Rat Bone Marrow Mononuclear Cells Research Articles

Identification of a distinct cluster of GDF15high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities.

Macrophage-mediated inflammatory response may have crucial roles in the pathogenesis of a variety of human diseases. Growth differentiation factor 15 (GDF15) is a cytokine of the transforming growth factor-β superfamily, with potential anti-inflammatory activities. Previous studies observed in human lungs some macrophages which expressed a high level of GDF15. In the present study, we employed multiple techniques, including immunofluorescence, flow cytometry, and single-cell RNA sequencing, in order to further clarify the identity of such GDF15high macrophages. We demonstrated that macrophages derived from human peripheral blood mononuclear cells and rat bone marrow mononuclear cells by in vitro differentiation with granulocyte-macrophage colony stimulating factor contained a minor population (~1%) of GDF15high cells. GDF15high macrophages did not exhibit a typical M1 or M2 phenotype, but had a unique molecular signature as revealed by single-cell RNA sequencing. Functionally, the in vitro derived GDF15high macrophages were associated with reduced responsiveness to pro-inflammatory activation; furthermore, these GDF15high macrophages could inhibit the pro-inflammatory functions of other macrophages via a paracrine mechanism. We further confirmed that GDF15 per se was a key mediator of the anti-inflammatory effects of GDF15high macrophage. Also, we provided evidence showing that GDF15high macrophages were present in other macrophage-residing human tissues in addition to the lungs. Further scRNA-seq analysis in rat lung macrophages confirmed the presence of a GDF15high sub-population. However, these data indicated that GDF15high macrophages in the body were not a uniform population based on their molecular signatures. More importantly, as compared to the in vitro derived GDF15high macrophage, whether the tissue resident GDF15high counterpart is also associated with anti-inflammatory functions remains to be determined. We cannot exclude the possibility that the in vitro priming/induction protocol used in our study has a determinant role in inducing the anti-inflammatory phenotype in the resulting GDF15high macrophage cells. In summary, our results suggest that the GDF15high macrophage cells obtained by in vitro induction may represent a distinct cluster with intrinsic anti-inflammatory functions. The (patho)physiological importance of these cells in vivo warrants further investigation.

NR4A2 alleviates cardiomyocyte loss and myocardial injury in rats by transcriptionally suppressing CCR5 and inducing M2 polarization of macrophages.

CC chemokine receptor 5 (CCR5) has been demonstrated to be correlated to activation of pro-inflammatory immune cells and tissue injury. This study focused on the role of CCR5 in myocardial injury in rats with diabetic cardiomyopathy (DCM) and the mechanism of action. A rat model of DCM was induced by streptozotocin (STZ). CCR5 was knocked down in rats to determine its role in myocardial injury and immune cell infiltration. The upstream regulators of CCR5 were bioinformatically predicted and the binding between nuclear receptor subfamily 4 group A member 2 (NR4A2) and CCR5 was validated. The portion of M1 and M2 macrophages in tissues was determined by flow cytometry or double-labeling immunofluorescence. Rat bone marrow mononuclear cells (BMMCs) were treated with granulocyte/macrophage colony stimulating factor (GM-CSF/M-CSF) and co-cultured with H9C2 cells for in vitro experiments. STZ-treated rats had impaired cardiac function and increased levels of creatine kinase-MB, cardiac troponin I and lactate dehydrogenase. CCR5 inhibition significantly alleviated myocardial injury in rats and reduced the portion of M1 macrophages in rat cardiac tissues. NR4A2, which could suppress CCR5 transcription, was poorly expressed in rats with DCM. NR4A2 overexpression played a similar myocardium-protective role in rats. In vitro, overexpression of NR4A2 induced M2 polarization of macrophages, which protected the co-cultured H9C2 cells from high glucose-induced damage, but the protective role was blocked after CCR5 overexpression. This study demonstrated that NR4A2 suppresses CCR5 expression and promotes M2 polarization of macrophages to alleviate cardiomyocyte loss and myocardial injury.

Effect of procyanidine on VEGFR-2 expression and transduction pathway in rat endothelial progenitor cells under high glucose conditions.

The protective effect of procyanidine and its oligomers against high glucose-mediated oxidative stress injury in endothelial progenitor cells (EPCs), and effect of procyanidin on vascular endothelial growth factor receptor-2 (VEGFR-2) expression and downstream signal pathway were analyzed in vitro. Rat bone marrow mononuclear cells were isolated, cultured under normal and high glucose (HG) conditions, and the changes in cell morphology observed. The EPCs were identified, and the oxidative stress products produced by EPCs (under normal and HG conditions) were quantified. Subsequently, an appropriate number of EPCs were cultured with and without procyanidin (OPC), and the MDA concentration and relative expression of VEGFR-2, AKT, IκB-α, and nuclear factor (NF)-κB were detected 1, 3, 5, and 7 days post-culture. We observed minor (round, translucent, gradually adhering) and significant (fusiform morphology/pebble distribution) cell morphological changes 3 and 7 days post-culture, respectively. Apoptosis and oxidative stress product release in EPCs cultured with HG increased significantly compared to the control group (P < 0.05). The oxidative stress product generation and relative expression of VEGFR-2, AKT, IkB-α, and NF-κB were not significantly affected by OPC addition in normal glucose conditions (P > 0.05); alternately, products generated as a result of oxidative stress were significantly reduced, the relative expression of VEGFR-2, AKT, and NF-κB protein was upregulated, and that of IκB-α was downregulated (P < 0.05) in HG + OPC EPCs. Therefore, procyanidin may promote cell proliferation by alleviating oxidative damage to EPCs under HG conditions, and upregulating VEGFR-2 expression and its downstream signal pathway.

REVASCULARIZATION OF ACELLULAR RAT LUNG SCAFFOLDS USING THE ENDOTHELIAL COLONY FORMING CELLS

s S157 revascularization of acellular lung scaffolds using ECFCs as a critical first step in engineering truly functional grafts. OBJECTIVES: 1) Compare decellularization Methods to achieve optimal decellularization with maintained vascular perfusion. 2) Regenerate vascular endothelium using ECFCs. METHODS: Acellular rat lung scaffolds were generated by static incubation and perfusions Methods of decellularization. For static incubation, lungs were submerged into Triton-X 100 (0.1%) and sodium deoxycholate (2%) based decellularization buffer. For perfusion decellularization, isolated rat lungs were perfused using CHAPS buffer (8mM CHAPS, 1M NaCl, 25mM EDTA in 1X PBS) or SDS-Triton-X buffer (0.1% SDS followed by 1% Triton-X). Decellularization of lungs was evaluated by tissue histology, total DNA measurement and immuno-blotting. For revascularization, ECFCs were generated from human peripheral blood or rat bone marrow derived mononuclear cells and characterized for ECFCmarkers. Further, we also studied initial engraftment of human ECFCs in the rat lung scaffolds. RESULTS: Static incubation method of decellularization generated acellular lung scaffolds with no evidence of intact cells; however, presence of intracellular protein (GAPDH) was evident. Vascular perfusion could not be established in these scaffolds. On the other side, perfusion decellularization using CHAPS buffer produced acellular lung scaffolds with maintained extracellular structure. Moreover, perfusion decellularization with CHAPS buffer consistently demonstrated complete absence of cells, below detectable DNA remnants and intracellular protein, with more optimal vascular perfusion. ECFCs were successfully derived from human peripheral blood and rat bone-marrow mononuclear cells. ECFCs were positive for endothelial specific markers such as Ac-LDL uptake, lectin binding and expressed CD31, von Willbrand Factor (vWF) and VEGFR2. Moreover, these cells did not express pan-leukocyte marker CD45. Initial engraftment study of human ECFCs in rat lung scaffolds showed engraftment of human ECFCs in vascular compartment of the lung scaffolds. CONCLUSION: We successfully prepared acellular lung scaffolds and identified ECFC, which can be utilized to generate truly functional grafts. HSF, CIHR 204 GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION M Nakhaei-Nejad, AG Murray, N Jahroudi

Biological properties of bone marrow-derived early and late endothelial progenitor cells in different culture media

Ex vivo expansion of endothelial progenitor cells (EPCs) may be a promising strategy to overcome the clinical problem of limited cell numbers. As the culture medium is the key for the cell characteristics, the effects of different culture media on EPCs were investigated in the present study. Rat bone marrow mononuclear cells were cultured in different media, including M-199 media with 20% fetal bovine serum (FBS) and bovine pituitary extract (M1); M-199 media with 10% FBS, 20 ng/ml vascular endothelial growth factor (VEGF) and 10 ng/ml basic fibroblast growth factor (bFGF; M2) or epidermal growth medium (EGM)-2MV media. The cell morphology and biological functions, such as proliferation, adhesion, migration, tube formation and nitric oxide (NO) production were subsequently assayed in vitro. Moreover, endothelial biomarkers and apoptosis were also analyzed. The results showed that endothelial‑like cells appeared in all of the culture systems. First‑passage cells, namely early EPCs, tended to form colonies in M2 and EGM-2MV media but showed a fusiform shape in M1 media. The 3rd or 4th generation EPCs, namely late EPCs, cultured in EGM-2MV media exhibited increased adhesion, migration, tube formation and NO production as compared with EPCs in M1 or M2 media. Furthermore, late EPCs cultured in EGM-2MV expressed higher levels of endothelial cell markers, such as von Willibrand factor (vWF)and CD31, but relatively greater levels of apoptosis were observed. In conclusion, cell culture conditions, for example the medium used, affects the biological properties of bone marrow-derived early and late EPCs.

Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25 × 10(6) cells/mL, containing an estimated 5 × 10(4) MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2 × 10(5) cells/mL) were added to a 65-35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering.

Mesenchymal stem cell therapy in a rat model of osteoarthritis

Mesenchymal stem cell therapy in a rat model of osteoarthritis

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2-EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine-sensitive K(+) current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.

Intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in endothelial progenitor cells from rat bone marrow mononuclear cells

Bone marrow endothelial progenitor cells (BMEPCs) are believed to be a promising cell source for regenerative medicine; however, their electrophysiology properties have not been fully clarified, which is important to the clinical application of BMEPCs. The current study was designed to determine the transmembrane ion currents and mRNA expression levels of related ion channel subunits in rat BMEPCs. Bone marrow mononuclear cells were isolated by density gradient separation and cultured in EPC medium. The transmembrane ion currents were determined using whole-cell patch-voltage clamp technique, and the levels of mRNA and protein expressions of functional ionic channels were measured using RT-PCR and western immunoblot analysis. We observed two types of ionic currents inundifferentiated rat BMEPCs. One was Ca(2+) -activated potassium current (I(kca) ), which was seen in approx. 90% of cells when 1μm Ca(2+) was employed in pipette solution, and it was predominantly inhibited by intermediate-conductance I(kca) inhibitor clotrimazole. The other one was volume-sensitive chloride current (I(cl) ), which was detected in 85.7% of cells when BMEPCs were subjected to K(+) -free hypotonic extracellular solution, whose currents could be inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). The corresponding ion channel genes and proteins, KCNN4 for I(kca) and Clcn3 for I(cl) , were confirmed by RT-PCR and western immunoblot analysis of BMEPCs. Our results demonstrated for the first time that rat BMEPCs expressed intermediate-conductance Ca(2+) -activated potassium currents and volume-sensitive chloride currents, and corresponding genes and proteins of these two channels are KCNN4 and Clcn3 respectively.

Influence of Angiogenesis by Implantation of Bone Marrow Mononuclear Cells in the Rat Ischemic Heart

Bone marrow implantation (BMI) enhances angiogenesis in several animal models of ischemic diseases, and it is currently applied in the clinical treatment of humans. However, the mechanisms of this effect have not yet been fully described. Rat bone marrow mononuclear cells (BM-MNCs) were obtained by Histopaque density gradient centrifugation and injected directly into the ischemic myocardium of the test rats (BMI group), which were then examined and compared with the groups that received surgery only (Controls) or surgery and an injection of phosphate buffered saline (PBS group). Cardiac function was evaluated by echocardiography, and neovascularization was examined both histologically and immunohistochemically before, 1 day after, and 7 or 28 days after the operation. BM-MNCs were analyzed by fluorescence staining for the endothelial cell marker CD31 and alkaline phosphatase (ALP). The mechanisms of angiogenesis were examined by gene expression analysis. In the BMI group, cardiac function parameters at 7 days after operation were significantly improved and the number of capillaries in the myocardium was significantly larger than that in the PBS and Control groups. Gene analysis showed the expression of 12 genes in the BMI group 7 days after operation. The implantation of BM-MNCs into the myocardiumin cases of acute infarction enhances cytoprotection and angiogenesis by affecting gene expression.

Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium

Neovascularization may improve cardiac function and prevent further scar tissue formation in infarcted myocardium. A number of studies have demonstrated that bone marrow-derived cells have the potential to induce neovascularization in ischemic tissues. In this study, we hypothesized that implantation of bone marrow mononuclear cells (BMMNCs) using injectable fibrin matrix further enhances neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. To test this hypothesis, infarction was induced in rat myocardium by cryoinjury. Three weeks later, rat BMMNCs were mixed with fibrin matrix and injected into the infarcted myocardium. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Eight weeks after the treatments, histological analyses indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium compared to BMMNC implantation without matrix. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye prior to implantation survived in the infarcted myocardium at 8 weeks of implantation. Importantly, implantation of BMMNCs using fibrin matrix resulted in much more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. The microvessel density in infarcted myocardium was significantly higher ( p<0.05) when BMMNCs were implanted using fibrin matrix (350±22 microvessels/mm 2) compared to BMMNC implantation without matrix (262±13 microvessels/mm 2) and medium injection (76±9 microvessels/mm 2). In addition, average internal diameter of microvessels was significantly larger ( p<0.05) in BMMNC implantation with fibrin matrix group (14.6±1.2 μm) than BMMNC implantation without matrix group (10.2±0.7 μm) and medium injection group (7.3±0.5 μm). These results suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy for myocardial infarction treatment.

Erythroid accelerating factor detected in serum from rats with drug induced hemolysis.

We have previously observed that an erythroid enhancing activity presents in rat serum in the early stage of drug induced hemolytic anemia. The further studies on biological and physicochemical aspects of this erythroid accelerating factor (EAF) is described in this paper. Hemolytic anemia was induced in rats by single intraperitoneal injection of acetylphenylhydrazine (APH) and serum was obtained from the rats on day 1 after APH injection. It was first fractionated by ultrafiltration on Amicon Diaflo membranes to give a series of fractions lying in the following ranges of molecular weight: 10-30 kDa, 30-50 kDa, 50-100 kDa, and >100 kDa. Among those fractions, largest increase in the number of colony forming unit erythroid CFU-E) colonies was shown in the fraction of >100 kDa that was subsequently fractionated by fast protein liquid chromatography (FPLC) system. EAF activity for CFU-E proliferation was detected in a FPLC fraction corresponding to a molecular weight of about 160 kDa. An addition of EAF significantly increased with dose dependent manner in the number of CFU-E colonies from rat bone marrow mononuclear cells. EAF alone had no burst promoting activity and exhibited no distinct activity to proliferate burst forming unit-erythroid even when interleukin-3 (IL-3) and high concentration (2 U/ml) of erythropoietin (Epo) were added together to the culture. The stimulating effect of EAF on CFU-E was markedly dependent on the presence of adherent cells in the culture. Partially purified protein was relatively heat-unstable (60% at 75 degrees C, 30 minutes) and sensitive to treatment with trypsin and alpha-galactosidase. These results suggest that EAF is a novel factor, possible glycoprotein to reinforce Epo function and is different from various cytokines previously documented because of differences of approximate molecular weight.

Erythroid accelerating activity of rat serum in early stage of drug induced hemolysis.

An increase in the number of erythroblasts can be seen to some extent in the bone marrow of rats in the early stage of experimentally induced hemolytic anemia prior to any elevation in the plasma erythropoietin (Epo) level. This observation suggests that there is another erythroid stimulating factor present other than Epo. We studied the enhancing effect of serum, taken sequentially during experimentally induced hemolysis in rats, on erythroid proliferation, differentiation and maturation in vitro. Single intraperitoneal injection of 60 mg/kg of acetylphenylhydrazine (APH) induced self-limited hemolytic anemia in rats, in which the hematocrit dropped rapidly with a nadir at day 4 after APH injection, followed by a gradual increase with return to normal level by day 8. Serum obtained consecutively every day after APH injection from day 1 to day 7 was applied to an in vitro culturing system of erythroid progenitors. Addition of day 1 serum, in which an elevation of Epo level had not occurred, to a conventional methyl-cellulose culture of rat bone marrow mononuclear cells (BM-MNCs) resulted in a significant increase in the number of colonies derived from colony forming unit erythroid, but not in burst forming unit erythroid. This erythropoietic activity of the serum was particularly evident in the presence of Epo. In the liquid culture of BM-MNCs, day 1 serum also showed some enhancing effect on erythroblast formation. We were able to see significant differences in these erythroid enhancing activities induced by serum drawn on day 1 in comparison to the serum drawn on subsequent days. These results suggest that an unknown erythroid enhancing factor besides Epo stimulates erythropoiesis in the early stage of hemolytic anemia or sudden hypoxia before there is a measurable rise in the serum Epo level. We propose that this factor be termed erythroid accelerating factor (EAF).

Differential posttranslational processing of proenkephalin in rat bone marrow and spleen mononuclear cells: evidence for synenkephalin cleavage.

Proenkephalin (PENK) messenger RNA was reported to be present in bone marrow mononuclear cells (BMMC) and spleen mononuclear cells (SMC). Nevertheless, the pattern of PENK products in normal cells of the rat immune system, which is important for defining the physiological role of PENK gene expression, has not been well established. In this work we have characterized the processing of the opioid portion (met-enkephalin-containing peptides) and nonopioid portion (synenkephalin-derived peptides) of PENK in rat BMMC and SMC. Met-enkephalin-containing peptides were detected in mononuclear cells of both hematopoietic tissues. In BMMC, free immunoreactive (IR)-met-enkephalin corresponded only to the 15% of total met-enkephalin-IR, whereas in SMC it represented the 66.5%. Gel filtration chromatography showed that BMMC contained partially processed PENK-derived peptides of high and intermediate molecular weight, whereas SMC displayed fully processed products containing met-enkephalin and/or the carboxyterminal portion of synenkephalin. HPLC purification of low molecular weight products showed that free IR-met-enkephalin in SMC mainly corresponded to met-enkephalin and oxidized met-enkephalin. In addition we have characterized in SMC three peptides lower than 3.0 kilodalton containing the C-terminal sequence of synenkephalin. These peptides were purified by gel filtration, affinity chromatography, ion exchange chromatography, and HPLC. These results show that PENK was processed in mononuclear cells of the primary (bone marrow) and secondary (spleen) organs of the rat hematopoietic system, as occurs in neural and endocrine tissues. Nevertheless, the precursor was cleaved only in the latter tissue to low molecular weight peptides. Furthermore we demonstrated that synenkephalin (proenkephalin 1-70) in SMC was processed to low molecular weight peptides containing the C-terminus free. This last result suggests that a dibasic Lys-Lys and monobasic (Lys) sites were cleaved.