Migration Of Bone Marrow Cells Research Articles

Human adipose ECM alleviates radiation-induced skin fibrosis via endothelial cell-mediated M2 macrophage polarization

Radiation therapy can lead to late radiation-induced skin fibrosis (RISF), causing movement restriction, pain, and organ dysfunction. This study evaluated adipose-derived extracellular matrix (Ad-ECM) as a mitigator of RISF. Female C57BL/6J mice that were irradiated developed fibrosis, which was mitigated by a single local Ad-ECM injection, improving limb movement and reducing epithelium thickness and collagen deposition. Ad-ECM treatment resulted in decreased expression of pro-inflammatory and fibrotic genes, and upregulation of anti-inflammatory cytokines, promoting M2 macrophage polarization. Co-culture of irradiated human fibroblasts with Ad-ECM down-modulated fibrotic gene expression and enhanced bone marrow cell migration. Ad-ECM treatment also increased interleukin (IL)-4, IL-5, and IL-15 expression in endothelial cells, stimulating M2 macrophage polarization and alleviating RISF. Prophylactic use of Ad-ECM showed effectiveness in mitigation. This study suggests Ad-ECM's potential in treating chronic-stage fibrosis.

Mechanism underlying Fanmugua () leaf multicomponent synergistic therapy for anemia: data mining based on hematopoietic network.

To investigate the underlying mechanism of Fanmugua (Fructus Caricae) Leaf (CPL) multicomponent synergistic therapy for anemia. The components were identified in the literature. Six databases were searched for targets of CPL. Enrichment analysis was used to determine the targets associated with anemia and in bone marrow. Based on the Kyoto Encyclopedia of Genes and Genomes database, pathways and targets related to hematopoiesis were obtained. The key targets were obtained by protein-protein interaction analysis. Molecular docking was used to analyze the binding ability of key targets and active components. Bone marrow cells were used as an experimental model to verify the drug efficacy. A total of 139 components and 1868 targets of CPL were retrieved from the literature. By disease enrichment analysis, 543 targets for hemorrhagic anemia, 223 targets for aplastic anemia, and 126 targets for sickle cell anemia were obtained. Target organ enrichment yielded 27, 29, and 20 targets of bone marrow. Based on KEGG pathway enrichment, a total of 47 shared hematopoietic pathways and 42 related targets were found. The key targets were vascular endothelial growth factor A (VEGFA), interleukin 10 (IL-10), platelet-endothelial cell adhesion molecule-1 (PECAM1), C-C motif chemokine 2 (CCL2), and vascular cell adhesion molecule 1 (VCAM1). The CPL active components included ursolic acid, quercetin, and hesperidin. The expression of VEGFA was significantly increased after CPL treatment. Quercetin and ursolic acid acted on VEGFA. Quercetin and Hesperidin acted on VCAM1. Quercetin acted on IL-10, CCL2, VCAM1, and VEGFA. Cell experiments revealed that CPL could promote the proliferation and migration of bone marrow cells. CPL has the synergistic efficacy of treating anemia through multiple components, targets, and pathways.

Matrix Metalloproteinase 1 as a Marker of Tonsil-Derived Mesenchymal Stem Cells to Assess Bone Marrow Cell Migration.

To achieve optimal bone marrow engraftment during bone marrow transplantation, migration of donor bone marrow cells (BMCs) toward the recipient's bone marrow is critical. Despite the enhanced engraftment of BMCs by co-administration of mesenchymal stem cells (MSCs), the efficiency can be variable depending on MSC donor. The purpose of this study is to examine the functional heterogeneity of tonsil-derived MSCs (TMSCs) and to identify a marker to evaluate efficacy for the enhancement of BMC migration. To examine the donor-to-donor variation of TMSCs in potentiating BMC migration, we isolated TMSCs from 25 independent donors. Transcriptome of TMSCs and proteome of conditioned medium derived from TMSC were analyzed. Enhanced BMC migration by conditioned medium derived from TMSCs was variable depending on TMSC donor. The TMSCs derived from 25 donors showed distinct expression profiles compared with other cells, including fibroblasts, adipose-derived MSCs and bone marrow-derived MSCs. TMSCs were distributed in two categories: high- and low-efficacy groups for potentiating BMC migration. Transcriptome analysis of TMSCs and proteome profiles of conditioned medium derived from TMSCs revealed higher expression and secretion of matrix metalloproteinase (MMP) 1 in the high-efficacy group. MMP1 knockdown in TMSCs abrogated the supportive efficacy of conditioned medium derived from TMSC cultures in BMC migration. These data suggest that secreted MMP1 can be used as a marker to evaluate the efficacy of TMSCs in enhancing BMC migration. Furthermore, the strategy of analyzing transcriptomes and proteomes of the MSCs may be useful to set the standard for donor variation.

Tonsil-derived mesenchymal stem cells enhance allogeneic bone marrow engraftment via collagen IV degradation

BackgroundCo-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified.MethodsMice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration.ResultsMice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro.ConclusionsCollectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Safety and Demonstrated Efficacy of Placenta-Derived Cell Therapy PLX-R18 in Subjects with Incomplete Hematopoietic Recovery Following Hematopoietic Cell Transplantation: A Phase I International Multi-Center Study

Introduction: Failure to engraft donor cells is a devastating complication after allogeneic hematopoietic cell transplantation (HCT) while full engraftment remains a challenge in some recipients. Therapeutic approaches, such as recombinant hematopoietic growth factors, salvage only a minority of patients leaving affected patients at risk for recurrent infections and multiple blood products transfusions. PLX-R18 cells have been shown to mitigate bone marrow failure in rodent and non-human primate models. Further, these human placenta-derived mesenchymal-like adherent stromal cells secrete many cytokines after intramuscular (IM) injection that have been shown to stimulate growth and migration of bone marrow cells in vitro, e.g. MCP-1, IL-6, and IL-8. The initial results of the first cohort of this phase I trial were previously reported (ASH 2018 abstract 3379). Methods: Enrollment for this multi-center, open label, dose-escalation, phase I trial (ClinicalTrials.gov: NCT03002519) began in October 2017. Eligibility criteria included patients age ≥18 years who underwent either autologous (auto) or allogeneic (allo) HCT but suffered from incomplete hematopoietic recovery at least 3 months post-transplant, defined as failure to maintain either hemoglobin (Hb) >8g/dL and/or neutrophil count (ANC) >1,000/μL and/or platelet (PLT) count >50,000/μL. Other eligibility criteria included no evidence of underlying malignancy at time of enrollment, no active infection and no evidence of grade 3-4 acute or severe chronic GvHD. Subjects were enrolled into 3 dose-escalating cohorts: low-dose at 1M cells/kg (n=3), intermediate-dose at 2M cells/kg (n=6), and high-dose at 4M cells/kg (n=10). The specified dose of PLX-R18 cells was injected IM in multiple locations and given twice, 1 week apart. Cohorts were recruited sequentially, and progression to the next cohort was based on the safety assessment of its predecessor. Adverse event grading used the NCI CTCAE criteria. As of July 2020, all patients in cohorts 1 and 2 (3 & 6 patients, respectively) and 10 patients in cohort 3 were treated. Data were analyzed using the lmerTest package (ver. 3.1.0) in R (ver. 3.6.1) to produce baseline-adjusted least square means. As study is in progress and data collection ongoing, not all subjects have data on all parameters for all time points. Results: Data on 19 subjects (29-75 yrs) with either allo (n=17) or auto (n=2) HCT are reported herein. Not all patients reached the 9 month time point. Four patients died while on study and 2 withdrew consent within the 1 year follow up period. All deaths and other severe adverse events (SAEs) occurred due to progression of primary disease and were assessed as not related by investigators and sponsor. Most frequent related AEs were injection-site reactions. The table summarizes number of patients who improved from below the critical level, as defined above, at day 0 to above the critical level for a sustained period. Only those patients that were below the critical blood levels at day 0 are shown. To date, the majority of such patients improved to above the critical level after treatment. For example, 9 patients had ANC <1000/μL at day 0. Of the 4 patients for which data currently exists, all improved to ≥1000/μL at 9 months. The plots show changes in Hb, ANC and PLT up to 9 months following treatment. The dashed lines represent respective critical levels. Overall, there was improvement in the 3 parameters, with the greatest improvement observed in patients receiving the predicted optimal dose of 4M cells/kg. In this cohort, Hb levels increased from 9.29±0.5 to 13.19±1.2 (p=0.0019), ANC increased from 1.09±0.6 to 5.13±1.7 (p=0.018) and PLT increased from 45.7±10 to 175.9±28 (p=0.000012). Conclusions: Treatment with PLX-R18 is safe, well-tolerated and clearly effective in some patients. The most frequent AEs were reversible injection site reactions, all without sequelae. An overall improvement was observed in most patients, and among the high-dose cohort clinically impressive improvements in Hb, ANC and PLT were observed. The trial continues to follow up on patients. Upon completion, a large phase II trial, using the optimal dose, is considered for the same indication. Other attractive clinical areas for PLX-R18 therapy include acute radiation syndrome, bone marrow failure, aplastic anemia, myelofibrosis and with HCT as a "pan" cytokine to enhance rapid count recovery in all cells lines. Figure Disclosures Lazarus: Pluristem ltd: Consultancy. McGuirk:Astellas: Research Funding; Bellicum Pharmaceutical: Research Funding; Gamida Cell: Research Funding; Pluristem Ltd: Research Funding; Kite Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Allo Vir: Consultancy, Honoraria, Research Funding; Juno Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Fresenius Biotech: Research Funding. Rowley:AbbVie: Current equity holder in publicly-traded company; FATE Therapeutics: Consultancy. Ofir:Pluristem: Current Employment. Shani:Pluristem ltd: Current Employment. Rowe:Pluristem ltd: Consultancy.

Abstract 5422: Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity

Abstract Doxorubicin (Dox) is one of the most effective drugs for the treatment of childhood cancer patients. However, there is a correlation between Dox dose/intensity and cardiotoxicity. We developed a juvenile mouse cardiotoxicity model and showed that Dox treatment affected the cardiac vessel morphology characterized by punctate vessels with decreased pericytes and endothelial cells. Cardiac blood flow and function (as quantified by echocardiography [Echo]) were also compromised. These abnormalities were not seen in mice treated with exercise (Ex) during Dox. We also demonstrated increased c-kit+ cells in the hearts of mice treated with Dox + Ex. To determine the origin of these c-kit+ cells we performed a bone marrow (BM) transplant using GFP+ BM cells from C57BL/6Tg(UBC-GFP)30Scha/J mice into C57BL/6 WT mice treated with Busulfan and anti-CD4 and anti-DC8 Abs. BM engraftment was documented 4 wks post-transplant. Transplanted mice were treated with Dox twice/wk x 2 wks; Dox + Ex, or control (no treatment). Cardiac function was assessed after treatment by Echo and the heart tissues were evaluated by immunohistochemistry (IHC) for cells expressing GFP (denoting cells derived from the BM); GFP+/NG2+ or GFP+/CD31+ (BM cells differentiated into pericytes or endothelial cells respectively). Echo data confirmed decreased cardiac function in Dox but not Dox + Ex-treated mice. Few GFP+ cells were found in the hearts from control or Dox-treated mice. By contrast, mice treated with Dox + Ex showed a significant increase in GFP+ cells indicating that exercise had induced the migration of BM cells into the heart. Co-localization of GFP/NG2 and GFP/CD31 indicated that these BM stem cells had differentiated into both endothelial cells and pericytes, and that these cells were incorporated into the cardiac vessels. Few GFP+cTnI+ cells were observed in the heart tissues from mice treated with Ex + Dox, indicating that the BM stem cells had not differentiated into cardiomyoctes. These studies suggest that acute Dox-induced cardiac damage may be partially mediated by decreased blood flow secondary to damaged vessels with decreased vascular endothelial cells and pericytes. Ex may inhibit this effect by inducing the migration of BM stem cells to repair the damaged vessels. We also showed that Dox therapy induced phosphorylation of MST1/2, LATS1 and YAP, decreased total YAP, and induced cleavage of caspase-3 and PARP. These changes were not seen in Dox + Ex hearts suggesting that Ex protected cardiomyocytes from Dox-induced apoptosis by suppressing Hippo-YAP signaling. Taken together, our data demonstrated that the protective effect of Ex may be mediated by restoring cardiac blood flow which may in turn inhibit Dox-induced Hippo-YAP signaling-mediated cardiomyocyte apoptosis. These data support the concept that exercise interventions have the potential to decrease Dox-induced cardiac damage and morbidity in childhood cancer patients. Citation Format: Rong-Hua Tao, Masato Kobayashi, Fei Wang, Yuanzheng Yang, Eugenie S. Kleinerman. Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5422.

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.

Loss of Dnmt3a Dysregulates Hematopoietic Homeostasis and Myeloid Skewing Via the PI3Kinase Pathway: PI3Kα/β Inhibition Corrects Extramedullary Hematopoiesis and Myeloid Skewing Due to Loss of Dnmt3a

Mutations in DNA methyltransferase type 3A (DNMT3A) are frequently associated with myeloid malignancies including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN) including systemic mastocytosis (SM). Presence of this mutation is associated with poor prognosis and reduced overall patient survival. Earlier studies have shown that hematopoietic specific loss of Dnmt3a in mice results in lethal, fully penetrant MPN with myelodysplasia (MDS/MPN) characterized by extramedullary hematopoiesis, peripheral cytopenias, splenomegaly and hepatomegaly. However, it's unclear how DNMT3A mutations contribute to myeloid skewing, and induce myeloid malignancies. Further, the mechanism(s) by which loss of DNMT3A impairs various mast cell functions leading to mastocytosis are also not defined. We show that loss of Dnmt3a in vitro results in accelerated and enhanced differentiation of mast cells from its bone marrow (BM) precursors and in vivo results in increased number of mast cells. Genome wide transcriptome analysis revealed elevated expression of the transcription factor MITF and a profound repression in the expression of C/EBPα in BM precursors derived from Dnmt3a-/- mice. Importantly, restoring the expression of Dnmt3a in Dnmt3a deficient BM cells, completely restored enhanced differentiation, along with correction in levels of MITF and C/EBPα, suggesting that early loss of C/EBPα in Dnmt3a-/- cells contributes to enhanced maturation of mast cells from its precursors. Furthermore, biochemical analysis revealed greater PI3Kinase activation in Dnmt3a-/- cells, and these findings were supported by genome wide transcriptome analysis. Importantly, mast cells derived from Dnmt3a-/- mice lacking the expression of p85α regulatory subunit of PI3Kinase or pharmacologic inhibition of PI3Kinase completely corrected cytokine induced hyperproliferation in these cells to near normal levels. Insights from mast cell studies led us to hypothesize that the aberrant hematopoietic regulation in Dnmt3a-/- mice described above may be due to hyperactivation of the PI3Kinase signaling in hematopoietic stem and progenitors (HSCPs). We investigated the role of PI3Kinase signaling in Dnmt3a loss induced myeloid malignancy using pharmacological inhibitor, GDC0941 (PI3Kα/δ inhibitor) and BAY1082439 (PI3Kα/β inhibitor). PI3Kα/β inhibitor treatment of Dnmt3a-/- mice markedly reduced monocytosis, neutrophilia, enhanced WBC counts, and improved RBCs, hematocrits and platelet numbers compared to control mice. In contrast, PI3Kα/δ inhibition moderately corrected the monocytosis and WBC aberrancy, however correction in erythroid dysregulation was not observed. Inhibiting PI3Kα/β signaling dramatically reduced splenomegaly and hepatomegaly in Dnmt3a-/- mice. In contrast, PI3Kα/δ inhibition moderately reduced the spleen size, however the correction in hepatomegaly was insignificant. Importantly, clonal hematopoiesis due to loss of Dnmt3a in a competitive transplant setting was rescued by inhibiting the PI3Kinase pathway. In the BM, PI3Kα/β inhibition moderately decreased LSK cells, however a marked decrease in GMP, and significant increase in MEP, and CLP was observed compared to controls. Flow cytometry analysis revealed that PI3Kα/β inhibition reduced Dnmt3a-/- BM cell migration to liver, and completely corrected the extramedullary hematopoiesis in the spleen compared to controls. Further, Dnmt3a loss induced alteration in erythroid development in the BM, spleen and PB was corrected upon PI3Kα/β blockade compared to controls. Genome wide transcriptome analysis revealed that PI3Kα/β inhibition markedly reduced the expression of GMP related genes, fetal liver hematopoiesis transcription program, and expression levels of inflammatory cytokines, growth factors, and chemokines in Dnmt3a depleted malignant mice. However PI3Kα/β inhibition increased the expression of genes involved in erythroid development in Dnmt3a-/- mice compared to controls. These results demonstrate that Dnmt3a ablation induces liver specific expansion of hematopoietic cells, and extramedullary hematopoiesis in spleen via aberrant activation of PI3Kinase signaling in HSCPs. Consistent with this notion, PI3K inhibitor treatment of malignant Dnmt3a-/- bearing mice showed significantly improved overall survival compared to controls. Disclosures No relevant conflicts of interest to declare.

Bone Marrow-Derived CD44+ Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair.

Background and Aims Host-derived cells play crucial roles in the regeneration process of tissue-engineered constructs (TECs) during the treatment of large segmental bone defects (LSBDs). However, their identity, source, and cell recruitment mechanisms remain elusive. Methods A complex model was created using mice by combining methods of GFP+ bone marrow transplantation (GFP-BMT), parabiosis (GFP+-BMT and wild-type mice), and femoral LSBD, followed by implantation of TECs or DBM scaffolds. Postoperatively, the migration of host BM cells was detected by animal imaging and immunofluorescent staining. Bone repair was evaluated by micro-CT. Signaling pathway repressors including AMD3100 and SP600125 associated with the migration of BM CD44+ cells were further investigated. In vitro, transwell migration and western-blotting assays were performed to verify the related signaling pathway. In vivo, the importance of the SDF-1/CXCR4-JNK pathway was validated by ELISA, fluorescence-activated cell sorting (FACS), immunofluorescent staining, and RT-PCR. Results First, we found that host cells recruited to facilitate TEC-mediated bone repair were derived from bone marrow and most of them express CD44, indicating the significance of CD44 in the migration of bone marrow cells towards donor MSCs. Then, the predominant roles of SDF-1/CXCR4 and downstream JNK in the migration of BM CD44+ cells towards TECs were demonstrated. Conclusion Together, we demonstrated that during bone repair promoted by TECs, BM-derived CD44+ cells were essential and their migration towards TECs could be regulated by the SDF-1/CXCR4-JNK signaling pathway.

Prior β-blocker treatment decreases leukocyte responsiveness to injury.

Following injury, leukocytes are released from hematopoietic organs and migrate to the site of damage to regulate tissue inflammation and repair, however leukocytes lacking β2-adrenergic receptor (β2AR) expression have marked impairments in these processes. β-blockade is a common strategy for the treatment of many cardiovascular etiologies, therefore the objective of our study was to assess the impact of prior β-blocker treatment on baseline leukocyte parameters and their responsiveness to acute injury. In a temporal and βAR isoform-dependent manner, chronic β-blocker infusion increased splenic vascular cell adhesion molecule-1 (VCAM-1) expression and leukocyte accumulation (monocytes/macrophages, mast cells and neutrophils) and decreased chemokine receptor 2 (CCR2) expression, migration of bone marrow cells (BMC) and peripheral blood leukocytes (PBL), as well as infiltration into the heart following acute cardiac injury. Further, CCR2 expression and migratory responsiveness was significantly reduced in the PBL of patients receiving β-blocker therapy compared to β-blocker-naïve patients. These results highlight the ability of chronic β-blocker treatment to alter baseline leukocyte characteristics that decrease their responsiveness to acute injury and suggest that prior β-blockade may act to reduce the severity of innate immune responses.

The role of the transcription factor Sox2 and cytokeratins in the formation and development of the gastroesophageal junction epithelial cell differon

The source of the origin of the epithelium of the cardiac part stomach mucosa has been repeatedly discussed in the literature and different variants of the transformation of the epithelium as manifestation of normal anatomical peculiarities of a man and as a result of changing the program of stem cell differentiation, migration of bone marrow cells, transdifferentiation of simple columnar epithelium have been proposed. Probably it is related to difficulties of studying insignificant in size epithelium of the cardiac mucosa itself and establishment of connection of the duodenogastroesophageal reflux with the development of metaplasia in the epithelium of the terminal department of the esophagus mucosa, which resembles its structure in the cardiac part of the stomach. The purpose of the research was to study the expression of the transcription factor Sox2 and the distribution of cytokeratins in the epithelium of the gastroesophageal zone during the stages of the embryonic and fetal periods of ontogenesis. According to the purpose of the research, an immunohistochemical analysis of the epithelial differon of the esophageal-gastric junction (GEJ) was used. The current study was carried out on 169 human embryos and fetuses of gestational age from 4-5 till 38 weeks. It was established that the transcription factor Sox2 is expressed in basal epitheliocytes of GEJ in all terms of observation and plays a major role in the development, differentiation and formation of the epithelial cell lineage of GEJ. The peculiarity of expression of cytokeratin 7 was positive marking in the cytoplasm of spinosum epitheliocytes, despite the negative expression in the basal layer. It showed weak expression in the epitheliocytes of the esophageal part of the GEJ in the embryonic period with an increased reaction in the embryo-fetal period and with subsequent disappearance, starting at 14 weeks in the early fetal period. For the cardiac mucous membrane GEJ was characterized by its moderate expression on all terms of observation. Cytokeratin 8/18 is embryo-fetal for the esophageal part of the esophagus, as it is defined in early periods of embryogenesis and disappears in the late period (28-38 weeks). For the cardiac mucous membrane GEJ was characterized by its moderate expression on all terms of observation. Cytokeratin 14, unlike CK7 and CK8/18, was localized in the cytoplasm and membranes of basal epitheliocytes of the esophageal part of the mucosa from the 17 gestational weeks and was absent in the gastrointestinal part of the GEJ throughout the prenatal period. Thus, our data on the expression of the transcription factor Sox2 and cytokeratins in the GEJ epithelial differon in the prenatal period of ontogenesis will improve the diagnostic accuracy in determining tissue or organ belonging and can be widely used in various GEJ diseases.

Suppression of angiotensin II-induced pathological changes in heart and kidney by the caveolin-1 scaffolding domain peptide.

Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 μg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased β1 and β3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure.

Bone marrow cell homing to sites of acute tibial fracture: 89Zr-oxine cell labeling with positron emission tomographic imaging in a mouse model

BackgroundBone fracture healing is dependent upon the rapid migration and engraftment of bone marrow (BM) progenitor and stem cells to the site of injury. Stromal cell-derived factor-1 plays a crucial role in recruiting BM cells expressing its receptor CXCR4. Recently, a CXCR4 antagonist, plerixafor, has been used to mobilize BM cells into the blood in efforts to enhance cell migration to sites of injury presumably improving healing. In this study, we employed zirconium-89 (89Zr)-oxine-labeled BM cells imaged with positron emission tomography (PET)/computed tomography (CT) to visualize and quantitate BM cell trafficking following acute bone injury and to investigate the effect of plerixafor on BM cell homing. Unilateral 1-mm incisions were created in the distal tibia of mice either on the same day (d0) or 24 h (d1) after 89Zr-oxine-labeled BM cell transfer (n = 4–6, 2–2.3 × 107 cells at 9.65–15.7 kBq/106 cells). Serial microPET/CT imaging was performed and migration of 89Zr-labeled cells to the bone injury was quantified. The effects of three daily doses of plerixafor on cell trafficking were evaluated beginning on the day of fracture generation (n = 4–6). The labeled cells localizing to the fracture were analyzed by flow cytometry and immunohistochemistry.ResultsIn d0- and d1-fracture groups, 0.7% and 1.7% of administered BM cells accumulated within the fracture, respectively. Plerixafor treatment reduced BM cell migration to the fracture by approximately one-third (p < 0.05 for both fracture groups). Flow cytometry analysis of donor cells collected from the injured site revealed a predominance of CD45+ stem/progenitor cell populations and subsequent histological analysis demonstrated the presence of donor cells engrafted within sites of fracture repair.Conclusion89Zr-oxine labeling enabled visualization and quantitation of BM cell recruitment to acute fractures and further demonstrated that plerixafor plays an inhibitory role in this recruitment.

Prospective, Multi-Center, Phase I Clinical Trial of PLX-R18 Placental Expanded Adherent Stromal Cells in Subjects with Incomplete Hematopoietic Recovery after Hematopoietic Cell Transplantation

Background: Successful engraftment remains a challenge in hematopoietic cell transplantation (HCT) for some patients. Salvage approaches, including use of recombinant hematopoietic growth factors, cytokine therapy, CD34+-selected progenitor cell boosts, or second or subsequent HCT, are successful only in a minority of patients, and patients are exposed to prolonged blood products transfusions. With our successful pre-clinical rodent and Rhesus Macaques total body irradiation injury-induced marrow failure models (Metheny, Front Med 2018; Sher, Cell Transplantation 2018; Sher, Blood 2017), this prospective, multi-center, phase I clinical trial is evaluating the safety of PLX-R18 cells used to improve peripheral blood counts in patients who have experienced incomplete hematopoietic recovery after HCT. PLX-R18 cells are human placental-derived adherent stromal cells that are predominantly of fetal origin, expressing CD105, CD73 and CD29, and lacking surface expression of CD45, CD34, CD14, CD19, HLA-DR, CD31 (endothelial marker) and GlyA (erythrocyte cell marker). PLX-R18 also do not express HLA class II molecules (HLA-DP, DQ, and DR) and co-stimulatory markers (CD80, CD86, and CD40). PLX-R18 cells secrete MCP-1, IL-6, and IL-8 and had been shown to stimulate migration of bone marrow cells in vitro. Cells are grown ex vivo, under GMP conditions, using a 3-dimensional system, and are administered as several intramuscular (IM) injections. PLX-R18 cells already are approved for treatment of acute radiation syndrome (ARS) under an IND.Methods: This is a multi-center, open label, dose-escalation, phase 1 trial (ClinicalTrials.gov: NCT03002519). Eligibility criteria include patients age ≥18 years who underwent either an autologous (auto) or an allogeneic (allo) HCT but had incomplete hematopoietic recovery at 4 months post-transplant, defined as failure to maintain either hemoglobin (Hb) >8 g/dL and/ or neutrophil count (ANC) >1,000/µL and/or platelet count (Plt) (unsupported) >50,000/µL. Other eligibility criteria include ECOG performance status of 0-2, no evidence of underlying malignancy at time of enrollment, no active infection, and no evidence of grade 3-4 acute or severe chronic GvHD. The PLX-R18 cells, cryopreserved in DMSO and human serum albumin in Plasma-Lyte, are thawed and injected IM in multiple locations - half into the gluteus medius muscle on one side and half to the thigh muscles on the contralateral side. A total of 24 subjects are planned to be enrolled into 3 dose escalating cohorts: low (n=3) given 1 million cells/kg; intermediate (n=6) at 2 million cells/kg and high (n=15) dosed at 4 million cells/kg each. The assigned dose is given twice, 1 week apart; cohorts are recruited sequentially and progression to the next cohort is based on the safety assessment of its predecessor. Adverse event is graded using the CTCAE criteria of the NCI. As of July 2018, all 3 patients of cohort 1 were treated (with sufficient follow-up), as well as 3 out of the 6 subjects planned for the 2nd cohort, and data of the first cohort are presented herein.Results: Cohort 1 (low-dose) - 3 subjects (ages 43, 48 and 55 years old) who had an allo (n=2) or auto (n=1) HCT received two IM administrations of PLX-R18, 1 million cells/kg each within a 1-week interval. 67 Adverse Events (AEs) were recorded. 64.3% of the AEs were grade 1, 30.9% were grade 2, 3.6% were grade 3 and 1.2% were grade 4. One patient suffered from 3 Serious AEs (immune thrombocytopenic purpura, bacteremia and migraine), all assessed as not related to treatment. Most frequent related AE was transient injection site pain, experienced by all 3 patients. See patients' details and blood counts in the table and graphs.Conclusions: Treatment with PLX-R18 generally was well-tolerated and most frequent adverse events were injection site reactions, which were all reversible without sequelae. The trial continues to accrue patients and the next dosing cohort (intermediate dose) is at 2 million cells/kg.Upon completion, a larger phase II trial will be considered in the same indication using the optimal dose. Other potential uses include PLX-R18 therapy in Acute Radiation Syndrome, Bone marrow failure, and in HCT as a “pan” cytokine for rapid count recovery. [Display omitted] DisclosuresLazarus:Pluristem Ltd.: Consultancy. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy. McGuirk:Novartis Pharmaceuticals Corporation: Honoraria, Other: speaker, Research Funding; Fresenius Biotech: Research Funding; Kite Pharma: Honoraria, Other: travel accommodations, expenses, speaker ; Gamida Cell: Research Funding; Astellas Pharma: Research Funding; Pluristem Ltd: Research Funding; Bellicum Pharmaceuticals: Research Funding. Rosen:Pluristem: Employment.

Cytokines, hepatic cell profiling and cell interactions during bone marrow cell therapy for liver fibrosis in cholestatic mice.

Bone marrow cells (BMC) migrate to the injured liver after transplantation, contributing to regeneration through multiple pathways, but mechanisms involved are unclear. This work aimed to study BMC migration, characterize cytokine profile, cell populations and proliferation in mice with liver fibrosis transplanted with GFP+ BMC. Confocal microscopy analysis showed GFP+ BMC near regions expressing HGF and SDF-1 in the fibrotic liver. Impaired liver cell proliferation in fibrotic groups was restored after BMC transplantation. Regarding total cell populations, there was a significant reduction in CD68+ cells and increased Ly6G+ cells in transplanted fibrotic group. BMC contributed to the total populations of CD144, CD11b and Ly6G cells in the fibrotic liver, related to an increment of anti-fibrotic cytokines (IL-10, IL-13, IFN-γ and HGF) and reduction of pro-inflammatory cytokines (IL-17A and IL-6). Therefore, HGF and SDF-1 may represent important chemoattractants for transplanted BMC in the injured liver, where these cells can give rise to populations of extrahepatic macrophages, neutrophils and endothelial progenitor cells that can interact synergistically with other liver cells towards the modulation of an anti-fibrotic cytokine profile promoting the onset of liver regeneration.

Bone marrow cell migration to the heart in a chimeric mouse model of acute chagasic disease.

BACKGROUND Chagas disease is a public health problem caused by infection with the protozoan Trypanosoma cruzi. There is currently no effective therapy for Chagas disease. Although there is some evidence for the beneficial effect of bone marrow-derived cells in chagasic disease, the mechanisms underlying their effects in the heart are unknown. Reports have suggested that bone marrow cells are recruited to the chagasic heart; however, studies using chimeric mouse models of chagasic cardiomyopathy are rare.OBJECTIVES The aim of this study was to investigate the migration of bone marrow cells to the heart after T. cruzi infection in a model of chagasic disease in chimeric mice.METHODS To obtain chimerical mice, wild-type (WT) C57BL6 mice were exposed to full body irradiation (7 Gy), causing bone marrow ablation. Then, bone marrow cells from green fluorescent protein (GFP)-transgenic mice were infused into the mice. Graft effectiveness was confirmed by flow cytometry. Experimental mice were divided into four groups: (i) infected chimeric (iChim) mice; (ii) infected WT (iWT) mice, both of which received 3 × 104 trypomastigotes of the Brazil strain; (iii) non-infected chimeric (Chim) mice; and (iv) non-infected WT mice.FINDINGS At one-month post-infection, iChim and iWT mice showed first degree atrioventricular block with decreased heart rate and treadmill exercise parameters compared to those in the non-infected groups.MAIN CONCLUSIONS iChim mice showed an increase in parasitaemia, myocarditis, and the presence of amastigote nests in the heart tissue compared to iWT mice. Flow cytometry analysis did not detect haematopoietic progenitor cells in the hearts of infected mice. Furthermore, GFP+ cardiomyocytes were not detected in the tissues of chimeric mice.

Investigating the Abscopal Effects of Radioablation on Shielded Bone Marrow in Rodent Models Using Multimodality Imaging.

The abscopal effect is the response to radiation at sites that are distant from the irradiated site of an organism, and it is thought to play a role in bone marrow (BM) recovery by initiating responses in the unirradiated bone marrow. Understanding the mechanism of this effect has applications in treating BM failure (BMF) and BM transplantation (BMT), and improving survival of nuclear disaster victims. Here, we investigated the use of multimodality imaging as a translational tool to longitudinally assess bone marrow recovery. We used positron emission tomography/computed tomography (PET/CT), magnetic resonance imaging (MRI) and optical imaging to quantify bone marrow activity, vascular response and marrow repopulation in fully and partially irradiated rodent models. We further measured the effects of radiation on serum cytokine levels, hematopoietic cell counts and histology. PET/CT imaging revealed a radiation-induced increase in proliferation in the shielded bone marrow (SBM) compared to exposed bone marrow (EBM) and sham controls. T2-weighted MRI showed radiation-induced hemorrhaging in the EBM and unirradiated SBM. In the EBM and SBM groups, we found alterations in serum cytokine and hormone levels and in hematopoietic cell population proportions, and histological evidence of osteoblast activation at the bone marrow interface. Importantly, we generated a BMT mouse model using fluorescent-labeled bone marrow donor cells and performed fluorescent imaging to reveal the migration of bone marrow cells from shielded to radioablated sites. Our study validates the use of multimodality imaging to monitor bone marrow recovery and provides evidence for the abscopal response in promoting bone marrow recovery after irradiation.

Methods for Studying the Role of RAAS in the Modulation of Vascular Repair-Relevant Functions of Stem/Progenitor Cells.

In recent years, previously unknown functions have been conferred to the RAAS and have been explored in mechanistic studies and disease models. Implication of bone marrow stem/progenitor cells in the cardiovascular protective or detrimental effects of RAAS is a prominent advancement because of the translational significance. Selected members of RAAS are now known to modulate migration, proliferation, and mobilization of bone marrow cells in response to ischemic insult, which are sensitive indicators of vascular repair-relevant functions. In this Chapter, protocols for most frequently used, in vitro, ex vivo, and in vivo assays to explore the potential of RAAS members to stimulate vascular repair-relevant functions of bone marrow stem/progenitor cells of human and murine origin.

Antiangiogenic and Antitumor Activities of Aflibercept, a Soluble VEGF Receptor-1 and -2, in a Mouse Model of Hepatocellular Carcinoma

BACKGROUND & AIM: Aflibercept known as ziv-aflibercept in the United States is a soluble decoy receptor of both vascular endothelial growth factor (VEGF) receptor-1 and -2 known to inhibit the binding of VEGF and placental growth factor (PlGF) to VEGF receptor-1 and -2. Here, we analyzed the mechanisms of the antitumor effects of aflibercept in mouse hepatoma models. METHODS: In in vitro studies, we determined the effects of aflibercept on human umbilical vein cell (HUVEC) proliferation and bone marrow (BM) cell differentiation to endothelial progenitor cells (EPCs). In in vivo experiments, aflibercept was injected intraperitoneally in hepatoma cell tumor-bearing mice, and its inhibitory effects on tumor growth and BM cell migration to tumor tissues were evaluated. RESULTS: Aflibercept suppressed phosphorylation of VEGF receptor-1 and -2 in HUVEC and dose-dependently inhibited VEGF-induced HUVEC proliferation. It suppressed the differentiation of BM cells to EPCs and migration of BM cells to tumor tissues. It also suppressed tumor growth and prolonged survival time of tumor-bearing mice without side effects. In tumor tissues, aflibercept upregulated the expression of hypoxia inducible factor1-α, VEGF, PlGF, fibroblast growth factor-2, platelet derived growth factor-BB, and transforming growth factor-α and reduced microvascular density. It also reduced sinusoidal density in noncancerous liver tissues. CONCLUSIONS: Our results demonstrated potent antitumor activity for aflibercept in a mouse model of hepatocellular carcinoma. These effects were mediated through inhibition of neovascularization, caused by inhibition of endothelial cell proliferation, EPC differentiation, and BM cell migration to tumor tissues.

Effect of Irradiation on Tumor Microenvironment and Bone Marrow Cell Migration in a Preclinical Tumor Model

To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts. Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2Gy/d for a total dose of 20Gy using a 5days on, 2days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40Gy in 4weeks using the same 2-Gy/d regimens. Over the 2weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31±0.02mm(3)/d and 0.55±0.04mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5%±3%), hypoxia (10%±3.5%), vascular endothelial growth factor (2.3%±1%), and stromal-derived factor-1α (2.5%±1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8%±2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6%±0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT. Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs.