Mouse Bone Marrow Transplantation Model Research Articles

Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease

To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

CD155 is essential for skeletal muscle regeneration by regulating satellite cell proliferation and differentiation.

CD155, a member of the immunoglobulin superfamily, is closely related to cell proliferation, adhesion, and migration. CD155 is overexpressed on the surface of cancer cells to promote cell proliferation and is upregulated in damaged tissues as a stress-induced molecule. The process of skeletal muscle regeneration after injury is complex and involves injurious stimulation and subsequent satellite cell proliferation. However, the role of CD155 in this process remains unelucidated. This study aimed to explore the role of CD155 in injured skeletal muscle regeneration and to clarify its effect on satellite cell proliferation and differentiation. Here, quantitative real-time polymerase chain reaction (RT-qPCR) and immunofluorescence results indicated that CD155 expression in satellite cells increased after skeletal muscle injury. CD155 knockout in mice impaired the regeneration of skeletal muscle. A bone marrow transplantation mouse model was constructed and revealed that CD155 on skeletal muscle tissues, not immune cells, affected muscle regeneration. Invitro, CD155 knockdown in myoblasts inhibited their proliferation and differentiation. The transcriptomic analysis also indicated that CD155 absence can impair the biological proliferation and differentiation process of myoblasts. Our research demonstrates that CD155 directly promotes injured muscle regeneration by regulating satellite cell proliferation and differentiation, which may be a potential therapeutic molecule for skeletal muscle injury.

Novel Anti-TMPRSS6 Monoclonal Antibody Provides a Potential Therapeutic Approach for the Treatment of Polycythemia Vera

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) triggered by activating mutations in JAK2, an important kinase in the principal signaling pathway of the erythropoietin receptor. Most of the clinical manifestations of PV are caused by increased erythrocyte count, leading to increased risk of pulmonary hypertension and thrombosis. The mainstay of therapy for PV is therapeutic phlebotomy to reduce the hematocrit (HCT) level and minimize the risk of thrombosis, however, the effect of phlebotomy is transient until patients become iron deficient. Therefore, systemic iron restriction could be effective in treating PV patients. Transmembrane serine protease 6 (TMPRSS6) is a negative regulator hepcidin, the master regulator of iron homeostasis. Increasing genetic, preclinical and clinical evidence has demonstrated that inhibition of TMPRSS6 can potently elevated hepcidin levels, thereby achieving systemic iron restriction. Previously, we reported the generation of anti-TMPRSS6 antibody MWTx-003 and its efficacy in reducing iron overload and improving ineffective erythropoiesis in a mouse model of β-thalassemia. In this study, we explored therapeutic application of MWTx-003 in PV using a Jak2 V617F bone marrow transplantation mouse model. The mouse model exhibits typical PV phenotypes of erythrocytosis and elevated HCT levels. In the PV mice, treatment with MWTx-003 (2-10 mg/kg, i.p., every 4 days for 3 weeks) led to significantly reduced HCT levels, red blood cell (RBC) counts and hemoglobin (HGB) concentration that were comparable to that of wildtype control mice. These effects were evident in a dose-dependent manner and observed as early as two weeks into treatment. It is worth noting that in the 10 mg/kg dose group, a worsening splenomegaly and expansion of early erythroid progenitors were observed after 3 weeks of treatment, and these findings are typical of iron-restricted erythropoiesis in mice. Taken together, the anti-TMPRSS6 antibody offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes. A Phase I clinical trial with MWTx-003 in healthy volunteers has been initiated in China.

Impaired thymic iNKT cell differentiation at early precursor stage in murine haploidentical bone marrow transplantation with GvHD.

Early recovery of donor-derived invariant natural killer T (iNKT) cells are associated with reduced risk of graft-versus-host disease (GvHD) and overall survival. Patients with severe GvHD, however, had much slower iNKT cell reconstitution relative to conventional T cells. To characterize the delay of iNKT cell reconstitution and explore its possible causes, we used a haploidentical bone marrow transplantation (haplo-BMT) mouse model with GvHD. We found the delayed recovery of thymic and peripheral iNKT cell numbers with markedly decreased thymic NKT1 subset in GvHD mice. The defective generation of thymic iNKT precursors with egress capability contributed to the reduced peripheral iNKT cells in GvHD mice. We further identified intermediate NK1.1- NKT1 precursor subpopulations under steady-state conditions and found that the differentiation of these subpopulations was impaired in the thymi of GvHD mice. Detailed characterization of iNKT precursors and thymic microenvironment showed a close association of elevated TCR/co-stimulatory signaling provided by double positive thymocytes and macrophages with defective down-regulation of proliferation, metabolism, and NKT2 signature in iNKT precursor cells. Correspondingly, NKT2 but not NKT1 differentiation was favored in GvHD mice. These data underline the important roles of TCR and co-stimulatory signaling in the differentiation of thymic iNKT subsets under transplantation conditions.

Effects of different allo-Treg/allo-NK ratios on graft-versus-host disease in transplanted mice

To investigate the effects of allo-Treg cells, allo-NK cells, and their mixtures in different proportions on Graft-versus-host disease (GVHD) in bone marrow transplant mouse model. In this study, C57BL/6 mice were used as donors, and 6 Gy dose of 60Co γ was used as the receptor of BALB/c mice. The recipient mice were divided into NC (normal saline), CON (bone marrow cells), NK (bone marrow cells + NK cells), Treg (bone marrow cells + Treg cells), NK+ Treg (1:1) (bone marrow cells +1:1 ratio of Treg cells, NK cells), and NK+ Treg (6:1) (bone marrow cells +1:6 ratio of Treg cells, NK cells), according to the different injection mode through the tail vein. The differences of white blood cell (WBC), platelet (PLT), clinical manifestations, and GVHD score of target organs (liver, lung, small intestine) in each group after transplantation were observed, and the differences of chimerism rate and survival rate in each group at 28 days after transplantation were compared. The interaction between Treg cells and NK cells in different proportions (1:1, 1:2, 1:6, 1:12) was investigated in vitro in mouse erythroleukemia (MEL) cells of mouse erythroleukemia. The results showed that at the 28th day of transplantation, the clinical manifestations and GVHD scores of target organs of mice in NK+ Treg (1:1) group and NK+ Treg (6:1) group were significantly lower than other groups (P < 0.05); the WBC and PLT counts were significantly higher than other groups (P < 0.05), and the survival time was significantly longer than other groups (P < 0.05); the clinical manifestations and GVHD scores of each target organ in NK+ Treg (1:1) group were significantly lower than those in NK+ Treg (6:1) group (P < 0.05); the chimerism rate of each group was >90% on day 28 after transplantation. In vitro experiments showed that the inhibition of Treg cells on NK cell killing activity was dose-dependent, and the proportion of 1:6 and 1:12, killing activity of NK cell was significantly lower than that of groups 1:1 and 1:2 (P < 0.05), which showed that allo-NK and allo-Treg alone had a significant effect on the improvement of GVHD after transplantation, and Treg cells inhibited the killing activity of NK cells by direct contact and showed a dose-dependent effect.

Perivascular localized cells commit erythropoiesis in PDGF-B-expressing solid tumors.

Tumors possess incessant growth features, and expansion of their masses demands sufficient oxygen supply by red blood cells (RBCs). In adult mammals, the bone marrow (BM) is the main organ regulating hematopoiesis with dedicated manners. Other than BM, extramedullary hematopoiesis is discovered in various pathophysiological settings. However, whether tumors can contribute to hematopoiesis is completely unknown. Accumulating evidence shows that, in the tumor microenvironment (TME), perivascular localized cells retain progenitor cell properties and can differentiate into other cells. Here, we sought to better understand whether and how perivascular localized pericytes in tumors manipulate hematopoiesis. To test if vascular cells can differentiate into RBCs, genome-wide expression profiling was performed using mouse-derived pericytes. Genetic tracing of perivascular localized cells employing NG2-CreERT2:R26R-tdTomato mouse strain was used to validate the findings in vivo. Fluorescence-activated cell sorting (FACS), single-cell sequencing, and colony formation assays were applied for biological studies. The production of erythroid differentiation-specific cytokine, erythropoietin (EPO), in TME was checked using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA, magnetic-activated cell sorting and immunohistochemistry. To investigate BM function in tumor erythropoiesis, BM transplantation mouse models were employed. Genome-wide expression profiling showed that in response to platelet-derived growth factor subunit B (PDGF-B), neural/glial antigen 2 (NG2)+ perivascular localized cells exhibited hematopoietic stem and progenitor-like features and underwent differentiation towards the erythroid lineage. PDGF-B simultaneously targeted cancer-associated fibroblasts to produce high levels of EPO, a crucial hormone that necessitates erythropoiesis. FACS analysis using genetic tracing of NG2+ cells in tumors defined the perivascular localized cell-derived subpopulation of hematopoietic cells. Single-cell sequencing and colony formation assays validated the fact that, upon PDGF-B stimulation, NG2+ cells isolated from tumors acted as erythroblast progenitor cells, which were distinctive from the canonical BM hematopoietic stem cells. Our data provide a new concept of hematopoiesis within tumor tissues and novel mechanistic insights into perivascular localized cell-derived erythroid cells within TME. Targeting tumor hematopoiesis is a novel therapeutic concept for treating various cancers that may have profound impacts on cancer therapy.

The DNA-Sensor AIM2 Promotes BCR-Activated B Cells in Chronic Graft-Versus-Host-Disease

Chronic graft-versus-host-disease (cGVHD) remains a common and poorly understood cause of late non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HCT). T cells and IgG-producing B cells mediate cGVHD development and perpetuation, but molecular mechanisms driving immune pathology require further study. We have shown that B cells from cGVHD patients are B Cell Receptor (BCR)-activated and primed for survival. In a mouse model of bone-marrow transplantation (BMT) with cGVHD manifestations, BCR signaling is promoted by alloantigens and BAFF (Jia et al., Blood 2021). BCR signaling is a viable therapeutic target in cGVHD, but other targetable pathways that promote pathological BCR-activated B cells remain largely unknown. Our single cell RNA-sequencing study of B cells from the peripheral blood (PB) of HCT patients identified 'Absent in Melanoma 2' (AIM2) as a prime candidate for further study (ASH abstract 2019, Poe et al., manuscript submitted). AIM2 was a signature gene in a BCR-activated subset after HCT, and AIM2 transcripts were significantly increased in a pre-germinal center (GC) B cell subset in cGVHD vs. no cGVHD patients (adjusted p=0.049). AIM2 incites inflammasome-mediated pyroptotic cell death in myeloid cells, but the function of AIM2 in B cells is largely unknown. AIM2 is expressed by CD27+ antigen (Ag)-experienced B cells in healthy individuals (HI) (Svensson et al., PlosOne 2017) and emerging evidence suggests that AIM2 promotes antibody production (Yang et al., Signal Transduct Target Ther 2021). We hypothesize that AIM2 promotes pathological BCR-activated B cells and anti-host IgG production in cGVHD. Using intracellular flow cytometry (IFC) and PB samples from HI (n=27), we first confirmed that the AIM2 protein is mainly expressed by CD27+ Ag-experienced B cells. Specifically, 88% and 99% of the IgD+ pre- and IgD- post-GC CD27+ B cells expressed AIM2, respectively. In HCT, we found a significantly higher proportion of AIM2+ cells in the circulating CD27+ IgD- post-GC B cells from cGVHD patients (n=7) vs. no cGVHD patients (n=5) (p=0.02). We also found significant overexpression of AIM2 transcripts in B cells from mice with cGVHD manifestations vs. no cGVHD mice (n=5/group, p=0.03, Figure 1A). To determine if Ag-activation together with other extrinsic factors typically encountered by B cells after HCT induced AIM2 expression by purified CD27- B cells from HI, we used IFC. The proportion of AIM2+ CD27- B cells increased after BCR +/- Toll-like receptor 7 or 9 stimulation only when IFNγ was present. Next, we studied the function of AIM2 in B cells. Unlike myeloid cells, CD27+ B cells from HI did not undergo pyroptotic cell death after stimulation with double stranded DNA. This lack of pyroptosis was not due to a deficiency in the inflammasome components and B cells had the capacity to undergo pyroptosis after exposure to the NLRP1/3 activator, nigericin. To assess effects of AIM2 on specific IgG production, we used AIM2 KO mice in a hapten-carrier immunization model (NP-KLH + alum i.p.). AIM2-KO mice had larger splenic GC vs. WT controls at day 8 post-immunization (p<0.0001). GC enlargement paradoxically associated with lower anti-NP IgG levels (p=0.03). Finally, having previously shown that BM+Spl recipients have a significantly higher proportion of GL7+ BCR-activated blood B cells (Jia et al., Blood 2021), we asked whether AIM2 promoted GL7+ B cells in cGVHD. To address this, we used AIM2-floxed mice, kindly supplied by Genentech, to breed AIM2fl/fl Mb1Cre/+ conditional KO mice (AIM2-CKO), deficient in AIM2 only in B cells. We then used these AIM2-CKO vs. AIM2fl/fl Mb1+/+ control littermates mice which are AIM2-sufficient as BMT donors. At day 24 post-BMT, recipients of BM+Spl from AIM2-CKO mice had a significantly lower percentage of mature (CD19+ CD93-) GL7+ B cells in PB vs. recipients of BM+Spl from the AIM2-sufficient littermates (n=10/group, p=0.004, Figure 1B). This was not due to baseline B cell differences in donor CKO mice. These results are being confirmed at later time points in ongoing, additional experiments. Our data show that AIM2 promotes IgG-producing B cells and circulating GL7+ B cells, suggesting that AIM2 plays a novel inflammasome-independent role in cGVHD B cell pathology. We are actively investigating how AIM2 effects alloantibody production and cGVHD development, so that therapeutic targets can be identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effective and Safe Immune Checkpoint Blockade Induces Complete Responses in Murine PTCL Model of ALCL

Anaplastic large cell lymphoma (ALCL) is a rare T-cell non-Hodgkin lymphoma. In the majority of cases the disease is driven by a chromosomal translocation t(2;5)(p23;35) leading to the fusion of the anaplastic lymphoma kinase (ALK) gene with the nucleophosmin gene (NPM), constitutively activating ALK kinase. In the last decade immunotherapy turned out to be a real game-changer in cancer treatment, especially in the field of solid tumors. Regarding T-cell lymphomas (TCL), the significance of immune checkpoint inhibition is still highly debated. The outcomes in preclinical models as well as clinical trials are ranging from response to hyperprogression. Consequently, to provide a better understanding of the role and underlying mechanisms of targeting immune checkpoint axes, preclinical models of TCL are urgently needed to identify patients that benefit from immunotherapy as well as potential biomarkers. To examine disease biology and therapy effects of ALCL in vivo, we use a syngeneic retroviral bone marrow transplantation mouse model of ALK+ ALCL that closely resembles the disease in humans. In order to study the effect of blocking the PD-1/PD-L1-axis in ALCL in vivo, serial transplantations of lymphoma cells from a murine thymus were performed. We further investigated both spontaneous and immunotherapy-induced immune responses and characterized the interaction of innate and adaptive immune master players with flow cytometry, cytokine analysis, histology and transcriptome analysis. In our mouse model of ALK+ ALCL we confirmed the upregulation of PD-L1 on ALK+ lymphoma cells in vivo. Immune checkpoint inhibition of PD-L1 in vivo leads to high complete response rates, prolonged survival and significantly reduced lymphoma burden of treated animals. Flow cytometry analysis revealed that PD-L1 blockade reverses Treg-mediated immunosuppression and causes augmented circulation of CD8+ effector T-lymphocytes. Treatment with depletion antibodies of lymphocyte subpopulations unveils CD4+ T-cells and NK-cells but not CD8+ T-cells as master players of checkpoint-mediated immune response to ALK+ ALCL. Accordingly, we identified exhausted CD4+ T-cells in non-responders to checkpoint inhibition underlining the central role of CD4+ T-cells in ALCL immune surveillance and response. These results are contributing to a broader understanding of the immune surveillance and response in ALCL, with the outmost goal of better identifying patients that benefit from immunotherapy.

Cftr deletion in mouse epithelial and immune cells differentially influence the intestinal microbiota

Cystic fibrosis (CF) is a life-threatening genetic disorder, caused by mutations in the CF transmembrane-conductance regulator gene (cftr) that encodes CFTR, a cAMP-activated chloride and bicarbonate channel. Clinically, CF lung disease dominates the adult patient population. However, its gastrointestinal illness claims the early morbidity and mortality, manifesting as intestinal dysbiosis, inflammation and obstruction. As CF is widely accepted as a disease of epithelial dysfunction, it is unknown whether CFTR loss-of-function in immune cells contributes to these clinical outcomes. Using cftr genetic knockout and bone marrow transplantation mouse models, we performed 16S rRNA gene sequencing of the intestinal microbes. Here we show that cftr deletion in both epithelial and immune cells collectively influence the intestinal microbiota. However, the immune defect is a major factor determining the dysbiosis in the small intestine, while the epithelial defect largely influences that in the large intestine. This finding revises the current concept by suggesting that CF epithelial defect and immune defect play differential roles in CF intestinal disease.

AML-060 A Truncated Nucleus Isoform of FGFR1 Drives the Transformation of Leukemia Stem Cells in Leukemogenesis

Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated in the cytoplasm as a result of chromosome translocations. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. In this study, we used the mouse bone marrow transduction and transplantation model to demonstrate the leukemic transformation ability of this tnFGFR1 and identified its critical downstream targets, FLT3 and KIT, in mouse and acute myeloid leukemia (AML) patient samples. Stem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq were used to examine the related signaling pathways and regulated target genes. We now show that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1 induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expresses tnFGFR1, which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but sensitive to the FLT3 inhibitor quizartinib (AC220), suggesting an alternative approach for the treatment of SCLL. This study demonstrates a novel model for the transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by tnFGFR1. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

Anti-Sca-1 antibody-functionalized vascular grafts improve vascular regeneration via selective capture of endogenous vascular stem/progenitor cells

Small-diameter vascular grafts fabricated from synthetic biodegradable polymers exhibit beneficial mechanical properties but often face poor regenerative potential. Different tissue engineering approaches have been employed to improve tissue regeneration in vascular grafts, but there remains a requirement for a new generation of synthetic grafts that can orchestrate the host response to achieve robust vascular regeneration. Vascular stem/progenitor cells (SPCs) are mostly found in quiescent niches but can be activated in response to injury and participate in endothelium and smooth muscle regeneration during neo-artery formation. Here, we developed a functional vascular graft by surface immobilization of stem cell antigen-1 (Sca-1) antibody on an electrospun poly(ε-caprolactone) graft (PCL-Sca-1 Ab). PCL-Sca-1 Ab promoted capture and retainment of Sca-1+ SPCs in vitro. In rat abdominal aorta replacement models, PCL-Sca-1 Ab stimulated in vivo recruitment of Sca-1+ SPCs, and drove SPCs differentiation towards vascular cell lineages. The origin of infiltrated Sca-1+ SPCs was further investigated using a bone marrow transplantation mouse model, which revealed that Sca-1+ SPCs originating from the resident tissues and bone marrow contributed to rapid vascular regeneration of vascular grafts. Our data indicated that PCL-Sca-1 Ab vascular grafts may serve as a useful strategy to develop next generation cell-free vascular grafts.

Promotion of Platelet Production by Co-Transplantation of Mesenchymal Stem Cells in Bone Marrow Transplantation.

Therapeutic strategies that can promote platelet production are in demand to enhance clinical outcomes of bone marrow transplantation (BMT). Our research group has studied human tonsil-derived mesenchymal stem cells (T-MSCs) and their effectiveness in promoting bone marrow (BM) engraftment. Here, we analyzed the effects of T-MSCs on platelet production and hemostasis. Donor BM cells (BMCs) were isolated from C57BL/6 mice and transplanted with or without T-MSCs to BALB/c recipient mice. Mice were sacrificed and blood cells were counted using an Auto Hematology Analyzer. Femur sections were stained with CD41 antibody to analyze megakaryocytes in the BM. Growth factor secretion from MSCs was analyzed using the Quantibody Array. Effects of T-MSC conditioned medium (CM) on megakaryopoiesis were investigated using the MegaCult assay. In a mouse model of BMT, T-MSC CM was injected with or without anti-placental growth factor (α-PlGF) blocking antibody, and blood cell numbers and coagulation were analyzed. T-MSC co-transplantation increased percent survival of BMT mice. Platelet numbers were significantly lower in the BMC-only group, whereas T-MSC co-transplantation restored circulating platelets to levels similar to those of the control group. Significantly reduced numbers of CD41 + megakaryocytes in Bu-Cy and BMC groups were increased by T-MSC co-transplantation. PlGF secretion from T-MSCs were detected and enhanced megakaryopoiesis, platelet production, and coagulation by T-MCS CM were disrupted in the presence of the α-PlGF blocking antibody. We demonstrated the effectiveness of T-MSC co-transplantation in promoting platelet production and coagulation after BMT. These findings highlight the potential therapeutic relevance of T-MSCs for preventing thrombocytopenia after BMT.

Mesenchymal Stem Cell-Derived Exosomes Protect Muscle Loss by miR-145-5p Activity Targeting Activin A Receptors.

Skeletal muscle mass is decreased under a wide range of pathologic conditions. In particular, chemotherapy is well known for inducing muscle loss and atrophy. Previous studies using tonsil-derived mesenchymal stem cells (T-MSCs) or a T-MSC-conditioned medium showed effective recovery of total body weight in the chemotherapy-preconditioned bone marrow transplantation mouse model. This study investigated whether extracellular vesicles of T-MSCs, such as exosomes, are a key player in the recovery of body weight and skeletal muscle mass in chemotherapy-treated mice. T-MSC exosomes transplantation significantly decreased loss of total body weight and muscle mass in the busulfan-cyclophosphamide conditioning regimen in BALB/c recipient mice containing elevated serum activin A. Additionally, T-MSC exosomes rescued impaired C2C12 cell differentiation in the presence of activin A in vitro. We found that T-MSC exosomes possess abundant miR-145-5p, which targets activin A receptors, ACVR2A, and ACVR1B. Indeed, T-MSC exosomes rescue muscle atrophy both in vivo and in vitro via miR-145-5p dependent manner. These results suggest that T-MSC exosomes have therapeutic potential to maintain or improve skeletal muscle mass in various activin A elevated pathologic conditions.

Oncogenic cooperation between TCF7-SPI1 and NRAS(G12D) requires \u03b2-catenin activity to drive T-cell acute lymphoblastic leukemia

Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the β-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/β-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/β-catenin interaction.

Increased Galectin-9 expression, a prognostic biomarker of aGVHD, regulates the immune response through the Galectin-9 induced MDSC pathway after allogeneic hematopoietic stem cell transplantation

Galectin-9 (Gal-9) is a β-galactoside-binding soluble lectin family member that exerts its primary biological functions via specific glycoconjugate interactions. Gal-9 expression is closely related to tumor occurrence, development, metastasis and prognosis. In transplant immunology, a high level of Gal-9 expression has been shown to markedly reduce the severity of acute graft rejection and effectively prolong survival time in organ and bone marrow transplantation (BMT) models. The main mechanism of Gal-9-mediated immunoregulation involves the Tim-3/Gal-9 axis in T cells. However, myeloid-derived suppressor cell (MDSC) accumulation in transgenic mice with persistently high Gal-9 expression was observed in a model of lung inflammation, indicating that a potential immunosuppressive mechanism distinct from the Gal-9/Tim-3 axis might exist. In the present study, increased Gal-9 expression and MDSC frequencies before acute graft-versus-host disease (aGVHD) onset were observed in patients who developed aGVHD. Patients with higher Gal-9 expression (≥14.8417 ng/ml) exhibited reduced overall survival and increased cumulative incidences of GVHD at +100 day. We considered the elevated Gal-9 expression before aGVHD onset a secondary inflammatory response. This increase might be part of a negative feedback pathway corresponding to aGVHD pathogenesis. Additionally, a high Gal-9 concentration induced MDSC proliferation in vivo and in vitro. Gal-9-induced MDSCs (G9-MDSCs) suppressed T cell proliferation and activation. An infusion of G9-MDSCs into a graft contributed to the successful control of severe aGVHD and long-term survival in an allogeneic (allo)-BMT mouse model. Thus, we speculated that increased Gal-9 expression after allo-hematopoietic stem cell transplantation is a potential prognostic biomarker of aGVHD. The Gal-9-associated immunosuppressive effects on aGVHD development might occurr through G9-MDSCs and were independent of the Gal-9/Tim-3 axis.

Radiation causes tissue damage by dysregulating inflammasome-gasdermin D signaling in both host and transplanted cells.

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.

Large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established Graft-versus-Host-Disease model in batch dependent manners

Background & Aim Extracellular vesicles (EVs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (MSCs) can suppress acute inflammatory cues in a variety of different diseases, including Graft-versus-Host Disease (GvHD) and ischemic stroke. Furthermore, they can promote regeneration of affected tissues. Following a successful clinical treatment attempt of a steroid refractory GvHD patient, we intend to optimize MSC-EV production strategies for further clinical applications. As we observed functional differences of independent MSC-EV preparations in vitro, we aimed to adopt an in vivo GvHD model for the more advanced functional testing of different MSC-EV preparations. Methods, Results & Conclusion To this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (IIR). GvHD was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine T cells. If not treated otherwise, myeloablated mice developed severe GvHD symptoms. The GvHD symptoms were effectively suppressed, when MSC-EV preparations were applied at 3 consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. MSC-EV preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the GvHD mice. Thus, our results demonstrate that not all MSC-EV preparations harvested from adult bone marrow-derived MSCs contain the same therapeutic potential. Thus, successful transplantation of MSC-EVs into the clinics requires a platform allowing identification of MSC-EV preparations with sufficient therapeutic, most probably immune modulating activities.

CXCR4 or CXCR7 antagonists treat endometriosis by reducing bone marrow cell trafficking.

Adult stem cells have a major role in endometrial physiology, including remodelling and repair. However, they also have a critical role in the development and progression of endometriosis. Bone marrow‐derived stem cells engraft eutopic endometrium and endometriotic lesions, differentiating to both stromal and epithelial cell fates. Using a mouse bone marrow transplantation model, we show that bone marrow‐derived cells engrafting endometriosis express CXCR4 and CXCR7. Targeting either receptor by the administration of small molecule receptor antagonists AMD3100 or CCX771, respectively, reduced BM‐derived stem cell recruitment into endometriosis implants. Endometriosis lesion size was decreased compared to vehicle controls after treatment with each antagonist in both an early growth and established lesion treatment model. Endometriosis lesion size was not effected when the local effects of CXCL12 were abrogated using uterine‐specific CXCL12 null mice, suggesting an effect primarily on bone marrow cell migration rather than a direct endometrial effect. Antagonist treatment also decreased hallmarks of endometriosis physiopathology such as pro‐inflammatory cytokine production and vascularization. CXCR4 and CXCR7 antagonists are potential novel, non‐hormonal therapies for endometriosis.

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair

Vascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

Megakaryocyte Ablation Ameliorates Bone Marrow Fibrosis By Reducing Myofibroblast Differentiation in a Mouse Model of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a chronic myeloproliferative disorder characterized by excessive bone marrow (BM) fibrosis that can lead to ineffective hematopoiesis and reduced survival. PMF patients develop several megakaryocyte (MK) abnormalities including increased proliferation, abnormal morphology and upregulated expression of pro-fibrotic genes. Although several studies support the role of MK in BM fibrosis, the significance of MK in the development of BM fibrosis has not been demonstrated in vivo. Here, we investigated the in vivo role of MK in the pathogenesis of BM fibrosis. First, we reproduced an established bone marrow transplantation (BMT) mouse model of PMF. To achieve this, lethally irradiated wild-type mice were transplanted with wild-type hematopoietic stem cells (HSC) transduced with either MPLW515L gene construct (MPLW515L BMT) or MIGR1 empty vector (MIGR1 BMT). Consistent with previous studies, starting at day 14 post-BMT, MPLW515L BMT mice gradually developed increased number of leukocytes and platelets, increased proliferation of abnormal megakaryocytes in the BM and spleen, hepatosplenomegaly with extra-medullary hematopoiesis, and reticulin fibrosis in the BM. Notably, robust fibrosis was also seen in the spleen and liver. Next, to study the significance of MK in the development of BM fibrosis, we developed a MPLW515L BMT mouse model in which MK lineage could be selectively ablated. We accomplished this using PF4-Cre inducible diphtheria toxin receptor transgenic mice (iDTR+/-/PF4-Cre) as BMT donors. HSCs from iDTR+/-/PF4-Cre mice were transduced with MPLW515L or MIGR1 empty vector and transplanted as described above. Beginning day 14 post-BMT, recipient mice were injected with diphtheria toxin (DT) or water every 48 hours. At day 20 and day 29, DT injection significantly depleted MK in both MPLW515L BMT and MIGR1 BMT groups compared to water injection. Importantly, DT-injected MPLW515L BMT mice displayed attenuated fibrosis in the BM, spleen, and liver compared to water-injected MPLW515L BMT mice. In addition, DT treatment decreased the level of alpha smooth muscle actin in both BM and spleen of MPLW515L BMT mice, which suggests that MKs are critical for myofibroblast differentiation. MK ablation did not leukocytosis, thrombocytosis or hepatosplenomegaly. Together, our study show that successful MK ablation in vivo reduces fibrosis development in the BM, spleen, and liver of the MPLW515L mouse model of PMF. In summary, these results support the essential role of MK in the pathogenesis of BM fibrosis in PMF. Further studies are underway to elucidate the mechanisms by which MK contribute to fibrosis in PMF. Disclosures No relevant conflicts of interest to declare.