Allogenic inhibition of the stem hematopoietic cells in the bone marrow of adult mice and in the embryonal liver]

The maternal effect was shown to influence the degree of allogenic inhibition of stem hemopoietic cells of the embryonic liver and adult bone marrow in CBA and C57Bl/6 mice. The display of allogenic inhibition of stem cells of the embryonic liver and adult bone marrow proved to be similar in C57Bl/6 mice and dissimilar in CBA.     

Analysis of knockout mice suggests a role for VGF in the control of fat storage and energy expenditure

Background  

Previous studies of mixed background mice have demonstrated that targeted deletion of Vgf produces a lean, hypermetabolic mouse that is resistant to diet-, lesion-, and genetically-induced obesity. To investigate potential mechanism(s) and site(s) of action of VGF, a neuronal and endocrine secreted protein and neuropeptide precursor, we further analyzed the metabolic phenotypes of two independent VGF knockout lines on C57Bl6 backgrounds.     

Allogeneic Bone Marrow Transplant from MRL/MpJ Super-Healer Mice Does Not Improve Articular Cartilage Repair in the C57Bl/6 Strain

Background

Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ) “super-healer” mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant.

Methodology

The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ “super-healer” mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints.

Principal Findings

Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair.

Conclusion and Significance

The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.     

Bone marrow stromal cell transplantation mitigates radiation-induced gastrointestinal syndrome in mice

Background

Nuclear accidents and terrorism presents a serious threat for mass casualty. While bone-marrow transplantation might mitigate hematopoietic syndrome, currently there are no approved medical countermeasures to alleviate radiation-induced gastrointestinal syndrome (RIGS), resulting from direct cytocidal effects on intestinal stem cells (ISC) and crypt stromal cells. We examined whether bone marrow-derived adherent stromal cell transplantation (BMSCT) could restitute irradiated intestinal stem cells niche and mitigate radiation-induced gastrointestinal syndrome.

Methodology/Principal Findings

Autologous bone marrow was cultured in mesenchymal basal medium and adherent cells were harvested for transplantation to C57Bl6 mice, 24 and 72 hours after lethal whole body irradiation (10.4 Gy) or abdominal irradiation (16–20 Gy) in a single fraction. Mesenchymal, endothelial and myeloid population were characterized by flow cytometry. Intestinal crypt regeneration and absorptive function was assessed by histopathology and xylose absorption assay, respectively. In contrast to 100% mortality in irradiated controls, BMSCT mitigated RIGS and rescued mice from radiation lethality after 18 Gy of abdominal irradiation or 10.4 Gy whole body irradiation with 100% survival (p<0.0007 and p<0.0009 respectively) beyond 25 days. Transplantation of enriched myeloid and non-myeloid fractions failed to improve survival. BMASCT induced ISC regeneration, restitution of the ISC niche and xylose absorption. Serum levels of intestinal radioprotective factors, such as, R-Spondin1, KGF, PDGF and FGF2, and anti-inflammatory cytokines were elevated, while inflammatory cytokines were down regulated.

Conclusion/Significance

Mitigation of lethal intestinal injury, following high doses of irradiation, can be achieved by intravenous transplantation of marrow-derived stromal cells, including mesenchymal, endothelial and macrophage cell population. BMASCT increases blood levels of intestinal growth factors and induces regeneration of the irradiated host ISC niche, thus providing a platform to discover potential radiation mitigators and protectors for acute radiation syndromes and chemo-radiation therapy of abdominal malignancies.     

Increases in tumor necrosis factor-alpha following transient global cerebral ischemia do not contribute to neuron death in mouse hippocampus

The actions of tumor necrosis factor-alpha (TNF-alpha) produced by resident brain cells and bone marrow-derived cells in brain following a transient global ischemia were evaluated. In wild-type mice (C57Bl/6J) following 20 min ischemia with bilateral common carotid artery occlusion (BCCAo), TNF-alpha mRNA expression levels in the hippocampus were significantly increased at 3 h and 36 h and exhibited a biphasic expression pattern. There were no hippocampal TNF-alpha mRNA expression levels at early time points in either wild-type mice bone marrow transplanted (BMT)-chimeric-TNF-alpha gene-deficient (T/W) or TNF-alpha gene-deficient mice BMT-TNF-alpha gene-deficient mice (T/T), although TNF-alpha mRNA levels were detectable in T/W BMT mice at 36 h. Histopathological findings showed no intergroup differences between wild-type and TNF-alpha gene-deficient mice at 4 and 7 days after transient ischemia. In addition, nuclear factor-kappaB (NF-kappaB) was activated within 12 h after global cerebral ischemia, but electrophoretic mobility shift assays (EMSA) showed no intergroup differences between wild type and TNF-alpha gene-deficient mice. In summary, early hippocampal TNF-alpha mRNA expression may not be related to bone marrow-derived cells, and secondary TNF-alpha expression as early as 36 h after ischemia probably resulted mainly from endogenous brain cells and possibly a few bone marrow-derived cells. Although we cannot exclude the possibility of the TNF-alpha contribution to the physiologic changes of hippocampus after transient global ischemia, these results indicate that TNF-alpha does not influence the morphological changes of the hippocampal neurons under our study condition.     

Innate resistance to experimental African trypanosomiasis: differences in cytokine (TNF-alpha, IL-6, IL-10 and IL-12) production by bone marrow-derived macrophages from resistant and susceptible mice

Resistance to African trypanosomiasis is under multigenic control. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant. Macrophages eliminate opsonized trypanosomes from the bloodstream and are involved in immunosuppression. We therefore investigated the production of a number of cytokines (IL-10, IL-6, TNF-alpha and IL-12) by bone marrow-derived macrophages (BMDM) from C57Bl/6 and BALB/c mice following challenge with either Trypanosoma congolense or Trypanosoma brucei. BMDM from C57Bl/6 mice, upon challenge with whole cell extracts (WCE) of T. congolense or T. brucei, produced significantly more TNF-alpha and IL-12 than those from BALB/c mice. The production of these cytokines was significantly enhanced by pretreatment of the cells with IFN-gamma. BMDM from BALB/c mice, however, produced significantly more IL-6 and IL-10 than those from C57Bl/6 mice. In contrast to LPS stimulation, simultaneous treatment of cells with WCE and IFN-gamma enhanced IL-10 synthesis by BMDM from BALB/c mice. These results indicate that cytokine genes are differentially regulated in macrophages from trypanosome-susceptible and -resistant mice and are consistent with our previous findings wherein retrovirus-immortalized macrophage cell lines from BALB/c and C57Bl/6 mice produce differential amounts of cytokines after phagocytosis of trypanosomes.     

Adoptive transfer of mesenchymal stromal cells accelerates intestinal epithelium recovery of irradiated mice in an interleukin-6-dependent manner

Background aimsApoptosis of radiosensitive cells in the bone marrow and gut is a serious, at times life-threatening, complication arising from radiation exposure.MethodsWe investigated whether adoptive transfer of allogeneic bone marrow-derived mesenchymal stromal cells (MSC) could exert cytoprotective and life-sparing effects in a mouse model of sublethal total body irradiation (TBI).ResultsWe demonstrated that a single intraperitoneal injection of C57Bl/6 MSC given to major histocompatibility complex (MHC)-mismatched Balb/c mice within 24 h of sublethal TBI significantly reduced mortality in a dose-dependent manner. Histologic analysis and Ki67 immunostaining of jejunum sections collected 3 and 6 days post-TBI indicated that MSC protected the gastrointestinal epithelium from TBI-induced damage and significantly accelerated recovery of the gut by stimulating proliferation of the crypt cell pool. Using interleukin-6^–/– (IL-6) MSC, we demonstrated that IL-6 expressed by MSC played a role in gastrointestinal epithelium regeneration.ConclusionsOur results suggest that allogeneic MHC-mismatched MSC may be exploited to reduce gastrointestinal complications and mortality arising from ionizing radiation exposure.     

Sex and Genetic Factors Determine Osteoblastic Differentiation Potential of Murine Bone Marrow Stromal Cells

Sex and genetic factors determine skeletal mass, and we tested whether bone histomorphometric parameters were sexually dimorphic in femurs from 1 to 6 month old C57BL/6 mice. Trabecular bone volume declined more rapidly in female mice than in male littermates because of enhanced bone resorption. Although bone formation was not different between sexes, female mice exhibited a higher number of osteoblasts than male littermates, suggesting that osteoblasts from female mice may have a reduced ability to form bone. To determine the impact of sex on osteoblastogenesis, we investigated the potential for osteoblastic differentiation of bone marrow stromal cells from C57BL/6, Friend leukemia virus-B (FVB), C3H/HeJ and BALB/c mice of both sexes. Bone marrow stromal cells from female FVB, C57BL/6 and C3H/HeJ mice exhibited lower Alpl and Osteocalcin expression and alkaline phosphatase activity, and formed fewer mineralized nodules than cells from male littermates. Proliferative capacity was greater in cells from male than female C57BL/6, but not FVB, mice. Sorting of bone marrow stromal cells from mice expressing an α-Smooth muscle actin-green fluorescent protein transgene, revealed a higher yield of mesenchymal stem cells in cultures from male mice than in those from female littermates. Sex had a modest impact on osteoblastic differentiation of mesenchymal stem cells. To determine the influence of sex and genetic factors on osteoblast function, calvarial osteoblasts were harvested from C57BL/6, FVB, C3H/HeJ and BALB/c mice. Alpl expression and activity were lower in osteoblasts from C57BL/6 and C3H/HeJ, but not FVB or BALB/c, female mice than in cells from littermates. Sex had no effect on osteoclastogenesis of bone marrow cultures of C57BL/6 mice, but osteoblasts from female mice exhibited higher Rankl and lower Opg expression than cells from male littermates. In conclusion, osteoblastogenesis is sexually dimorphic and influenced by genetic factors.     

Infusion of Endothelial Progenitor Cells Accelerates Hematopoietic and Immune Reconstitution, and Ameliorates the Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation

Hematopoietic stem cells transplantation (HSCT) causes endothelial cell damage, disrupting hematopoietic microenviroment and leading to various complications. We hypothesized that infusion of endothelial progenitor cells (EPCs) may improve endothelium repair, facilitate hematopoietic reconstitution, and alleviate complications associated with HSCT. C57Bl6, and BALB/c mice received total body irradiation followed by infusion of C57Bl6-derived bone marrow (BM) cells, with or without concomitant infusion of C57Bl6-derived EPCs. The time course of hematopoietic and immune reconstitution and the severity of the graft-versus-host disease (GVHD) were monitored. Further, to confirm that EPCs promote endothelial cell recovery, HSCT mice were treated with anti-VE-cadherin antibody targeting the endothelium. The EPCs-treated mice exhibited accelerated recovery of BM vasculature, cellularity, hematopoietic stem and progenitor cell recovery, improved counts of lymphocyte subsets in peripheral blood, and facilitated spleen structure reconstruction. EPCs infusion also ameliorated the GVHD in the C57Bl6????BALB/c allo-HSCT model. Systemic administration of anti-VE-cadherin antibody significantly delayed hematological and immune reconstitution in the EPCs-infused mice. In conclusion, our data demonstrate that infusion of EPCs augments the hematopoietic and immune reconstitution, and alleviates the GVHD. These findings further highlight the relationship between the microvascular recovery, hematopoietic and immune reconstitution, and the GVHD.     

The synthetic analogue of mycoplasmal lipoprotein FSL-1 induces dendritic cell maturation through Toll-like receptor 2

Granulocyte-macrophage colony-stimulating factor-differentiated bone marrow-derived dendritic cells were stimulated with the synthetic lipopeptide S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1) or the Escherichia coli lipopolysaccharide. FSL-1 induced the production of TNF-alpha and IL-12 by C57BL/6-derived bone marrow-derived dendritic cells but not by bone marrow-derived dendritic cells from Toll-like receptor 2-deficient (TLR2(-/-)) mice. Lipopolysaccharide induced the production of TNF-alpha and IL-12 by bone marrow-derived dendritic cells derived from either type of mice. FSL-1 did not induce production of IL-10 by bone marrow-derived dendritic cells from either type of mice, whereas lipopolysaccharide induced small amounts of IL-10 by bone marrow-derived dendritic cells from both types of mice. The upregulation by FSL-1 of the expression of CD80, CD86 and the MHC class II molecule IA(b) was dose- and time-dependent on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells but not on the surface of TLR2(-/-)-derived bone marrow-derived dendritic cells. Lipopolysaccharide upregulated the expression of these molecules on the surfaces of bone marrow-derived dendritic cells from both types of mice. The expression of CD11c on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells was upregulated by stimulation with both FSL-1 and lipopolysaccharide up to 12 h; thereafter, the expression was downregulated. The results suggest that FSL-1 can accelerate maturation of bone marrow-derived dendritic cells and this FSL-1 activity is mediated by TLR2.     

Orp8 Deficiency in Bone Marrow-Derived Cells Reduces Atherosclerotic Lesion Progression in LDL Receptor Knockout Mice

Introduction

Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown.

Methods and Results

LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity.

Conclusions

Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.     

Inhibition of the CXCL12/CXCR4-Axis as Preventive Therapy for Radiation-Induced Pulmonary Fibrosis

Background

A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process.

Methodology/Principal Findings

The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process.

Conclusions/Significance

CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.     

CD34 mediates intestinal inflammation in Salmonella‐infected mice

CD34 is a highly glycosylated sialomucin expressed on a variety of cells, ranging from vascular endothelial cells to haematopoietic stem cells. Depending on its glycosylation state, CD34 has been shown to promote or inhibit cell adhesion and migration; however, a functional role for CD34 in the gut has not been determined. Using a model of Salmonella‐induced gastroenteritis, we investigated the role of CD34 in the context of infection. Upon oral infection, the number of CD34+ cells detected in the submucosa, vascular endothelium and lamina propria significantly increased in S. Typhimurium‐infected C57Bl/6 mice. The pathology of S. Typhimurium‐infected C57Bl/6 mice was characterized by recruitment of neutrophils to the site of inflammation, submucosal oedema and crypt destruction. In contrast, Cd34^?/? mice showed a delayed pathology, a defect in inflammatory cell migration into the intestinal tissue and enhanced survival. Importantly, this was not due to a lack of chemotactic signals in Cd34^?/? mice as these mice had either similar or significantly higher levels of pro‐inflammatory cytokines and chemokines post infection when compared with infected C57/Bl6 control mice. In summary, we demonstrate a novel role for CD34 in enhancing migration of inflammatory cells and thereby exacerbating host‐mediated immunopathology in the intestine of S. Typhimurium‐infected mice.     

Experimental model for studying the participation of bone marrow cells in antibody formation

Participation of bone marrow cells in the production of IgM antibody forming cells (AFC) in the primary immune response to sheep red blood cells (SRBC) in C57Bl/6 and BDA/2 mice was studied. The animals of this line differed in sensitivity to preoral benz(a)pyren (BP) injection. After BP injection a toxical injury of bone marrow cells was observed for two days in DBA 2 mice but was not marked in C57Bl/6 mice. In the former it was followed by a 10-fold decrease of IgMAFC, while no profound changes were noticed in the immune response of the latter. A new model is offered for the evaluation of bone marrow cell participation. A suggestion is made concerning some connection of immunodepression in the bone marrow with the change of the stem hemopoietic precursor differentiation.     

Specific Humoral Immunity versus Polyclonal B Cell Activation in Trypanosoma cruzi Infection of Susceptible and Resistant Mice

Background

The etiologic agent of Chagas Disease is Trypanosoma cruzi. Acute infection results in patent parasitemia and polyclonal lymphocyte activation. Polyclonal B cell activation associated with hypergammaglobulinemia and delayed specific humoral immunity has been reported during T. cruzi infection in experimental mouse models. Based on preliminary data from our laboratory we hypothesized that variances in susceptibility to T. cruzi infections in murine strains is related to differences in the ability to mount parasite-specific humoral responses rather than polyclonal B cell activation during acute infection.

Methodology/Principal Findings

Relatively susceptible Balb/c and resistant C57Bl/6 mice were inoculated with doses of parasite that led to similar timing and magnitude of initial parasitemia. Longitudinal analysis of parasite-specific and total circulating antibody levels during acute infection demonstrated that C57Bl/6 mice developed parasite-specific antibody responses by 2 weeks post-infection with little evidence of polyclonal B cell activation. The humoral response in C57Bl/6 mice was associated with differential activation of B cells and expansion of splenic CD21^highCD23^low Marginal Zone (MZ) like B cells that coincided with parasite-specific antibody secreting cell (ASC) development in the spleen. In contrast, susceptible Balb/c mice demonstrated early activation of B cells and early expansion of MZ B cells that preceded high levels of ASC without apparent parasite-specific ASC formation. Cytokine analysis demonstrated that the specific humoral response in the resistant C57Bl/6 mice was associated with early T-cell helper type 1 (Th1) cytokine response, whereas polyclonal B cell activation in the susceptible Balb/c mice was associated with sustained Th2 responses and delayed Th1 cytokine production. The effect of Th cell bias was further demonstrated by differential total and parasite-specific antibody isotype responses in susceptible versus resistant mice. T cell activation and expansion were associated with parasite-specific humoral responses in the resistant C57Bl/6 mice.

Conclusions/Significance

The results of this study indicate that resistant C57Bl/6 mice had improved parasite-specific humoral responses that were associated with decreased polyclonal B cell activation. In general, Th2 cytokine responses are associated with improved antibody response. But in the context of parasite infection, this study shows that Th2 cytokine responses were associated with amplified polyclonal B cell activation and diminished specific humoral immunity. These results demonstrate that polyclonal B cell activation during acute experimental Chagas disease is not a generalized response and suggest that the nature of humoral immunity during T. cruzi infection contributes to host susceptibility.     

Genetic and spectrally distinct <Emphasis Type="Italic">in vivo</Emphasis>imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein

Background  

DsRed the red fluorescent protein (RFP) isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP) for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems.     

Characterisation of eGFP-transgenic BALB/c mouse strain established by lentiviral transgenesis

Lentiviral technology is a powerful tool for the creation of stable transgenic animals. However, uncertainties have remained whether constitutive promoters resist long-term silencing. We used concentrated HIV-1 based lentiviral vectors to create stable transgenic BALB/c mice by perivitelline injection. In our vectors eGFP expression was driven by the human EF1α promoter. The established transgenic animals were analyzed for eGFP expression by in vivo fluorescence imaging, PCR, histology and flow-cytometry. eGFP expression showed even distribution without mosaicism; however, tissue-dependent differences of eGFP expression were observed. Up to the sixth generation only one newborn showed eGFP inactivation. eGFP + transgenic bone marrow cells efficiently provided long-term haemopoietic repopulation in radiation chimeras, regenerating all bone marrow-derived lineages with eGFP + cells with distinct eGFP expression profiles. The established eGFP + BALB/c mouse strain is expected to be extremely useful in various immunological experiments.     

Isolation murine mesenchymal stem cells by positive selection

Isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures is arduous because of the unwanted growth of hematopoietic cells and non-MSCs. In this work, homogenous populations of CD34⁺ MSCs from mouse bone marrow were isolated via positive selection. For this purpose, C57Bl/6 mice were killed and bone marrow cells were aspirated before incubation with magnetic bead conjugated to anti-CD34 antibody. A sample of positively selected CD34⁺ cells were prepared for flow cytometry to examine the expression of CD34 antigen and others were subcultured in a 25-cm² culture flask. To investigate the mesenchymal nature, the plastic adherent cultivated cells were induced to differentiate along osteoblastic and adipogenic lineages. Furthermore, the expression of some surface markers was investigated by flow cytometry. According to the result, purified populations of fibroblast-like CD34⁺ cells were achieved in the first passage (1 wk after culture initiation). The cells expressed CD34, CD44, Sca-1, and Vcam-1 antigens (markers) but not CD11b and CD45. They were capable of differentiating into osteocytes and adipocytes. This study indicated that our protocol can result in the efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We have shown that murine bone marrow-derived CD34⁺ cells with plastic adherent properties and capability of differentiating into skeletal lineages in vitro are MSCs.     

Synergistic targeting with bone marrow-derived cells and PDGF improves diabetic vascular function

Diabetes mellitus is associated with an increased risk of vascular disease, with significant alterations in systemic endothelial progenitor cells (EPCs) and peripheral vascular function. To identify the contribution of the different vascular compartments in the diabetic impairment of vascularization, we employed streptozotocin- and control-treated 3-mo-old C57Bl/6 mice in an isogeneic pinnal cardiac allograft model, revealing a significant delay in vascularization of wild-type cardiac tissue transplanted into diabetic mice. To investigate the basis of this impairment, the function of diabetic bone marrow cells was tested by transplantation of bone marrow cells isolated from diabetic and control mice into intact, unirradiated 18-mo-old C57Bl/6 mice, which have impaired function of both EPCs and peripheral endothelial cells. Importantly, cells derived from control, but not diabetic, bone marrow integrated into transplanted cardiac allografts. To assess the contribution of diabetic changes in the local vasculature, diabetic mice were treated with pinnal injections of platelet-derived growth factor (PDGF)-AB, which promotes cardiac angiogenesis in wild-type mice. However, whereas PDGF-AB enhanced allograft function in control mice, the activity of the cardiac transplants in the PDGF-AB-treated diabetic mice was significantly decreased. To decipher the potential interactions between systemic bone marrow-derived cells and local vascular pathways, diabetic mice were transplanted with wild-type bone marrow cells with or without PDGF-AB pinnal pretreatment, resulting in improved allograft function and donor cell recruitment only in the combination treatment arm. Overall, these studies show that the diabetic impairment in cardiac angiogenesis can be reversed by targeting the synergism between local trophic pathways and systemic cell function.