of the bone marrow (BM) niche and hematopoietic stem cells (HSC) in steady state conditions and malignancies, but HSC-niche interactions are still underexplored in inherited disorders. We have recently provided the first demonstration of impaired HSC function caused by an altered BM stromal niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a congenital severe anemia and it is corrected by HSC transplantation from normal donors or by gene therapy with autologous HSC. In addition to the BM stroma, we found altered levels of multiple local and systemic factors, including reduction of systemic thrombopoietin (TPO).
Aims
Further investigation of the BM alterations is pivotal to develop supportive therapies to ameliorate the BM niche and preserve long-term HSC. To this aim, we defined the role of defective TPO on HSC and BM microenvironment.
Methods
Gene expression profiling of HSC and megakaryocytes (Mk) from Hbbth3/+ (th3) BT mice was assessed by RNA-seq analysis. Flow cytometry characterization, in vitro Mk maturation, histological analysis on the BM of BT mice and patients, in vivo biogenesis, half-life and in vitro phagocytosis assays were performed. In vivo stimulation of TPO was evaluated.
Results
Since TPO is a key regulator of both HSC and Mk, we investigated the dual role of TPO defect in BT. RNA-seq profiling revealed a downregulation of TPO signaling and target stemness genes in th3 HSC, including Cdkn1a, Hoxa9 and Hoxb4, negatively affecting HSC function. The decreased TPO causes a reduced commitment of HSC towards the Mk lineage, with under-expression of Mk-biased genes and lower frequency of CD41+CD9high HSC. In vivo stimulation of TPO axis in th3 mice restored the pool of quiescent HSC, thus demonstrating the contribution of defective TPO signaling in altering BT HSC.
Consistently, histopathological analyses of th3 mice showed dysmegakaryopoiesis and this defect was confirmed in BM sections from BT patients. Evaluation of Mk nuclear content revealed a decreased maturation of Mk, with loss of the mature polyploid profile. These findings correlate with a reduced in vivo Plt biogenesis and impaired in vitro differentiation of th3 Mk. Sorted BT Mk showed the downregulation of niche factors, as Pf4, Cxcl12, TnC, relevant for HSC maintenance. We highlighted a reduced expression of extracellular matrix molecules by BT Mk, whereas overexpression of IFN response genes. Since Mk play a direct role on HSC activity, we are investigating by in vivo and imaging studies their contribution to the impaired HSC-niche crosstalk in BT.
We explored the origin of TPO defect: TPO levels fluctuate in response to platelet (Plt) number and its reduction in th3 mice is associated to the increased count of Plt. A negative correlation between Plt and TPO was confirmed in BT patients. In vivo labeling revealed a higher half-life of BT Plt, which accumulate in the circulation because of a reduced clearance by th3 spleen phagocytes, as assessed by phagocytosis assays. Iron overload and extramedullary erythropoiesis in BT spleen might cause the impaired phagocytic activity.
Conclusion
Our results uncovered the underexplored contribution of TPO alteration to HSC and BM niche defects in BT by unraveling the dual role of TPO on HSC and Mk in a condition of chronically reduced stimulation. This research elucidates the multifactorial alterations in the BM microenvironment, with a potential relevance in improving HSC transplantation approaches for BT.
Keyword(s): Hematopoietic stem cell, Megakaryocyte, Thalassemia, Thrombopoietin (TPO)