The Co-Transplantation of Bone Marrow Derived Mesenchymal Stem Cells Reduced Inflammation in Intramuscular Islet Transplantation

Aims/HypothesisAlthough the muscle is one of the preferable transplant sites in islet transplantation, its transplant efficacy is poor. Here we attempted to determine whether an intramuscular co-transplantation of mesenchymal stem cells (MSCs) could improve the outcome.MethodsWe co-cultured murine islets with MSCs and then analyzed the morphological changes, viability, insulin-releasing function (represented by the stimulation index), and gene expression of the islets. We also transplanted 500 islets intramuscularly with or without 5 × 10⁵ MSCs to diabetic mice and measured their blood glucose level, the glucose changes in an intraperitoneal glucose tolerance test, and the plasma IL-6 level. Inflammation, apoptosis, and neovascularization in the transplantation site were evaluated histologically.ResultsThe destruction of islets tended to be prevented by co-culture with MSCs. The stimulation index was significantly higher in islets co-cultured with MSCs (1.78 ± 0.59 vs. 7.08 ± 2.53; p = 0.0025). In terms of gene expression, Sult1c2, Gstm1, and Rab37 were significantly upregulated in islets co-cultured with MSCs. Although MSCs were effective in the in vitro assays, they were only partially effective in facilitating intramuscular islet transplantation. Co-transplanted MSCs prevented an early inflammatory reaction from the islets (plasma IL-6; p = 0.0002, neutrophil infiltration; p = 0.016 inflammatory area; p = 0.021), but could not promote neovascularization in the muscle, resulting in the failure of many intramuscular transplanted islets to engraft.ConclusionsIn conclusion, co-culturing and co-transplanting MSCs is potentially useful in islet transplantation, especially in terms of anti-inflammation, but further augmentation for an anti-apoptosis effect and neovascularization is necessary.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation I. Regeneration and Quantification of Thymocyte Progenitor Cells In the Bone Marrow

Growth kinetics of the donor-type thymus cell population after transplantation of bone marrow into irradiated syngeneic recipient mice is biphasic. During the first rapid phase of regeneration, lasting until day 19 after transplantation, the rate of development of the donor cells is independent of the number of bone marrow cells inoculated. the second slow phase is observed only when low numbers of bone marrow cells (2.5 × 10⁴) are transplanted. the decrease in the rate of development is attributed to an efflux of donor cells from the thymus because, at the same time, the first immunologically competent cells are found in spleen. After bone marrow transplantation the regeneration of thymocyte progenitor cells in the marrow is delayed when compared to regeneration of CFUs. Therefore, regenerating marrow has a greatly reduced capacity to restore the thymus cell population. One week after transplantation of 3 × 10⁶ cells, 1% of normal capacity of bone marrow is found. It is concluded that the regenerating thymus cells population after bone marrow transplantation is composed of the direct progeny of precursor cells in the inoculum.     

Metabolic and Pancreatic Effects of Bone Marrow Mesenchymal Stem Cells Transplantation in Mice Fed High-Fat Diet

The purpose of this study was to investigate the effects of multiple infusions of allogeneic MSCs on glucose homeostasis and morphometry of pancreatic islets in high- fat diet (HFD) fed mice. Swiss mice were fed standard diet (C group) or HFD (HFD group). After 8 weeks, animals of HFD group received sterile phosphate-buffered saline infusions (HFD-PBS) or four infusions of MSCs one week apart (HFD-MSCs). Fasting glycemia (FG) was determined weekly and glucose (GTT) and insulin (ITT) tolerance tests were performed 4, 8, 12, and 16 weeks after the infusions of MSCs. The MSCs transplanted mice were classified as responder (FG < 180 mg/dL, 72.2% of transplanted mice) or non-responder (FG > 180mg/dL, 28.8%) Seven weeks after MSCs infusions, FG decreased in HFD-MSCs responder mice compared with the HFD-PBS group. Sixteen weeks post MSCs infusions, GTT and ITT areas under the curve (AUC) decreased in HFD-MSCs responder mice compared to HFD-PBS group. Serum insulin concentration was higher in HFD-PBS group than in control animals and was not different compared with the other groups. The relative volume of α-cells was significantly smaller in HFD-PBS group than in C group and significantly higher in HFD-MSCs-NR than in HFD-PBS and HFD-MSCs-R groups. Cell apoptosis in the islets was higher in HFD-PBS group than in C group, and lower in HFD-MSCs responder mice than in HFD-PBS group and non-responder animals. The results demonstrate the ability of multiple infusions of MSCs to promote prolonged decrease in hyperglycemia and apoptosis in pancreatic islets and increase in insulin sensitivity in HFD fed mice.     

Infusion of Bone Marrow Mononuclear Cells Reduces Lung Fibrosis but Not Inflammation in the Late Stages of Murine Silicosis

We hypothesized that infusion of bone marrow mononuclear cells (BMMCs) in the late stages of silica-induced damage would reduce the remodelling process in a murine model of silicosis. C57BL/6 mice were assigned to 2 groups. In the SIL group, mice were instilled with a silica particle suspension intratracheally. Control (C) mice received saline under the same protocol. On the 40th day, some of the animals from both groups were killed. The others were treated with either saline or BMMCs (1×10⁶cells) intravenously (C+BMMC and SIL+BMMC), and the mice were killed 70 days after the start of the protocol. In the mice in the SIL+BMMC group, collagen deposition, the presence of silica particles inside nodules, the presence of macrophages and cells reactive for inducible nitric oxide synthase were reduced. Lung parameters also improved. Beyond that, the total and differential cellularity of bronchoalveolar lavage fluid, immunoexpression of transforming growth factor-β, the number of T regulatory cells and apoptosis were increased. However, the presence of male donor cells in lung tissue was not observed using GFP+ cells (40d) or Y chromosome DNA (70d). Therefore, BMMC therapy in the late stages of experimental silicosis improved lung function by diminishing fibrosis but inflammatory cells persisted, which could be related to expansion of T regulatory cells, responsible for the beneficial effects of cell therapy.     

Ultrasound-Microbubble Transplantation of Bone Marrow Stromal Cells Improves Neurological Function after Forebrain Ischemia in Adult Mice

In this study, bone marrow stromal cells (MSCs) were transplanted into the brain of adult rats after forebrain ischemia induced by 4VO. SD rats (n = 60) were randomly divided into three groups: (I) rats (n = 20) were subjected to 4VO and transplanted with MSCs into the ischemic region using ultrasound-microbubble method, (2) rats (n = 20) were subjected to 4VO and transplanted with MSCs into the ischemic region (n = 20), and (3) 4VO alone (n = 20). Rats were sacrificed 28 days after treatment. Neurological functions of rats were evaluated by Morris Water Maze. The current findings suggest that the ultrasound microbubble transplanted MSCs survived in the ischemic brain and significantly improved functional recovery of adult rats compared to regular transplantation.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation Ii. Physical Characteristics of Thymocyte Progenitor Cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H × AKR)F₁ bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, thymocyte progenitor cells show the same distribution as CFUs. the relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Allergen-Induced Bone Marrow Eosinophilopoiesis and Airways Eosinophilic Inflammation in Leptin-Deficient ob/ob Mice

Asthma and obesity are growing epidemics in the world. It is well established that obesity worsens the asthma outcomes. High-fat diet-induced obesity in mice exacerbates the pulmonary eosinophilic inflammation. We have used wild-type (WT) and ob/ob mice to further explore the mechanisms by which obesity aggravates the pulmonary eosinophilic inflammation. The eosinophil (EO) number in bronchoalveolar lavage (BAL) fluid, lung tissue, blood, and bone marrow were evaluated at 24, 48, and 72 h after ovalbumin (OVA) challenge in sensitized mice. The basal EO number (phosphate-buffered saline (PBS)-instilled mice) in lung tissue was about 3.5-fold greater in ob/ob compared with WT mice. OVA challenge in ob/ob mice promoted an EO accumulation into the lung that was accompanied by a lower emigration to airways lumen (BAL fluid) in comparison with WT mice. OVA challenge also markedly elevated the number of mature and immature EO in bone marrow of ob/ob mice at 24 h compared with WT group. Blood EO at 48 h was markedly greater in ob/ob mice. Tumor necrosis factor (TNF)-α and interleukin (IL)-10 levels in BAL fluid were significantly higher in ob/ob mice, whereas no changes for IL-5 and eotaxin were found. The IL-6 levels were significantly lower in ob/ob mice. In conclusion, OVA challenge in ob/ob obese mice potentiates eosinophilopoiesis and promotes an accumulation of EO into the lung tissue, delaying their transit to airways lumen. The longer EO remain into the lung tissue is likely to contribute, at least in part, to the asthma worsened by obesity.     

Bone Marrow Mesenchymal Stromal Cell-Derived Periostin Promotes B-ALL Progression by Modulating CCL2 in Leukemia Cells

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骨髓单个核细胞抗体及外周血B淋巴细胞与IRP的相关性研究

采用流式细胞术双标法检测拟诊免疫相关性全血细胞减少症患者(A组)、非免疫相关性恶性血液病患者(B组)及正常人(正常对照组)的骨髓单个核细胞结合的自身抗体,同时检测B组、确诊免疫相关性全血细胞减少症(C组)及正常对照组外周血B淋巴细胞及CD5+B淋巴细胞比率;A组中16例骨髓造血细胞自身抗体阳性,阳性率88.88%;B组1例骨髓造血细胞自身抗体阳性,阳性率9.09%。C组外周血B淋巴细胞、CD5+B淋巴细胞比率显著高于B组及正常对照组(P均<0.05),而正常对照组外周血B淋巴细胞、CD5+B淋巴细胞显著高于B组(P均<0.05);IRP患者骨髓单个核细胞自身抗体表达显著增高,B淋巴细胞总数及CD5+B淋巴细胞数量显著增高可能是IRP发病的重要因素之一;利用流式细胞术检测骨髓造血细胞自身抗体及B淋巴细胞数可以为IRP提供科学可靠的依据,优于骨髓Coomb’s实验。     

Single Cell Gene Profiling Revealed Heterogeneity of Paracrine Effects of Bone Marrow Cells in Mouse Infarcted Hearts

It is now recognized that transplantation of bone marrow cells (BMCs) into infarcted hearts has the capacity to improve the cardiac function through paracrine effects. However, detailed expression levels of paracrine factors in BMCs in infarcted hearts are poorly described. By use of laser capture microdissection combined with real-time PCR, we depicted the expression profiles of paracrine factors in infarcted hearts versus normal hearts. Consistent with the in vivo observation, a similar expression pattern was evidenced in cultured BMCs. Furthermore, BMCs displayed heterogeneity of paracrine effects in infarcted hearts as analyzed at the single cell level using single cell PCR. Interestingly, the CD45⁺ subpopulation showed higher expression levels of angiogenic factors compared to other subpopulations. Finally, most angiogenic factors were induced under the microenvironment of infarction. Our study demonstrated the heterogeneity of paracrine effects in BMCs at single cell level in infarcted hearts, highlighting preferential expression of angiogenic factors in the CD45⁺ subpopulation. These findings broaden our understanding of paracrine effects of BMCs in vivo, and offer new insights into BMCs therapy in myocardial infarction (MI).     

G-CSF动员外周血单个核细胞移植治疗大鼠缺血缺氧性脑病

目的:应用人外周血单个核细胞移植治疗新生大鼠缺血缺氧性脑病,探讨干细胞移植治疗神经系统损伤性疾病的有效性和安全性。方法:7 d龄SD大鼠随机分为3组(n=12),即正常组、手术组、细胞移植组;在征得同意的情况下利用粒细胞集落刺激因子(G-CSF)对捐献者的外周血进行动员,采用血细胞分离仪分离外周血单个核细胞,并进行鉴定及荧光标记;通过尾静脉注射的方法将标记细胞植入经免疫抑制剂处理的大鼠体内,利用HE染色法观察模型建立后大鼠大脑的损伤情况,利用荧光显微镜观察移植细胞在宿主体内的存活、迁移及分化情况,利用斜板实验明确细胞移植对宿主神经功能损伤情况的影响。结果:移植细胞可在宿主脑内存活,向损伤部位迁移,细胞移植可显著改善缺血缺氧引发的大脑功能损伤;细胞移植后,动物未现不良反应。结论:外周血单个核细胞移植治疗中枢系统损伤性疾病具有较高的安全性与有效性,有望成为一种临床治疗方案。     

Reduction of Acute Rejection by Bone Marrow Mesenchymal Stem Cells during Rat Small Bowel Transplantation

Background

Bone marrow mesenchymal stem cells (BMMSCs) have shown immunosuppressive activity in transplantation. This study was designed to determine whether BMMSCs could improve outcomes of small bowel transplantation in rats.

Methods

Heterotopic small bowel transplantation was performed from Brown Norway to Lewis rats, followed by infusion of BMMSCs through the superficial dorsal veins of the penis. Controls included rats infused with normal saline (allogeneic control), isogeneically transplanted rats (BN-BN) and nontransplanted animals. The animals were sacrificed after 1, 5, 7 or 10 days. Small bowel histology and apoptosis, cytokine concentrations in serum and intestinal grafts, and numbers of T regulatory (Treg) cells were assessed at each time point.

Results

Acute cellular rejection occurred soon after transplantation and became aggravated over time in the allogeneic control rats, with increase in apoptosis, inflammatory response, and T helper (Th)1/Th2 and Th17/Treg-related cytokines. BMMSCs significantly attenuated acute cellular rejection, reduced apoptosis and suppressed the concentrations of interleukin (IL)-2, IL-6, IL-17, IL-23, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ while upregulating IL-10 and transforming growth factor (TGF)-β expression and increasing Treg levels.

Conclusion

BMMSCs improve the outcomes of allogeneic small bowel transplantation by attenuating the inflammatory response and acute cellular rejection. Treatment with BMMSCs may overcome acute cellular rejection in small bowel transplantation.     

Donor NK Cells and IL-15 Promoted Engraftment in Nonmyeloablative Allogeneic Bone Marrow Transplantation

Donor NK cells could promote engraftment by suppressing host alloreactive responses during allogeneic bone marrow transplantation (allo-BMT). The biological activity of NK cells could be significantly enhanced by IL-15. The current study attempted to evaluate the effect of donor NK cells and IL-15 administration on engraftment and immune reconstitution in a murine nonmyeloablative allo-BMT model. Mice infused with donor NK cells and treated with IL-15 during nonmyeloablative allo-BMT resulted in increased donor engraftment compared with either treatment alone. The number of donor-derived cell subsets also increased in the spleen of the recipient mice with combination treatment. The alloreactivity to donor type Ags was significantly reduced in the recipient mice with donor NK cell infusion and IL-15 treatment, which was manifested by decreased proliferation and IL-2 secretion of splenocytes from recipient mice in response to donor type Ags in MLR and decreased capacity of the splenocytes killing donor type tumor targets. We subsequently exposed recipient mice to reduced irradiation conditioning and showed that donor NK cell infusion and hydrodynamic injection-mediated IL-15 expression could synergistically promote donor engraftment and suppress alloreactivity during nonmyeloablative allo-BMT. Infusion of CFSE-labeled donor CD45.1(+) NK cells demonstrated that IL-15 could enhance the infused donor NK cell proliferation and function in vivo. IL-15 treatment also promoted donor bone marrow-derived NK cell development and function. Thus, donor NK cell infusion and IL-15 treatment could synergistically promote the engraftment and the development of donor-derived cell subsets and suppress the host alloresponse in a murine nonmyeloablative allo-BMT model.     

Evidences of Early Senescence in Multiple Myeloma Bone Marrow Mesenchymal Stromal Cells

Background

In multiple myeloma, bone marrow mesenchymal stromal cells support myeloma cell growth. Previous studies have suggested that direct and indirect interactions between malignant cells and bone marrow mesenchymal stromal cells result in constitutive abnormalities in the bone marrow mesenchymal stromal cells.

Design and Methods

The aims of this study were to investigate the constitutive abnormalities in myeloma bone marrow mesenchymal stromal cells and to evaluate the impact of new treatments.

Results

We demonstrated that myeloma bone marrow mesenchymal stromal cells have an increased expression of senescence-associated β-galactosidase, increased cell size, reduced proliferation capacity and characteristic expression of senescence-associated secretory profile members. We also observed a reduction in osteoblastogenic capacity and immunomodulatory activity and an increase in hematopoietic support capacity. Finally, we determined that current treatments were able to partially reduce some abnormalities in secreted factors, proliferation and osteoblastogenesis.

Conclusions

We showed that myeloma bone marrow mesenchymal stromal cells have an early senescent profile with profound alterations in their characteristics. This senescent state most likely participates in disease progression and relapse by altering the tumor microenvironment.     

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.     

Paracrine-Mediated Neuroprotection and Neuritogenesis of Axotomised Retinal Ganglion Cells by Human Dental Pulp Stem Cells: Comparison with Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

We have investigated and compared the neurotrophic activity of human dental pulp stem cells (hDPSC), human bone marrow-derived mesenchymal stem cells (hBMSC) and human adipose-derived stem cells (hAMSC) on axotomised adult rat retinal ganglion cells (RGC) in vitro in order to evaluate their therapeutic potential for neurodegenerative conditions of RGC. Using the transwell system, RGC survival and length/number of neurites were quantified in coculture with stem cells in the presence or absence of specific Fc-receptor inhibitors to determine the role of NGF, BDNF, NT-3, VEGF, GDNF, PDGF-AA and PDGF-AB/BB in stem cell-mediated RGC neuroprotection and neuritogenesis. Conditioned media, collected from cultured hDPSC/hBMSC/hAMSC, were assayed for the secreted growth factors detailed above using ELISA. PCR array determined the hDPSC, hBMSC and hAMSC expression of genes encoding 84 growth factors and receptors. The results demonstrated that hDPSC promoted significantly more neuroprotection and neuritogenesis of axotomised RGC than either hBMSC or hAMSC, an effect that was neutralized after the addition of specific Fc-receptor inhibitors. hDPSC secreted greater levels of various growth factors including NGF, BDNF and VEGF compared with hBMSC/hAMSC. The PCR array confirmed these findings and identified VGF as a novel potentially therapeutic hDPSC-derived neurotrophic factor (NTF) with significant RGC neuroprotective properties after coculture with axotomised RGC. In conclusion, hDPSC promoted significant multi-factorial paracrine-mediated RGC survival and neurite outgrowth and may be considered a potent and advantageous cell therapy for retinal nerve repair.     

Bone Marrow Transplantation Concurrently Reconstitutes Donor Liver and Immune System across Host Species Barrier in Mice

Liver immunopathologic mechanisms during hepatotropic infection, malignant transformation, and autoimmunity are still unclear. Establishing a chimeric mouse with a reconstituted liver and immune system derived from a single donor across species is critical to study regional donor immune responses in recipient liver. Using a strain of mice deficient in tyrosine catabolic enzyme fumarylacetoacetate hydrolase (fah^-/-) and bone marrow transplantation (BMT), we reconstituted the donor''s hepatocytes and immune cells across host species barrier. Syngeneic, allogeneic or even xenogeneic rat BMT rescued most recipient fah^-/- mice against liver failure by donor BM-derived FAH⁺ hepatocytes. Importantly, immune system developed normally in chimeras, and the immune cells together with organ architecture were intact and functional. Thus, donor BM can across host species barrier and concurrently reconstitutes MHC-identical response between immune cells and hepatocytes, giving rise to a new simple and convenient small animal model to study donor''s liver immune response in mice.