ResearchPad - bone-marrow-cells https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SULF1 suppresses Wnt3A-driven growth of bone metastatic prostate cancer in perlecan-modified 3D cancer-stroma-macrophage triculture models]]> https://www.researchpad.co/article/elastic_article_14741 Bone marrow stroma influences metastatic prostate cancer (PCa) progression, latency, and recurrence. At sites of PCa bone metastasis, cancer-associated fibroblasts and tumor-associated macrophages interact to establish a perlecan-rich desmoplastic stroma. As a heparan sulfate proteoglycan, perlecan (HSPG2) stores and stabilizes growth factors, including heparin-binding Wnt3A, a positive regulator of PCa cell growth. Because PCa cells alone do not induce CAF production of perlecan in the desmoplastic stroma, we sought to discover the sources of perlecan and its growth factor-releasing modifiers SULF1, SULF2, and heparanase in PCa cells and xenografts, bone marrow fibroblasts, and macrophages. SULF1, produced primarily by bone marrow fibroblasts, was the main glycosaminoglycanase present, a finding validated with primary tissue specimens of PCa metastases with desmoplastic bone stroma. Expression of both HSPG2 and SULF1 was concentrated in αSMA-rich stroma near PCa tumor nests, where infiltrating pro-tumor TAMs also were present. To decipher SULF1’s role in the reactive bone stroma, we created a bone marrow biomimetic hydrogel incorporating perlecan, PCa cells, macrophages, and fibroblastic bone marrow stromal cells. Finding that M2-like macrophages increased levels of SULF1 and HSPG2 produced by fibroblasts, we examined SULF1 function in Wnt3A-mediated PCa tumoroid growth in tricultures. Comparing control or SULF1 knockout fibroblastic cells, we showed that SULF1 reduces Wnt3A-driven growth, cellularity, and cluster number of PCa cells in our 3D model. We conclude that SULF1 can suppress Wnt3A-driven growth signals in the desmoplastic stroma of PCa bone metastases, and SULF1 loss favors PCa progression, even in the presence of pro-tumorigenic TAMs.

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<![CDATA[Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity]]> https://www.researchpad.co/article/elastic_article_14593 Mesenchymal stem cells have the ability to transdifferentiate into neurons and therefore one of the potential adult stem cell source for neuronal tissue regeneration applications and understanding neurodevelopmental processes. In many studies on human mesenchymal stem cell (hMSC) derived neurons, success in neuronal differentiation was limited to neuronal protein expressions which is not statisfactory in terms of neuronal activity. Established neuronal networks seen in culture have to be investigated in terms of synaptic signal transmission ability to develop a culture model for human neurons and further studying the mechanism of neuronal differentiation and neurological pathologies. Accordingly, in this study, we analysed the functionality of bone marrow hMSCs differentiated into neurons by a single step cytokine-based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (≥75%) as demonstrated by Ca++ imaging. Furthermore, when electrically stimulated, hMSC derived neurons (hMd-Neurons) matched the response of a typical neuron in the process of maturation. Our results reveal that a combination of neuronal inducers enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. Conceptually, we suggest these functional hMd-Neurons to be used as a tool for disease modelling of neuropathologies and neuronal differentiation studies.

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<![CDATA[Swainsonine, an alpha-mannosidase inhibitor, may worsen cervical cancer progression through the increase in myeloid derived suppressor cells population]]> https://www.researchpad.co/article/5c897787d5eed0c4847d2ef3

Cervical cancer, caused by high oncogenic risk Human Papillomavirus (HPV) infection, continues to be a public health problem, mainly in developing countries. Using peptide phage display as a tool to identify potential molecular targets in HPV associated tumors, we identified α-mannosidase, among other enriched sequences. This enzyme is expressed in both tumor and inflammatory compartment of the tumor microenvironment. Several studies in experimental models have shown that its inhibition by swainsonine (SW) led to inhibition of tumor growth and metastasis directly and indirectly, through activation of macrophages and NK cells, promoting anti-tumor activity. Therefore, the aim of this work was to test if swainsonine treatment could modulate anti-tumor immune responses and therefore interfere in HPV associated tumor growth. Validation of our biopanning results showed that cervical tumors, both tumor cells and leukocytes, expressed α-mannosidase. Ex vivo experiments with tumor associated macrophages showed that SW could partially modulate macrophage phenotype, decreasing CCL2 secretion and impairing IL-10 and IL-6 upregulation, which prompted us to proceed to in vivo tests. However, in vivo, SW treatment increased tumor growth. Investigation of the mechanisms leading to this result showed that SW treatment significantly induced the accumulation of myeloid derived suppressor cells in the spleen of tumor bearing mice, which inhibited T cell activation. Our results suggested that SW contributes to cervical cancer progression by favoring proliferation and accumulation of myeloid cells in the spleen, thus exacerbating these tumors systemic effects on the immune system, therefore facilitating tumor growth.

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<![CDATA[The pro-tumor effect of CD200 expression is not mimicked by agonistic CD200R antibodies]]> https://www.researchpad.co/article/5c605a9bd5eed0c4847cd2e3

Tumor-infiltrating immune cells can impact tumor growth and progression. The inhibitory CD200 receptor (CD200R) suppresses the activation of myeloid cells and lack of this pathway results in a reduction of tumor growth, conversely a tumorigenic effect of CD200R triggering was also described. Here we investigated the role of CD200R activation in syngeneic mouse tumor models. We showed that agonistic CD200R antibody reached tumors, but had no significant impact on tumor growth and minor effect on infiltration of immune myeloid cells. These effects were reproduced using two different anti-CD200R clones. In contrast, we showed that CD200-deficiency did decrease melanoma tumor burden. The presence of either endogenous or tumor-expressed CD200 restored the growth of metastatic melanoma foci. On the basis of these findings, we conclude that blockade of the endogenous ligand CD200 prevented the tumorigenic effect of CD200R-expressing myeloid cells in the tumor microenvironment, whereas agonistic anti-CD200R has no effect on tumor development.

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<![CDATA[Mutation, drift and selection in single-driver hematologic malignancy: Example of secondary myelodysplastic syndrome following treatment of inherited neutropenia]]> https://www.researchpad.co/article/5c3d00e3d5eed0c484036660

Cancer development is driven by series of events involving mutations, which may become fixed in a tumor via genetic drift and selection. This process usually includes a limited number of driver (advantageous) mutations and a greater number of passenger (neutral or mildly deleterious) mutations. We focus on a real-world leukemia model evolving on the background of a germline mutation. Severe congenital neutropenia (SCN) evolves to secondary myelodysplastic syndrome (sMDS) and/or secondary acute myeloid leukemia (sAML) in 30–40%. The majority of SCN cases are due to a germline ELANE mutation. Acquired mutations in CSF3R occur in >70% sMDS/sAML associated with SCN. Hypotheses underlying our model are: an ELANE mutation causes SCN; CSF3R mutations occur spontaneously at a low rate; in fetal life, hematopoietic stem and progenitor cells expands quickly, resulting in a high probability of several tens to several hundreds of cells with CSF3R truncation mutations; therapeutic granulocyte colony-stimulating factor (G-CSF) administration early in life exerts a strong selective pressure, providing mutants with a growth advantage. Applying population genetics theory, we propose a novel two-phase model of disease development from SCN to sMDS. In Phase 1, hematopoietic tissues expand and produce tens to hundreds of stem cells with the CSF3R truncation mutation. Phase 2 occurs postnatally through adult stages with bone marrow production of granulocyte precursors and positive selection of mutants due to chronic G-CSF therapy to reverse the severe neutropenia. We predict the existence of the pool of cells with the mutated truncated receptor before G-CSF treatment begins. The model does not require increase in mutation rate under G-CSF treatment and agrees with age distribution of sMDS onset and clinical sequencing data.

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<![CDATA[Preclinical IV busulfan dose-finding study to induce reversible myeloablation in a non-human primate model]]> https://www.researchpad.co/article/5c2400ced5eed0c48409930e

In this study we utilized a large animal model to identify a dose of intravenous busulfan that can cause reversible myelosuppression. Nine baboons (Papio anubis) were treated with IV busulfan at 6.4 (Group A), 8 (Group B), or 9.6 mg/kg (Group C). Peripheral blood counts were measured up to 90 days after treatment and serial bone marrow samples were obtained to analyze CD34+ cell content and colony forming units. Overall, the highest grade of peripheral blood cytopenia was observed 15 days after treatment in all three groups (n = 3/group). In particular, we observed a notable reduction of neutrophil and platelet counts in the blood and the number of marrow CD34+ cells and colony forming units. In contrast, the effect of busulfan on hemoglobin levels was mild. Baboons who received the highest dose of busulfan showed only a 25–35% recovery of marrow CD34+ cells and colony forming units after 90 days of busulfan administration. However, all three groups of animals showed a full recovery of peripheral blood counts and normal marrow cellularity and tri-lineage hematopoiesis after treatment. Notably, all three doses of busulfan were tolerated well without significant extra-medullary toxicity. These results validate the hierarchy of blood cells likely targeted by busulfan, and based on these findings, clinical trials using myelotoxic but not myeloablative doses of intravenous busulfan will be designed for patients with myeloid malignancies.

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<![CDATA[Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells]]> https://www.researchpad.co/article/5ab0848b463d7e2faeab8ab9

X-chromosome inactivation (XCI) in female lymphocytes is uniquely regulated, as the inactive X (Xi) chromosome lacks localized Xist RNA and heterochromatin modifications. Epigenetic profiling reveals that Xist RNA is lost from the Xi at the pro-B cell stage and that additional heterochromatic modifications are gradually lost during B cell development. Activation of mature B cells restores Xist RNA and heterochromatin to the Xi in a dynamic two-step process that differs in timing and pattern, depending on the method of B cell stimulation. Finally, we find that DNA binding domain of YY1 is necessary for XCI in activated B cells, as ex-vivo YY1 deletion results in loss of Xi heterochromatin marks and up-regulation of X-linked genes. Ectopic expression of the YY1 zinc finger domain is sufficient to restore Xist RNA localization during B cell activation. Together, our results indicate that Xist RNA localization is critical for maintaining XCI in female lymphocytes, and that chromatin changes on the Xi during B cell development and the dynamic nature of YY1-dependent XCI maintenance in mature B cells predisposes X-linked immunity genes to reactivation.

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<![CDATA[Pre-Osteoblasts Stimulate Migration of Breast Cancer Cells via the HGF/MET Pathway]]> https://www.researchpad.co/article/5989daa3ab0ee8fa60ba671e

Introduction

The occurrence of skeletal metastases in cancer, e.g. breast cancer (BC), deteriorates patient life expectancy and quality-of-life. Current treatment options against tumor-associated bone disease are limited to anti-resorptive therapies and aimed towards palliation. There remains a lack of therapeutic approaches, which reverse or even prevent the development of bone metastases. Recent studies demonstrate that not only osteoclasts (OCs), but also osteoblasts (OBs) play a central role in the pathogenesis of skeletal metastases, partly by producing hepatocyte growth factor (HGF), which promotes tumor cell migration and seeding into the bone. OBs consist of a heterogeneous cell pool with respect to their maturation stage and function. Recent studies highlight the critical role of pre-OBs in hematopoiesis. Whether the development of bone metastases can be attributed to a particular OB maturation stage is currently unknown.

Methods and Results

Pre-OBs were generated from healthy donor (HD)-derived bone marrow stromal cells (BMSC) as well as the BMSC line KM105 and defined as ALPlow OPNlow RUNX2high OSX high CD166high. Conditioned media (CM) of pre-OBs, but not of undifferentiated cells or mature OBs, enhanced migration of metastatic BC cells. Importantly, HGF mRNA was significantly up-regulated in pre-OBs versus mature OBs, and CM of pre-OBs activated the MET signaling pathway. Highlighting a key role for HGF, CM from HGF-negative pre-OBs derived from the BMSC line HS27A did not support migration of BC cells. Genetically (siMET) or pharmacologically (INCB28060) targeting MET inhibited both HGF- and pre-OB CM- mediated BC cell migration.

Conclusions

Our data demonstrate for the first time a role for pre-OBs in mediating HGF/MET- dependent migration of BC cells and strongly support the clinical evaluation of INCB28060 and other MET inhibitors to limit and/or prevent BC-associated bone metastases.

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<![CDATA[c-Fms Signaling Mediates Neurofibromatosis Type-1 Osteoclast Gain-In-Functions]]> https://www.researchpad.co/article/5989dadcab0ee8fa60bba2f3

Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1+/− mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1+/ osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1+/− osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1+/ osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1+/−-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.

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<![CDATA[REDD1 Protects Osteoblast Cells from Gamma Radiation-Induced Premature Senescence]]> https://www.researchpad.co/article/5989d9e2ab0ee8fa60b69f93

Radiotherapy is commonly used for cancer treatment. However, it often results in side effects due to radiation damage in normal tissue, such as bone marrow (BM) failure. Adult hematopoietic stem and progenitor cells (HSPC) reside in BM next to the endosteal bone surface, which is lined primarily by hematopoietic niche osteoblastic cells. Osteoblasts are relatively more radiation-resistant than HSPCs, but the mechanisms are not well understood. In the present study, we demonstrated that the stress response gene REDD1 (regulated in development and DNA damage responses 1) was highly expressed in human osteoblast cell line (hFOB) cells after γ irradiation. Knockdown of REDD1 with siRNA resulted in a decrease in hFOB cell numbers, whereas transfection of PCMV6-AC-GFP-REDD1 plasmid DNA into hFOB cells inhibited mammalian target of rapamycin (mTOR) and p21 expression and protected these cells from radiation-induced premature senescence (PS). The PS in irradiated hFOB cells were characterized by significant inhibition of clonogenicity, activation of senescence biomarker SA-β-gal, and the senescence-associated cytokine secretory phenotype (SASP) after 4 or 8 Gy irradiation. Immunoprecipitation assays demonstrated that the stress response proteins p53 and nuclear factor κ B (NFkB) interacted with REDD1 in hFOB cells. Knockdown of NFkB or p53 gene dramatically suppressed REDD1 protein expression in these cells, indicating that REDD1 was regulated by both factors. Our data demonstrated that REDD1 is a protective factor in radiation-induced osteoblast cell premature senescence.

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<![CDATA[The Influence of Therapeutic Radiation on the Patterns of Bone Marrow in Ovary-Intact and Ovariectomized Mice]]> https://www.researchpad.co/article/5989d9efab0ee8fa60b6dc11

Background

The functional components of bone marrow (i.e., the hematopoietic and stromal populations) and the adjacent bone have traditionally been evaluated incompletely as distinct entities rather than the integrated system. We perturbed this system in vivo using a medically relevant radiation model in the presence or absence of ovarian function to understand integrated tissue interaction.

Methodology/Principal Findings

Ovary-intact and ovariectomized mice underwent either no radiation or single fractional 16 Gy radiation to the caudal skeleton (I±R, OVX±R). Marrow fat, hematopoietic cellularity, and cancellous bone volume fraction (BV/TV %) were assessed. Ovariectomy alone did not significantly reduce marrow cellularity in non-irradiated mice (OVX−R vs. I−R, p = 0.8445) after 30 days; however it impaired the hematopoietic recovery of marrow following radiation exposure (OVX+R vs. I+R, p = 0.0092). The combination of radiation and OVX dramatically increases marrow fat compared to either factor alone (p = 0.0062). The synergistic effect was also apparent in the reduction of hematopoietic marrow cellularity (p = 0.0661); however it was absent in BV/TV% changes (p = 0.2520). The expected inverse relationship between marrow adiposity vs. hematopoietic cellularity and bone volume was observed. Interestingly compared with OVX mice, intact mice demonstrated double the reduction in hematopoietic cellularity and a tenfold greater degree of bone loss for a given unit of expansion in marrow fat.

Conclusions/Significance

Ovariectomy prior to delivery of a clinically-relevant focal radiation exposure in mice, exacerbated post-radiation adipose accumulation in the marrow space but blunted bone loss and hematopoietic suppression. In the normally coupled homeostatic relationship between the bone and marrow domains, OVX appears to alter feedback mechanisms. Confirmation of this non-linear phenomenon (presumably due to differential radiosensitivity) and demonstration of the mechanism of action is needed to provide strategies to diminish the effect of radiation on exposed tissues.

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<![CDATA[Diagnostic Value of Measuring Platelet Von Willebrand Factor in Von Willebrand Disease]]> https://www.researchpad.co/article/5989da54ab0ee8fa60b8e72a

Von Willebrand disease (VWD) may be caused by an impaired von Willebrand factor (VWF) synthesis, its increased clearance or abnormal function, or combinations of these factors. It may be difficult to recognize the different contributions of these anomalies. Here we demonstrate that VWD diagnostics gains from measuring platelet VWF, which can reveal a defective VWF synthesis. Measuring platelet VWF revealed that: severe type 1 VWD always coincided with significantly lower platelet and plasma VWF levels, whereas mild forms revealed low plasma VWF levels associated with low or normal platelet VWF levels, and the latter were associated with a slightly shorter VWF survival; type Vicenza (the archetype VWD caused by a reduced VWF survival) featured normal platelet VWF levels despite significantly reduced plasma VWF levels; type 2B patients could have either normal platelet VWF levels associated with abnormal multimer patterns, or reduced platelet VWF levels associated with normal multimer patterns; type 2A patients could have reduced or normal platelet VWF levels, the former associated mainly with type 2A-I, the latter with type 2A-II; plasma and platelet VWF levels were normal in type 2N, except when the defect was associated with a quantitative VWF mutation. Our findings show that measuring platelet VWF helps to characterize VWD, especially the ambiguous phenotypes, shedding light on the mechanisms underlying the disorder.

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<![CDATA[In Vitro Megakaryocyte Differentiation and Proplatelet Formation in Ph-Negative Classical Myeloproliferative Neoplasms: Distinct Patterns in the Different Clinical Phenotypes]]> https://www.researchpad.co/article/5989daffab0ee8fa60bc5f08

Background

Ph-negative myeloproliferative neoplasms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET). Although the pathogenesis of MPNs is still incompletely understood, an involvement of the megakaryocyte lineage is a distinctive feature.

Methodology/Principal Findings

We analyzed the in vitro megakaryocyte differentiation and proplatelet formation in 30 PMF, 8 ET, 8 PV patients, and 17 healthy controls (CTRL). Megakaryocytes were differentiated from peripheral blood CD34+ or CD45+ cells in the presence of thrombopoietin. Megakaryocyte output was higher in MPN patients than in CTRL with no correlation with the JAK2 V617F mutation. PMF-derived megakaryocytes displayed nuclei with a bulbous appearance, were smaller than ET- or PV-derived megakaryocytes and formed proplatelets that presented several structural alterations. In contrast, ET- and PV-derived megakaryocytes produced more proplatelets with a striking increase in bifurcations and tips compared to both control and PMF. Proplatelets formation was correlated with platelet counts in patient peripheral blood. Patients with pre-fibrotic PMF had a pattern of megakaryocyte proliferation and proplatelet formation that was similar to that of fibrotic PMF and different from that of ET.

Conclusions/Significance

In conclusion, MPNs are associated with high megakaryocyte proliferative potential. Profound differences in megakaryocyte morphology and proplatelet formation distinguish PMF, both fibrotic and prefibrotic, from ET and PV.

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<![CDATA[CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow]]> https://www.researchpad.co/article/5989dadaab0ee8fa60bb970c

Background

Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow.

Methodology/Principal Findings

CD34+ cells, c-Kit+/Sca-1+/Lin (KSL) cells, c-Kit+/Lin (KL) cells and Sca-1+/Lin (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34+ cells showed the lowest EPC colony forming activity, CD34+ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34+ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34+ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34+ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others.

Conclusion

These findings suggest that mouse CD34+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

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<![CDATA[Evaluating Tissue-Specific Recombination in a Pdgfrα-CreERT2 Transgenic Mouse Line]]> https://www.researchpad.co/article/5989d9daab0ee8fa60b6727a

In the central nervous system (CNS) platelet derived growth factor receptor alpha (PDGFRα) is expressed exclusively by oligodendrocyte progenitor cells (OPCs), making the Pdgfrα promoter an ideal tool for directing transgene expression in this cell type. Two Pdgfrα-CreERT2 mouse lines have been generated for this purpose which, when crossed with cre-sensitive reporter mice, allow the temporally restricted labelling of OPCs for lineage-tracing studies. These mice have also been used to achieve the deletion of CNS-specific genes from OPCs. However the ability of Pdgfrα-CreERT2 mice to induce cre-mediated recombination in PDGFRα+ cell populations located outside of the CNS has not been examined. Herein we quantify the proportion of PDGFRα+ cells that become YFP-labelled following Tamoxifen administration to adult Pdgfrα-CreERT2::Rosa26-YFP transgenic mice. We report that the vast majority (>90%) of PDGFRα+ OPCs in the CNS, and a significant proportion of PDGFRα+ stromal cells within the bone marrow (~38%) undergo recombination and become YFP-labelled. However, only a small proportion of the PDGFRα+ cell populations found in the sciatic nerve, adrenal gland, pituitary gland, heart, gastrocnemius muscle, kidney, lung, liver or intestine become YFP-labelled. These data suggest that Pdgfrα-CreERT2 transgenic mice can be used to achieve robust recombination in OPCs, while having a minimal effect on most PDGFRα+ cell populations outside of the CNS.

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<![CDATA[Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis]]> https://www.researchpad.co/article/5bd410bed5eed0c4847c6aba

Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFβ1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1.

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<![CDATA[Absence of genotoxic effects of the chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one) and its potential chemoprevention against DNA damage using in vitro and in vivo assays]]> https://www.researchpad.co/article/5989db51ab0ee8fa60bdc2a6

The chalcone (E)-1-(2-hydroxyphenyl)-3-(4-methylphenyl)-prop-2-en-1-one), or 2HMC, displays antileishmanial, antimalarial, and antioxidant activities. The aim of this study was to investigate the cytotoxic, genotoxic, mutagenic, and protective effects of 2HMC using the Ames mutagenicity test, the mouse bone marrow micronucleus test, and the comet assay in mice. In the assessment using the Ames test, 2HMC did not increase the number of His+ revertants in Salmonella typhimurium strains, demonstrating lack of mutagenicity. 2HMC showed no significant increase in micronucleated polychromatic erythrocyte frequency (MNPCE) in the micronucleus test, or in DNA strand breaks using the comet assay, evidencing absence of genotoxicity. Regarding cytotoxicity, 2HMC exhibited moderate cytotoxicity in mouse bone marrow cells by micronucleus test. 2HMC showed antimutagenic action in co-administration with the positive controls, sodium azide (SA) and 4-nitroquinoline-1-oxide (4NQO), in the Ames test. Co-administered and mainly pre-administered with cyclophosphamide (CPA), 2HMC caused a decrease in the frequency of MNPCE using the micronucleus test and in DNA strand breaks using the comet assay. Thus, 2HMC exhibited antimutagenic and antigenotoxic effects, displaying a DNA-protective effect against CPA, SA, and 4NQO carcinogens. In conclusion, 2HMC presented antimutagenic, antigenotoxic and moderate cytotoxic effects; therefore it is a promising molecule for cancer prevention.

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<![CDATA[The Immunologic Properties of Bone Morphogenic Protein Receptor IB Positive Subpopulation before and after Osteogenic Differentiation in Mouse Dermis]]> https://www.researchpad.co/article/5989d9d4ab0ee8fa60b65554

We have previously reported that human dermal bone morphogenic protein receptor (BMPR) IB positive subpopulation had a high osteogenic differentiation potential and may be a promising cell source for allogeneic bone tissue engineering. In this study, the immunologic properties of dermal BMPR-IB+ subpopulation before and after osteogenic differentiation were reported. The results confirmed that dermal BMPR-IB+ cells possessed a similar osteogenic differentiation potential with bone marrow mesenchymal stromal cells in a mouse model. Furthermore, the expression of immune rejection-related surface antigens such as major histocompatibility class II and co-stimulatory proteins (CD40, CD80, and CD86) were absent on dermal BMPRIB+ cells. Dermal BMPRIB+ cells elicited no proliferation of allogeneic splenocytes and suppressed the proliferation of stimulated immune cells. Interestingly, osteogenic differentiation in vitro had no adverse effect on the immunological features of these cells. Most importantly, inducible NO synthase (iNOS) was involved in immunoregulatory effects by undifferentiated BMPRIB+ fibroblasts, whereas indoleamine 2,3-dioxygenase (IDO) activity was related to mediating immunomodulatory function by osteogenic differentiated BMPRIB+ fibroblasts. In conclusion, dermal BMPRIB+ cells have a low immunogenicity and possess immunosuppressive capacity before and after osteogenic differentiation in vitro, which would facilitate the allotransplantation in the future. However, mechanisms mediating immunoregulatory property between undifferentiated and osteogenic differentiated BMPRIB+ fibroblasts may be different and need further investigation.

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<![CDATA[Rat Bone Marrow-Derived Schwann-Like Cells Differentiated by the Optimal Inducers Combination on Microfluidic Chip and Their Functional Performance]]> https://www.researchpad.co/article/5989d9ffab0ee8fa60b7389b

Numerous researches demonstrated the possibility of derivation of Schwann-like (SC-like) cells in vitro from bone marrow stromal cells (BMSCs). However, the concentration of the induce factors were different in those studies, especially for the critical factors forskolin (FSK) and β-heregulin (HRG). Here, we used a new and useful method to build an integrated microfluidic chip for rapid analyses of the optimal combination between the induce factors FSK and HRG. The microfluidic device was mainly composed of an upstream concentration gradient generator (CGG) and a downstream cell culture module. Rat BMSCs were cultured in the cell chambers for 11 days at the different concentrations of induce factors generated by CGG. The result of immunofluorescence staining on-chip showed that the group of 4.00 µM FSK and 250.00 ng/ml HRG presented an optimal effect to promote the derivation of SC-like cells. Moreover, the optimal SC-like cells obtained on-chip were further tested using DRG co-culture and ELISA to detect their functional performance. Our findings demonstrate that SC-like cells could be obtained with high efficiency and functional performance in the optimal inducers combination.

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<![CDATA[HMGA2 Moderately Increases Fetal Hemoglobin Expression in Human Adult Erythroblasts]]> https://www.researchpad.co/article/5989db44ab0ee8fa60bd7b80

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with beta-hemoglobin disorders. Previous studies showed that let-7 microRNAs (miRNAs) are highly regulated in erythroid cells during the fetal-to-adult developmental transition, and that targeting let-7 mediated the up-regulation of HbF to greater than 30% of the total globin levels in human adult cultured erythroblasts. HMGA2 is a member of the high-mobility group A family of proteins and a validated target of the let-7 family of miRNAs. Here we investigate whether expression of HMGA2 directly regulates fetal hemoglobin in adult erythroblasts. Let-7 resistant HMGA2 expression was studied after lentiviral transduction of CD34(+) cells. The transgene was regulated by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-OE). HMGA2-OE caused significant increases in gamma-globin mRNA expression and HbF to around 16% of the total hemoglobin levels compared to matched control transductions. Interestingly, no significant changes in KLF1, SOX6, GATA1, ZBTB7A and BCL11A mRNA levels were observed. Overall, our data suggest that expression of HMGA2, a downstream target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in adult human erythroblasts.

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