Annamaria Aprile

Many studies unraveled the reciprocal regulation between hematopoietic stem cells (HSC) and the bone marrow (BM) niche by using transgenic knock-out or reporter mice, often overlooking the pathophysiological relevance and the existence of compensatory mechanisms. Here, we exploit the th3 murine model of a genetic anemia, beta-thalassemia (BThal), to investigate the dual impact of chronically reduced thrombopoietin (TPO) production on HSC and megakaryocytic BM niche. Decreased systemic TPO downregulates stemness programs in HSC and impairs their commitment towards megakaryocytes (Mk). We uncover dysmegakaryopoiesis in the BM from BThal patients and mice with defective Mk maturation and reduced production of niche factors, altering the HSC-niche crosstalk. Notably, in vivo stimulation of the TPO signaling restores HSC quiescence, Mk maturation and niche support. These findings demonstrate that TPO regulates HSC through both direct and niche-mediated routes, underscoring the value of using a genetic disease model to disentangle general molecular mechanisms.

Iron is an essential element for most cellular processes, and recent evidence highlighted its role in regulating the function of hematopoietic stem cells (HSCs). Abnormal iron levels affect HSC quiescence and self-renewal; however, the mechanism by which iron overload (IO) influences HSC function is still unknown. Here, we show that intracellular IO impairs mitochondrial fitness and bioenergetics, inducing metabolic rewiring. In thalassemic mice, as a model of chronic IO, HSCs accumulate elevated mitochondrial reactive oxygen species (mtROS), low mitochondrial membrane potential, and reduced oxidative phosphorylation. Mitochondrial defects are confirmed in 2 other models of IO, sickle cell disease and iron-loaded wild-type mice, and in vivo iron reduction rescues HSC mitochondria. IO HSCs are highly proliferating and, in the presence of damaged mitochondria, rely on glycolysis for energy production. Notably, restoration of mitochondrial function by targeting in vivo mtROS improved the quiescence and self-renewal of IO HSCs. Our results unravel the critical interplay between iron, ROS, and mitochondrial activity in HSCs, revealing that IO shapes HSC metabolic programs.

The hematopoietic stem cell and multipotent progenitor (HSC/MPP) pool dynamically responds to stress to adapt blood output to specific physiological demands. In β-thalassemia (Bthal), severe anemia and ineffective erythropoiesis generate expansion of erythroid precursors and a chronic stress status in the bone marrow (BM) microenvironment. However, the response to the BM altered status at the level of the HSC/MPP compartment in terms of lineage commitment has not been investigated. Bulk and single-cell RNA-sequencing reveal that Bthal HSCs/MPPs are expanded and activated with enhanced priming along the whole Ery differentiation trajectory. Consistently, HSC/MPP showed an altered TGFβ expression and autophagy transcriptional signatures along with a declined dormancy state. We discovered that the altered TGFβ signaling fosters the Ery potential of HSCs by reducing their autophagic levels, and in vivo stimulation of autophagy is sufficient to rescue the imbalance of the HSC compartment. Our findings identify the interplay between TGFβ and HSC autophagy as a key driver in the context of non-malignant hematopoiesis.

Clinical evidence highlights a relationship between the blood and the bone, but the underlying mechanism linking these two tissues is not fully elucidated. Here, we used β-thalassemia as a model of congenital anemia with bone and bone marrow (BM) niche defects. We demonstrate that fibroblast growth factor 23 (FGF23) is increased in patients and mice with β-thalassemia because erythropoietin induces FGF23 overproduction in bone and BM erythroid cells via ERK1/2 and STAT5 pathways. We show that in vivo inhibition of FGF23 signaling by carboxyl-terminal FGF23 peptide is a safe and efficacious therapeutic strategy to rescue bone mineralization and deposition in mice with β-thalassemia, normalizing the expression of niche factors and restoring hematopoietic stem cell (HSC) function. FGF23 may thus represent a molecular link connecting anemia, bone, and the HSC niche. This study provides a translational approach to targeting bone defects and rescuing HSC niche interactions, with potential clinical relevance for improving HSC transplantation and gene therapy for hematopoietic disorders.

In the last decade, research on pathophysiology and therapeutic solutions for β-thalassemia (BThal) and sickle cell disease (SCD) has been mostly focused on the primary erythroid defect, thus neglecting the study of hematopoietic stem cells (HSCs) and bone marrow (BM) microenvironment. The quality and engraftment of HSCs depend on the BM microenvironment, influencing the outcome of HSC transplantation (HSCT) both in allogeneic and in autologous gene therapy settings. In BThal and SCD, the consequences of severe anemia alter erythropoiesis and cause chronic stress in different organs, including the BM. Here, we discuss the recent findings that highlighted multiple alterations of the BM niche in BThal and SCD. We point out the importance of improving our understanding of HSC biology, the status of the BM niche, and their functional crosstalk in these disorders towards the novel concept of combined therapies by not only targeting the genetic defect, but also key players of the HSC–niche interaction in order to improve the clinical outcomes of transplantation.

Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche that tune hematopoiesis at steady state and in hematologic disorders. To understand HSC-niche interactions in altered nonmalignant homeostasis, we selected β-thalassemia, a hemoglobin disorder, as a paradigm. In this severe congenital anemia, alterations secondary to the primary hemoglobin defect have a potential impact on HSC-niche cross talk. We report that HSCs in thalassemic mice (th3) have an impaired function, caused by the interaction with an altered BM niche. The HSC self-renewal defect is rescued after cell transplantation into a normal microenvironment, thus proving the active role of the BM stroma. Consistent with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. In vivo activation of PTH signaling through the reestablished Jagged1 and osteopontin levels correlated with the rescue of the functional pool of th3 HSCs by correcting HSC-niche cross talk. Reduced HSC quiescence was confirmed in thalassemic patients, along with altered features of the BM stromal niche. Our findings reveal a defect in HSCs in β-thalassemia induced by an altered BM microenvironment and provide novel and relevant insight for improving transplantation and gene therapy approaches.

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.

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (β⁺) or absent (β⁰) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life¹. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease². Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß⁰ or severe ß⁺ mutations in a phase 1/2 trial (NCT02453477) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6–76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10–1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.

For decades, bone marrow (BM) has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for transplants following myeloablative conditioning. At present, mobilized peripheral blood stem cells are commonly used for transplantation, particularly in the autologous setting.[1][1