Bone marrow drives central nervous system regeneration after radiation injury.
ABSTRACT: Nervous system injury is a frequent result of cancer therapy involving cranial irradiation, leaving patients with marked memory and other neurobehavioral disabilities. Here, we report an unanticipated link between bone marrow and brain in the setting of radiation injury. Specifically, we demonstrate that bone marrow-derived monocytes and macrophages are essential for structural and functional repair mechanisms, including regeneration of cerebral white matter and improvement in neurocognitive function. Using a granulocyte-colony stimulating factor (G-CSF) receptor knockout mouse model in combination with bone marrow cell transplantation, MRI, and neurocognitive functional assessments, we demonstrate that bone marrow-derived G-CSF-responsive cells home to the injured brain and are critical for altering neural progenitor cells and brain repair. Additionally, compared with untreated animals, animals that received G-CSF following radiation injury exhibited enhanced functional brain repair. Together, these results demonstrate that, in addition to its known role in defense and debris removal, the hematopoietic system provides critical regenerative drive to the brain that can be modulated by clinically available agents.
Project description:Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic factor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when allografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.
Project description:Granulocyte colony-stimulating factor (G-CSF) induces proliferation of bone marrow-derived cells. G-CSF is neuroprotective after experimental brain injury, but the mechanisms involved remain unclear. Stem cell factor (SCF) is a cytokine important for the survival and differentiation of hematopoietic stem cells. Its receptor (c-kit or CD117) is present in some endothelial cells. We aimed to determine whether the combination of G-CSF/SCF induces angiogenesis in the central nervous system by promoting entry of endothelial precursors into the injured brain and causing them to proliferate there. We induced permanent middle cerebral artery occlusion in female mice that previously underwent sex-mismatched bone marrow transplantation from enhanced green fluorescent protein (EGFP)-expressing mice. G-CSF/SCF treatment reduced infarct volumes by more than 50% and resulted in a 1.5-fold increase in vessel formation in mice with stroke, a large percentage of which contain endothelial cells of bone marrow origin. Most cells entering the brain maintained their bone marrow identity and did not transdifferentiate into neural cells. G-CSF/SCF treatment also led to a 2-fold increase in the number of newborn cells in the ischemic hemisphere. These findings suggest that G-CSF/SCF treatment might help recovery through induction of bone marrow-derived angiogenesis, thus improving neuronal survival and functional outcome.
Project description:Exogenously infused mesenchymal stem cells (MSCs) are thought to migrate to injury site through peripheral blood stream and participate in tissue repair. However, whether and how endogenous bone marrow MSCs mobilized to circulating and targeted to tissue injury has raised some controversy, and related studies were restricted by the difficulty of MSCs identifying in vivo. Nestin, a kind of intermediate filament protein initially identified in neuroepithelial stem cells, was recently reported as a credible criteria for MSCs in bone marrow. In this study, we used a green fluorescent protein (GFP) labeled bone marrow replacement model to trace the nestin positive bone marrow derived cells (BMDCs) of skin defected-mice. We found that after skin injured, numbers of nestin+ cells in peripheral blood and bone marrow both increased. A remarkable concentration of nestin+ BMDCs around skin wound was detected, while few of these cells could be observed in uninjured skin or other organs. This recruitment effect could not be promoted by granulocyte colony-stimulating factor (G-CSF), suggests a different mobilization mechanism from ones G-CSF takes effect on hematopoietic cells. Our results proposed nestin+ BMDCs as mobilized candidates in skin injury repair, which provide a new insight of endogenous MSCs therapy.
Project description:Acute high-dose radiation injury damages the bone marrow hematopoietic stem and progenitor cell compartment. This damage compromises the functional ability of the bone marrow to produce mature blood cells and results in an increased risk of death due to hematopoietic complications. Past work has shown that the bone marrow endothelium provides critical cues, which promote hematopoietic stem cell regeneration after injury. Additionally, transfusion of endothelial cells after radiation injury has been shown to promote recovery of both the bone marrow vasculature and hematopoietic systems. In this work, we examined the regenerative capacity of intravenous infusion of umbilical cord-blood derived endothelial progenitor cells (EPCs) since this is a cell source which is easy to obtain, expand and cryopreserve. We show that pre-treatment with the Wnt-antagonist Dickkopf1 (Dkk1) augments EPC regenerative function in an allogeneic mouse transplant model. Here, hematopoietic recovery was assessed in Balb/c mice after 5 Gy total-body irradiation and transplantation with C57/BL6-derived EPCs either with or without Dkk1 pre-treatment. The Dkk1-treated EPC group had significantly faster recovery of peripheral white blood cells, total bone marrow cellularity, bone marrow progenitors and BM endothelial cells compared to EPC treatment alone or saline controls. Importantly, after an LD50/30 dose of 8 Gy in the Balb/c mouse, Dkk1-treated EPCs were able to rescue 100% of irradiated mice versus 80% in the EPC control group and only 33% in the saline-treated group. To understand how Dkk1 induces regenerative function in the EPCs, we screened for pro-regenerative factors secreted by the EPC in response to Dkk1. Dkk1-treated EPCs were observed to secrete high levels of the anti-fibrotic protein follistatin as well as several proteins known to promote regeneration including EGF, VEGF and G-CSF. This work demonstrates the potential for Dkk1-treated EPCs as a rescue therapeutic for victims of acute radiation injury.
Project description:Preclinical studies using bone marrow derived cells to treat traumatic brain injury have demonstrated efficacy in terms of blood-brain barrier preservation, neurogenesis, and functional outcomes. Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe traumatic brain injury demonstrated safety and potentially a central nervous system structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Adults with severe traumatic brain injury (Glasgow Coma Scale 5-8) and without signs of irreversible brain injury were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6, 9, 12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re-infusion occurred within 48 hours after injury. Patients were monitored for harvest-related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included magnetic resonance imaging-based measurements of supratentorial and corpus callosal volumes as well as diffusion tensor imaging-based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months postinjury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pretreatment, posttreatment, and at 1 and 6 month follow-up. There were no serious adverse events. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were downregulated. Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by central nervous system structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are downregulated after cell infusion. Stem Cells 2016 Video Highlight: https://youtu.be/UiCCPIe-IaQ Stem Cells 2017;35:1065-1079.
Project description:Radiation therapy (RT) is a widely accepted treatment strategy for many central nervous system (CNS) pathologies. However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT. The pathophysiology and molecular alterations that culminate in the long-term detrimental effects of CR are poorly understood, though it is thought that endothelial injury plays a pivotal role in triggering cranial injury. We therefore explored the contribution of bone marrow derived cells (BMDCs) in their capacity to repair and contribute to neo-vascularization following CR. Using high-resolution in vivo optical imaging we have studied, at single-cell resolution, the spatio-temporal response of BMDCs in normal brain following CR. We demonstrate that BMDCs are recruited specifically to the site of CR, in a radiation dose and temporal-spatial manner. We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia. Most notably we provide evidence that more than 50% of the microglia in the irradiated region of the brain are not resident microglia but recruited from the bone marrow following CR. These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR. Identifying the critical steps involved in the sustained recruitment and differentiation of BMDCs into microglia at the site of CR can provide new insights into the mechanisms of injury following CR offering potential therapeutic strategies to counteract the long-term adverse effects of CR.
Project description:It proved that Zymosan-A protected the haematopoietic system from radiation-induced damage via Toll-Like Receptor2 in our previous study. In this study, we investigated the potential mechanism for the radioprotective effects of Zymosan-A. The mice were treated with Zymosan-A (50 mg/kg, dissolved in NS) via peritoneal injection 24 and 2 hours before ionizing radiation. Apoptosis of bone marrow cells and the levels of IL-6, IL-12, G-CSF and GM-CSF were evaluated by flow cytometry assay. DNA damage was determined by ?-H2AX foci assay. In addition, RNA sequencing was performed to identify differentially expressed genes (DEGs). Zymosan-A protected bone marrow cells from radiation-induced apoptosis, up-regulated IL-6, IL-12, G-CSF and GM-CSF in bone marrow cells. Zymosan-A also protected cells from radiation-induced DNA damage. Moreover, RNA sequencing analysis revealed that Zymosan-A induced 131 DEGs involved in the regulation of immune system process and inflammatory response. The DEGs were mainly clustered in 18 KEGG pathways which were also associated with immune system processes. Zymosan-A protected bone marrow cells from radiation-induced apoptosis and up-regulated IL-6, IL-12, G-CSF and GM-CSF. Moreover, Zymosan-A might also exhibit radioprotective effects through regulating immune system process and inflammatory response. These results provided new knowledge regarding the radioprotective effect of Zymosan-A.
Project description:Despite an important role in vascular development and repair, the origin of endothelial progenitors remains unknown. Accumulating evidence indicates that cells derived from the hematopoietic system participate in angiogenesis. However, the identity and functional role of these cells remain controversial. Here we show that vascular endothelial cells can differentiate from common myeloid progenitors and granulocyte/macrophage progenitors. Endothelial cells derived from transplanted bone marrow-derived myeloid lineage progenitors expressed CD31, von Willebrand factor, and Tie2 but did not express the hematopoietic markers CD45 and F4/80 or the pericyte markers desmin and smooth muscle actin. Lineage tracing analysis in combination with a Tie2-driven Cre/lox reporter system revealed that, in contrast to bone marrow-derived hepatocytes, bone marrow-derived endothelial cells are not the products of cell fusion. The establishment of both hematopoietic and endothelial cell chimerism after parabiosis demonstrates that circulating cells can give rise to vascular endothelium in the absence of acute radiation injury. Our findings indicate that endothelial cells are an intrinsic component of myeloid lineage differentiation and underscore the close functional relationship between the hematopoietic and vascular systems.
Project description:After kidney ischemia/reperfusion (I/R) injury, monocytes home to the kidney and differentiate into activated macrophages. Whereas proinflammatory macrophages contribute to the initial kidney damage, an alternatively activated phenotype can promote normal renal repair. The microenvironment of the kidney during the repair phase mediates the transition of macrophage activation from a proinflammatory to a reparative phenotype. In this study, we show that macrophages isolated from murine kidneys during the tubular repair phase after I/R exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4-stimulated STAT6 signaling. This unique activation profile can be reproduced in vitro by stimulation of bone marrow-derived macrophages with conditioned media from serum-starved mouse proximal tubule cells. Secreted tubular factors were found to activate macrophage STAT3 and STAT5 but not STAT6, leading to induction of the unique alternative activation pattern. Using STAT3-deficient bone marrow-derived macrophages and pharmacologic inhibition of STAT5, we found that tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and after renal I/R, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. Furthermore, administration of a neutralizing antibody against GM-CSF after renal I/R attenuated kidney macrophage alternative activation and suppressed tubular proliferation. Taken together, these data show that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phenotype that supports tubular proliferation after sterile ischemic injury.
Project description:The mononuclear phagocytic system is categorized in three major groups: monocyte-derived cells (MCs), dendritic cells and resident macrophages. During breast cancer progression the colony stimulating factor 1 (CSF-1) can reprogram MCs into tumor-promoting macrophages in the primary tumor. However, the effect of CSF-1 during colonization of the brain parenchyma is largely unknown. Thus, we analyzed the outcome of anti-CSF-1 treatment on the resident macrophage population of the brain, the microglia, in comparison to MCs, alone and in different in vitro co-culture models. Our results underline the addiction of MCs to CSF-1 while surprisingly, microglia were not affected. Furthermore, in contrast to the brain, the bone marrow did not express the alternative ligand, IL-34. Yet treatment with IL-34 and co-culture with carcinoma cells partially rescued the anti-CSF-1 effects on MCs. Further, MC-induced invasion was significantly reduced by anti-CSF-1 treatment while microglia-induced invasion was reduced to a lower extend. Moreover, analysis of lung and breast cancer brain metastasis revealed significant differences of CSF-1 and CSF-1R expression. Taken together, our findings demonstrate not only differences of anti-CSF-1 treatment on MCs and microglia but also in the CSF-1 receptor and ligand expression in brain and bone marrow as well as in brain metastasis.