Inhibition of Fibroblast Growth Factor-23 (FGF-23) As a Novel Strategy to Target Bone and Hematopoietic Stem Cell Niche Defects in Beta-Thalassemia

In the last decade many studies unraveled the bone marrow (BM) niche regulation and crosstalk with hematopoietic stem cells (HSC) in steady state conditions and malignancies, but HSC-niche interactions are still underexplored in hematological inherited disorders. We have recently provided the first demonstration of impaired HSC function caused by an altered BM niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a congenital hemoglobin disorder resulting in severe anemia, ineffective erythropoiesis and multi-organ secondary complications, such as bone defects. It is one of the most globally widespread monogenic diseases, which can be cured by transplantation of HSC from compatible healthy donors or autologous HSC from patients upon gene therapy. Cases of graft failure have been reported, but causes have not been deeply investigated and might include an impaired HSC function and a defective supporting activity of the BM niche, worsened by age and disease progression. We showed that the prolonged residence of HSC into an altered BM stromal niche in BT Hbbth3/+ (th3) mice negatively affects stem cell number, quiescence and self-renewal. Moreover, we demonstrated that correction of HSC-stromal niche crosstalk rescues BT HSC function by in vivo reactivation of parathyroid hormone (PTH) signaling. Consistently with the common finding of osteoporosis in BT patients, we found reduced bone deposition and low levels of PTH also in the murine model. We investigated the potential mechanisms underlying the decreased PTH and bone defect and we focused on the role of fibroblast growth factor-23 (FGF-23). FGF-23 is a systemic hormone mainly secreted by osteocytes, which acts as negative regulator of bone metabolism by inhibiting bone mineralization and PTH production by parathyroid glands. Since FGF-23 is positively modulated by the anemia-related factor erythropoietin (EPO), we hypothesized that the high EPO levels in BT, subsequent to ineffective erythropoiesis, might contribute to increase FGF-23. We measured high levels of circulating FGF-23 in th3 mice (wt vs. th3: 399.7±69.77 vs. 1975±209.3 pg/ml, p<0.01) and also in BT patients (HD vs. THAL: 94.2±3.8 vs. 125.8±9.2 RU/ml, p<0.05). To provide proof of concept data of the causative role of FGF-23 on BT bone and stromal niche defects, we inhibited FGF-23 signaling. FGF-23 inhibition by in vivo administration of FGF-23 blocking peptide rescued the bone defect in th3 mice, by increasing trabecular bone mineral density (th3 vs. th3+FGF23inh: 117.7±3.3 vs. 181.1±6.9 mg/cm3, p<0.0001). Importantly, the treatment restored the frequency of HSC to levels comparable to wild-type controls by expanding the pool of quiescent cells (th3 vs. th3+FGF23inh: 0.03±0.002 vs. 0.07±0.0% on Linneg BM cells, p<0.0001). Consistently, we found increased the expression of key molecules by bone cells, such as Jagged-1 and osteopontin, involved in the functional crosstalk between HSC and the stromal niche. Interestingly, FGF-23 inhibition had also a positive anti-apoptotic effect on the expanded BM erythroid compartment, promoting the maturation of erythroid precursors, as already shown in models of secondary anemias. Preliminary evidence in BT patients showed negative correlations between FGF-23 levels and markers of bone homeostasis (e.g. osteocalcin and vitamin D) and positive correlations with makers of ineffective erythropoiesis (e.g. reticulocytes), thus proposing FGF-23 as the molecule at the crossroads of erythropoiesis and bone metabolism in BT. In vivo studies and molecular analysis in th3 mice and patients' samples will better unravel the causative role of EPO on FGF-23 levels in BT and the negative impact of high FGF-23 on bone mineralization and BM stromal niche-HSC interactions. Our findings uncover an underexplored role of FGF-23 in bone and BM niche defects in BT, as a condition of severe anemia and chronic EPO stimulation. The inhibition of FGF-23 signaling might provide a novel strategy to ameliorate bone compartment and restore HSC-BM niche interactions in BT, with a potential translational relevance in improving HSC transplantation approaches. Disclosures Motta: Sanofi Genzyme: Honoraria. Cappellini:BMS: Honoraria; CRISPR Therapeutics, Novartis, Vifor Pharma: Membership on an entity's Board of Directors or advisory committees; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Inhibition of Fibroblast Growth Factor-23 (FGF-23) Rescues Bone and Hematopoietic Stem Cell Niche Defects in Beta-Thalassemia, Uncovering the Missing Link between Hematopoiesis and Bone

Blood ◽

10.1182/blood-2021-148366 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 572-572

Author(s):

Annamaria Aprile ◽

Laura Raggi ◽

Simona Bolamperti ◽

Mariangela Storto ◽

Isabella Villa ◽

...

Keyword(s):

Research Funding ◽

Fibroblast Growth Factor 23 ◽

Beta Thalassemia ◽

Erythroid Cells ◽

Ineffective Erythropoiesis ◽

Hematopoietic Stem ◽

Bm Niche ◽

Fgf 23

Abstract The bone marrow (BM) niche regulation and interactions with hematopoietic stem cells (HSC) have been extensively studied in steady state conditions and malignancies, but are still underexplored in hematological inherited disorders. We provided the first demonstration of impaired HSC function caused by an altered BM niche in a non-malignant disease, beta-thalassemia (BT) (Aprile et al., Blood 2020). BT is a globally widespread congenital hemoglobin disorder, resulting in severe anemia, ineffective erythropoiesis and multi-organ secondary complications, including bone alterations. Correction of the genetic defect is achieved by transplantation of HSC from healthy donors or autologous HSC from patients upon gene therapy. Since the quality and the engraftment of HSC depend on the BM microenvironment, niche-HSC crosstalk plays a crucial role for transplantation outcome. During the analysis of different components of the niche, we found reduced bone density in BT th3 mice, along with a defective HSC supporting activity by the BM stromal niche. Interestingly, osteoporosis is a constant hallmark in BT patients. We investigated the mechanisms underlying bone and HSC niche defects focusing on the role of fibroblast growth factor-23 (FGF-23), a hormone mainly secreted by osteocytes, but also by erythroid cells, which negatively modulates bone metabolism. Since FGF-23 is stimulated by the anemia-related factor erythropoietin (EPO), we hypothesized that the high EPO levels in BT might contribute to increase FGF-23, potentially affecting bone and BM niche homeostasis. We found high levels of circulating FGF-23 in th3 mice (wt vs. th3: 290.5±27.3 vs. 1823±136.1 pg/ml, p<0.0001) and in BT patients (HD vs. THAL: 94.7±1.8 vs. 117.2±5.3 RU/ml, p<0.01), associated to its increased expression by bone and BM erythroid cells. In vivo neutralization of EPO axis was sufficient to normalize FGF-23 levels (th3 vs. th3+anti-EPO: 1591±162.2 vs. 496.1±33.3 pg/ml, p<0.001), thus demonstrating the causative role of EPO. EPO stimulation and signaling inhibition strategies highlighted the involvement of Erk1/2 and Stat5 pathways in activating Fgf-23 transcription in bone and BM erythroid cells, respectively. To provide proof of concept data on the contribution of FGF-23 to BT bone and stromal niche alterations, we inhibited FGF-23 signaling. In vivo administration of FGF-23 blocking peptide rescued the trabecular bone density in th3 mice (th3 vs. th3+FGF23inh: 138.2±4.9 vs. 166.9±5.2 mg/cm 3, p<0.01). Short-term inhibition treatment (38 hours) was sufficient to enhance bone mineralization by acting on Alkaline Phosphatase and on the expression of the main regulators of mineralization Dmp1 and Mepe by osteocyte, whereas long-term administration (12 days) restored also osteoblast number and bone deposition. Importantly, FGF-23 inhibition normalized the expression of key niche molecules, such as Jagged-1 and osteopontin, involved in the functional crosstalk between HSC and the stromal niche. Consistently, the treatment restored the frequency of th3 HSC by expanding the pool of quiescent cells (th3 vs. th3+FGF23inh: 0.026 vs. 0.045% on Lin neg BM cells, p<0.01). FGF-23 inhibition had also a positive anti-apoptotic effect on the expanded BM erythroid compartment (th3 vs. th3+FGF23inh: 61.6±1.3 vs. 51.1±2.1% of BM Ter119 + cells, p<0.001), promoting the maturation of early erythroid precursors (th3 vs. th3+FGF23inh: 8.5±1 vs. 16.4±1.1% of BM Pro-Erythroblasts, p<0.0001), as already shown in models of secondary anemias. Evidence in BT patients showed negative correlations between FGF-23 levels and markers of bone homeostasis (e.g. osteocalcin R 2=0.88, p<0.05) and positive correlations with makers of ineffective erythropoiesis (e.g. circulating reticulocytes R 2=0.83, p<0.05), thus positioning FGF-23 as the molecule at the crossroads of erythropoiesis and bone metabolism in BT. Our findings uncover an underexplored role of FGF-23 in bone and BM niche defects in a primary anemia, as a condition of chronic EPO stimulation, and propose FGF-23 as the missing link between hematopoiesis and bone regulation. The inhibition of FGF-23 signaling might provide a novel strategy to ameliorate bone compartment and restore HSC-BM niche interactions in BT by a 'two birds with one stone' approach, with a potential translational relevance in improving HSC transplantation and gene therapy. Disclosures Cappellini: Celgene: Consultancy, Research Funding; Vifor: Consultancy; La Jolla: Research Funding; Protagonist Therapeutics: Research Funding; IONIS Pharmaceuticals: Consultancy; CRISPR Therapeutics: Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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“Phosphatonins” and the regulation of phosphorus homeostasis

AJP Renal Physiology ◽

10.1152/ajprenal.00072.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. F1170-F1182 ◽

Cited By ~ 143

Author(s):

Theresa J. Berndt ◽

Susan Schiavi ◽

Rajiv Kumar

Keyword(s):

Bone Mineralization ◽

Fibroblast Growth Factor 23 ◽

Enzyme Regulation ◽

Physiological Role ◽

Phosphate Transport ◽

Biological Properties ◽

Nucleotide Metabolism ◽

Fgf 23

Phosphate ions are critical for normal bone mineralization, and phosphate plays a vital role in a number of other biological processes such as signal transduction, nucleotide metabolism, and enzyme regulation. The study of rare disorders associated with renal phosphate wasting has resulted in the discovery of a number of proteins [fibroblast growth factor 23 (FGF-23), secreted frizzled related protein 4 (sFRP-4), matrix extracellular phosphoglycoprotein, and FGF 7 (FGF-7)] that decrease renal sodium-dependent phosphate transport in vivo and in vitro. The “phosphatonins,” FGF-23 and sFRP-4, also inhibit the synthesis of 1α,25-dihydroxyvitamin D, leading to decreased intestinal phosphate absorption and further reduction in phosphate retention by the organism. In this review, we discuss the biological properties of these proteins, alterations in their concentrations in various clinical disorders, and their possible physiological role.

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The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Nature Communications ◽

10.1038/s41467-021-24418-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Brianna J. Klein ◽

Anagha Deshpande ◽

Khan L. Cox ◽

Fan Xuan ◽

Mohamad Zandian ◽

...

Keyword(s):

Critical Role ◽

Cellular Transformation ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Nucleosome Core ◽

Stem And Progenitor Cells ◽

Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

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The Act of Controlling Adult Stem Cell Dynamics: Insights from Animal Models

Biomolecules ◽

10.3390/biom11050667 ◽

2021 ◽

Vol 11 (5) ◽

pp. 667

Author(s):

Meera Krishnan ◽

Sahil Kumar ◽

Luis Johnson Kangale ◽

Eric Ghigo ◽

Prasad Abnave

Keyword(s):

Stem Cells ◽

Animal Models ◽

Adult Stem Cells ◽

The Body ◽

In Vivo Studies ◽

Self Renewal ◽

Hematopoietic Stem ◽

Organ Systems

Adult stem cells (ASCs) are the undifferentiated cells that possess self-renewal and differentiation abilities. They are present in all major organ systems of the body and are uniquely reserved there during development for tissue maintenance during homeostasis, injury, and infection. They do so by promptly modulating the dynamics of proliferation, differentiation, survival, and migration. Any imbalance in these processes may result in regeneration failure or developing cancer. Hence, the dynamics of these various behaviors of ASCs need to always be precisely controlled. Several genetic and epigenetic factors have been demonstrated to be involved in tightly regulating the proliferation, differentiation, and self-renewal of ASCs. Understanding these mechanisms is of great importance, given the role of stem cells in regenerative medicine. Investigations on various animal models have played a significant part in enriching our knowledge and giving In Vivo in-sight into such ASCs regulatory mechanisms. In this review, we have discussed the recent In Vivo studies demonstrating the role of various genetic factors in regulating dynamics of different ASCs viz. intestinal stem cells (ISCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs), and epidermal stem cells (Ep-SCs).

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Fetal Hemoglobin Rescues Ineffective Erythropoiesis in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-137477 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15

Author(s):

Sara El Hoss ◽

Sylvie Cochet ◽

Auria Godard ◽

Hongxia Yan ◽

Michaël Dussiot ◽

...

Keyword(s):

Cell Death ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Ineffective Erythropoiesis ◽

Hypoxic Conditions ◽

Cd34 Cells ◽

F Cells

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a point mutation in the β globin gene resulting in a Glu-to-Val substitution at the 6th position of the β globin protein. The resulting abnormal hemoglobin (HbS) polymerizes under hypoxic conditions driving red blood cell (RBC) sickling (Pauling et al., 1949). While pathobiology of circulating RBCs has been extensively analyzed in SCD, erythropoiesis is surprisingly poorly documented. In β-thalassemia, ineffective erythropoiesis is characterized by high levels of apoptotic erythroblasts during the late stages of terminal differentiation, due to an accumulation of free β-globin chains (Arlet et al., 2016). Ineffective erythropoiesis is the major cause of anemia in β-thalassemia patients. In contrast, a marked decrease in life span of circulating red cells, a feature of sickle red cells, is considered to be the major determinant of chronic anemia in SCD. It is generally surmised that ineffective erythropoiesis contributes little to anemia. The bone marrow environment has been well documented to be hypoxic (0.1 to 6% O2) (Mantel et al., 2015). As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer formation in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques. In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1). In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2). In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction. Disclosures No relevant conflicts of interest to declare.

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Rac signaling in osteoblastic cells is required for normal bone development but is dispensable for hematopoietic development

Blood ◽

10.1182/blood-2011-07-368753 ◽

2012 ◽

Vol 119 (3) ◽

pp. 736-744 ◽

Cited By ~ 16

Author(s):

Steven W. Lane ◽

Serena De Vita ◽

Kylie A. Alexander ◽

Ruchan Karaman ◽

Michael D. Milsom ◽

...

Keyword(s):

Bone Growth ◽

Bone Development ◽

Long Bone ◽

Perivascular Niche ◽

Hematopoietic Stem ◽

Hsc Niche ◽

Rac1 Gtpase

Abstract Hematopoietic stem cells (HSCs) interact with osteoblastic, stromal, and vascular components of the BM hematopoietic microenvironment (HM) that are required for the maintenance of long-term self-renewal in vivo. Osteoblasts have been reported to be a critical cell type making up the HSC niche in vivo. Rac1 GTPase has been implicated in adhesion, spreading, and differentiation of osteoblast cell lines and is critical for HSC engraftment and retention. Recent data suggest a differential role of GTPases in endosteal/osteoblastic versus perivascular niche function. However, whether Rac signaling pathways are also necessary in the cell-extrinsic control of HSC function within the HM has not been examined. In the present study, genetic and inducible models of Rac deletion were used to demonstrate that Rac depletion causes impaired proliferation and induction of apoptosis in the OP9 cell line and in primary BM stromal cells. Deletion of Rac proteins caused reduced trabecular and cortical long bone growth in vivo. Surprisingly, HSC function and maintenance of hematopoiesis in vivo was preserved despite these substantial cell-extrinsic changes. These data have implications for therapeutic strategies to target Rac signaling in HSC mobilization and in the treatment of leukemia and provide clarification to our evolving concepts of HSC-HM interactions.

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Adenosine from Niche-Associated Tregs Maintains Hematopoietic Stem Cell Quiescence

Blood ◽

10.1182/blood.v130.suppl_1.91.91 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 91-91

Author(s):

Yuichi Hirata ◽

Kazuhiro Furuhashi ◽

Hiroshi Ishi ◽

Hao-Wei Li ◽

Sandra Pinho ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pool Size ◽

Immune Privilege ◽

Haematopoietic Stem Cell ◽

Hematopoietic Stem ◽

Conditional Deletion ◽

Radiation Induced

Abstract A crucial player in immune regulation, FoxP3+ regulatory T cells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. For example, specific subsets of Tregs in the adipose tissue control metabolic indices; muscle Tregs potentiate muscle repair, and lung Tregs prevent tissue damage. These studies, together with a previous finding that Tregs are enriched in the primary site for hematopoiesis, the bone marrow (BM), prompted us to examine whether there is a special Treg population which controls hematopoietic stem cells (HSCs). We showed that HSCs within the BM were frequently adjacent to distinctly activated FoxP3+ Tregs which highly expressed an HSC marker, CD150. Moreover, specific reduction of BM Tregs achieved by conditional deletion of CXCR4in Tregs, increased reactive oxygen species (ROSs) in HSCs. The reduction of BM Tregs further induced loss of HSC quiescence and increased HSC numbers in a manner inhibited by anti-oxidant treatment. Additionally, this increase in HSC numbers in mice lacking BM Tregs was reversed by transfer of CD150high BM Tregs but not of CD150low BM Tregs. These results indicate that CD150high niche-associated Tregs maintain HSC quiescence and pool size by preventing oxidative stress. We next sought to identify an effector molecule of niche Tregs which regulates HSCs. Among molecules highly expressed by niche Tregs, we focused on CD39 and CD73, cell surface ecto-enzymes which are required for generation of extracellular adenosine, because 1) CD39highCD73high cells within the BM were prevalent among CD150high Tregs and 2) HSCs highly expressed adenosine 2a receptors (A2AR). We showed that both conditional deletion of CD39 in Tregs and in vivo A2AR antagonist treatment induced loss of HSC quiescence and increased HSC pool size in a ROS-dependent manner, which is consistent with the findings in mice lacking BM Tregs. In addition, transfer of CD150high BM Tregs but not of CD150low BM Tregs reversed the increase in HSC numbers in FoxP3cre CD39flox mice. The data indicate that niche Treg-derived adenosine regulates HSCs. We further investigated the protective role of niche Tregs and adenosine in radiation injury against HSCs. Conditional deletion of CD39 in Tregs increased radiation-induced HSC apoptosis. Conversely, transfer of as few as 15,000 CD150high BM Tregs per B6 mouse (iv; day-1) rescued lethally-irradiated (9.5Gy) mice by preventing hematopoiesis failure. These observations indicate that niche Tregs protect HSCs from radiation stress. Finally, we investigated the role of niche Tregs in allogeneic (allo-) HSC transplantation. Our previous study showed that allo-hematopoietic stem and progenitor cells but not allo-Lin+ cells persisted in the BM of non-conditioned immune-competent recipients without immune suppression in a manner reversed by systemic Treg depletion1. This observation suggests that HSCs have a limited susceptibility to immune attack, as germline and embryonic stem cells are located within immune privileged sites. Because the study employed systemic Treg depletion and non-conditioned recipients, it remains unknown whether niche Tregs play a critical role in immune privilege of HSCs and in allo-HSC engraftment following conditioning. We showed here that the reduction of BM Tregs and conditional deletion of CD39 in Tregs abrogated allo-HSC persistence in non-conditioned immune-competent mice as well as allo-HSC engraftment following nonmyeloablative conditioning. Furthermore, transfer of CD150high BM Tregs but not of other Tregs (15,000 cells/recipient; day -2) significantly improved allo-HSC engraftment. This effect of niche Treg transfer is noteworthy given that 1-5 million Tregs per mouse were required in case of transfer of spleen or lymph node Tregs. These observations suggest that niche Tregs maintain immune privilege of HSCs and promote allo-HSC engraftment. In summary, our studies identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, numbers, stress response, engraftment, and immune privilege, further highlighting potential clinical utility of niche Treg transfer in radiation-induced hematopoiesis failure and in allo-HSC engraftment (under revision in Cell Stem Cell). 1 Fujisaki, J. et al. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature474, 216-219, doi:10.1038/nature10160 (2011). Disclosures No relevant conflicts of interest to declare.

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Abstract 5: Neovascularization By Substance-p- Mobilized Epc And Msc In Vitro And In Vivo

Circulation Research ◽

10.1161/res.115.suppl_1.5 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Hyun Sook Hong ◽

Suna Kim ◽

Youngsook Son

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Substance P ◽

Network Formation ◽

Skin Wound ◽

Hematopoietic Stem ◽

Vessel Structure

Bone marrow stem cells, especially, endothelial precursor cells (EPC), mesenchymal stem cells (MSC) or hematopoietic stem cell (HSC) are expected as reparative cells for the repair of a variety of tissue damages such as stroke and myocardial infarction, even though their role in the repair is not demonstrated. This report was investigated to find a role of Substance-p (SP) as a reparative agent in the tissue repair requiring EPC and MSC. In order to examine EPC (EPC SP ) and MSC (MSC SP ) mobilized by SP, we injected SP intravenously for consecutive 2 days and saline was injected as a vehicle. At 3 post injection, peripheral blood (PB) was collected.To get mesenchymal stem cells or endothelial progenitor cells, MNCs were incubated in MSCGM or EGM-2 respectively for 10 days. Functional characteristics of the EPC SP were proven by the capacity to form endothelial tubule network in the matrigel in vitro and in the matrigel plug assay in vivo. In contrast, MSC SP did not form a tube-like structure but formed a pellet-structure on matrigel. However, when both cells were premixed before the matrigel assay, much longer and branched tubular network was formed, in which a-SMA expressing MSC SP were decorating outside of the endothelial tube, especially enriched at the bifurcating point. MSC SP may contribute and reinforce elaborate vascular network formation in vivo by working as pericyte-like cells. Thus, the EPC SP and MSC SP were labeled with PKH green and PKH red respectively and their tubular network was examined. Well organized tubular network was formed, which was covered by PKH green labeled cells and was decorated in a punctate pattern by PKH red labeled cells. In order to investigate the role of EPC SP and MSC SP specifically in vivo, rabbit EPC SP and MSC SP were transplanted to full thickness skin wound. The vessel of EPC SP -transplanted groups was UEA-lectin+, which was not covered with a-SMA+ pericytes but EPC SP + MSC SP -transplanted groups showed, in part, a-SMA+ pericyte-encircled UEA-lectin+ vessels. This proved the specific role of MSC SP as pericytes. From these data, we have postulated that the collaboration of MSC and EPC is essential for normal vessel structure and furthermore, accelerated wound healing as ischemia diseases, which can be stimulated through by SP injection.

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Successful in vivo purging of CD34-containing peripheral blood harvests in mantle cell and indolent lymphoma: evidence for a role of both chemotherapy and rituximab infusion

Blood ◽

10.1182/blood.v96.3.864 ◽

2000 ◽

Vol 96 (3) ◽

pp. 864-869 ◽

Cited By ~ 158

Author(s):

Michele Magni ◽

Massimo Di Nicola ◽

Liliana Devizzi ◽

Paola Matteucci ◽

Fabrizio Lombardi ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Follicular Lymphoma ◽

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

Bone Marrow Involvement ◽

Hematopoietic Stem ◽

Mantle Cell

Abstract Elimination of tumor cells (“purging”) from hematopoietic stem cell products is a major goal of bone marrow–supported high-dose cancer chemotherapy. We developed an in vivo purging method capable of providing tumor-free stem cell products from most patients with mantle cell or follicular lymphoma and bone marrow involvement. In a prospective study, 15 patients with CD20+ mantle cell or follicular lymphoma, bone marrow involvement, and polymerase chain reaction (PCR)–detectable molecular rearrangement received 2 cycles of intensive chemotherapy, each of which was followed by infusion of a growth factor and 2 doses of the anti-CD20 monoclonal antibody rituximab. The role of rituximab was established by comparison with 10 control patients prospectively treated with an identical chemotherapy regimen but no rituximab. The CD34+ cells harvested from the patients who received both chemotherapy and rituximab were PCR-negative in 93% of cases (versus 40% of controls;P = .007). Aside from providing PCR-negative harvests, the chemoimmunotherapy treatment produced complete clinical and molecular remission in all 14 evaluable patients, including all 6 with mantle cell lymphoma (versus 70% of controls). In vivo purging of hematopoietic progenitor cells can be successfully accomplished in most patients with CD20+ lymphoma, including mantle cell lymphoma. The results depended on the activity of both chemotherapy and rituximab infusion and provide the proof of principle that in vivo purging is feasible and possibly superior to currently available ex vivo techniques. The high short-term complete-response rate observed suggests the presence of a more-than-additive antilymphoma effect of the chemoimmunotherapy combination used.

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Hematopoietic stem cell function in β-thalassemia is impaired and is rescued by targeting the bone marrow niche

Blood ◽

10.1182/blood.2019002721 ◽

2020 ◽

Vol 136 (5) ◽

pp. 610-622 ◽

Cited By ~ 6

Author(s):

Annamaria Aprile ◽

Alessandro Gulino ◽

Mariangela Storto ◽

Isabella Villa ◽

Stefano Beretta ◽

...

Keyword(s):

Bone Marrow ◽

Cross Talk ◽

Cell Function ◽

Active Role ◽

Common Finding ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Bm Niche ◽

Hsc Niche

Abstract 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.

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