Modeling Dynamics and Function of Bone Marrow Cells in Mouse Liver Regeneration

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Structure and Function in Aging Human Salivary Glands

Degenerative structural changes develop in the secretory tissues of most salivary glands in man with advancing age. Quantitative studies have shown losses of a third or more in the parenchymal content of submandibular and labial glands and mostly these changes accrue steadily across the adult life span. The parotid appears less prone to such extensive change but currently only limited data are available for this gland. Positive correlations are evident between the age-dependent decrements of secretory tissue and reductions in salivary flow rate for the submandibular and labial glands. The parotid, however, shows no functional correlation with the demonstrable tissue losses of old age. Future research should he directed at structural-functional anomalies in aging glands and should seek to examine the changing demands on salivary structure and function within the wider context of the maintenance of oral health in the elderly.     

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.     

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).     

The Compensating Function of Submandibular Salivary Glands in Diabetes and Their Possible Stimulation by Isoproterenol

A brief review is presented of experimental studies conducted over several years (1970s–1990s) on the incretory potential of salivary glands, specifically on their role in the maintenance of carbohydrate homeostasis; a review is also presented of data on the stimulation of this potential by isoproterenol, a -adrenergic agonist.     

Paracrine Effect of Mesenchymal Stem Cells Derived from Human Adipose Tissue in Bone Regeneration

Mesenchymal stem cell (MSC) transplantation has proved to be a promising strategy in cell therapy and regenerative medicine. Although their mechanism of action is not completely clear, it has been suggested that their therapeutic activity may be mediated by a paracrine effect. The main goal of this study was to evaluate by radiographic, morphometric and histological analysis the ability of mesenchymal stem cells derived from human adipose tissue (Ad-MSC) and their conditioned medium (CM), to repair surgical bone lesions using an in vivo model (rabbit mandibles). The results demonstrated that both, Ad-MSC and CM, induce bone regeneration in surgically created lesions in rabbit''s jaws, suggesting that Ad-MSC improve the process of bone regeneration mainly by releasing paracrine factors. The evidence of the paracrine effect of MSC on bone regeneration has a major impact on regenerative medicine, and the use of their CM can address some issues and difficulties related to cell transplants. In particular, CM can be easily stored and transported, and is easier to handle by medical personnel during clinical procedures.     

骨髓干细胞动员对动脉粥样硬化破裂斑块的稳定与修复作用

目的研究自体骨髓干细胞动员对兔动脉粥样硬化（AS）破裂斑块的稳定与修复作用。方法用液氮冻伤术创建兔AS破裂斑块模型,动员组注射重组人粒细胞刺激因子（rhG-CSF）动员自体骨髓干细胞,对照组注射等量生理盐水,连续5 d。动员第5天抽血分离获取单个核细胞,BrdU标记后经静脉注入动物体内;分别于动员后3d和4周末抽血,ELISA法检测兔血清MMP-9、hsC-RP及PAI-1水平;动员后4周处死兔,HE染色和Masson三色染色观察斑块病理形态,免疫组化染色观察BrdU在斑块区表达情况。结果动员5 d后,动员组兔外周血有核细胞计数及单核细胞比例明显增高;动员后4周,动员组新生内皮细胞及胶原纤维明显增多,在斑块区发现有BrdU标记的阳性细胞,动员组血清MMP-9、hsC-RP及PAI-1水平明显降低。结论应用rhG-CSF动员自体骨髓干细胞能通过促进血管内皮细胞和胶原纤维再生,降低炎症因子及凝血纤溶因子而稳定与修复AS破裂斑块。     

Induction of Unresponsiveness to Canine Renal Allografts by Total Body Irradiation and Bone Marrow Transplantation

RECENT progress in the definition of the DL-A antigens within a closely bred colony of beagles maintained at the Mary Imogene Basset Hospital (Cooperstown colony) has shown that these antigens are components of the principal system of histocompatibility (DL-A) in the dog^1–3. Dausset and associates⁴ have described the segregation of DL-A antigens in 679 offspring of 151 consecutive matings within the Coopers-town colony. Through methods of backcross analysis, 23 DL-A haplotypes (that is, specificities determined by the DL-A region of one single chromosome) have been identified and the DL-A genotypes of 1,302 offspring of 517 matings in this colony have been defined⁴. This combined genetic and serological information has provided an opportunity to reassess the reported survival rates of allogeneic bone marrow transplantation in serologically matched littermate dogs⁵. We describe here the long term survival of bone marrow transplants and renal allografts obtained from the same donors in nine littermate and three non-littermate recipients selected on the basis of genetic and serological criteria of DL-A compatibility in the Cooperstown colony. The results are contrasted with the survival of twenty-two renal allografts performed in untreated recipients selected on the basis of similar criteria.     

Development of a Model for the Role of Mitogens Initiating Repair Hyperplasia in Big Salivary Glands

It was shown in repeated experiments on 132 rats that proliferation in the corneal epithelium was nonspecifically enhanced after sialotomy. This effect appears to be due to the distant action of factors stimulating cell proliferation. The described phenomenon can be used for identification of mitogens formed during regeneration of the salivary glands.     

Early Transplantation of Bone Marrow Mononuclear Cells Promotes Neuroprotection and Modulation of Inflammation After Status Epilepticus in Mice by Paracrine Mechanisms

Status epilepticus (SE) is a severe clinical manifestation of epilepsy associated with intense neuronal loss and inflammation, two key factors involved in the pathophysiology of temporal lobe epilepsy. Bone marrow mononuclear cells (BMMC) attenuated the consequences of pilocarpine-induced SE, including neuronal loss, in addition to frequency and duration of seizures. Here we investigated the effects of BMMC transplanted early after the onset of SE in mice, as well as the involvement of soluble factors produced by BMMC in the effects of the cell therapy. Mice were injected with pilocarpine for SE induction and randomized into three groups: transplanted intravenously with 1 × 10⁷ BMMC isolated from GFP transgenic mice, injected with BMMC lysate, and saline-treated controls. Cell tracking, neuronal counting in hippocampal subfields and cytokine analysis in the serum and brain were performed. BMMC were found in the brain 4 h following transplantation and their numbers progressively decreased until 24 h following transplantation. A reduction in hippocampal neuronal loss after SE was found in mice treated with live BMMC and BMMC lysate when compared to saline-treated, SE-induced mice. Moreover, the expression of inflammatory cytokines IL-1β, TNF-α, IL-6 was decreased after injection of live BMMC and to a lesser extent, of BMMC lysate, when compared to SE-induced controls. In contrast, IL-10 expression was increased. Analysis of markers for microglia activation demonstrated a reduction of the expression of genes related to type 1-activation. BMMC transplantation promotes neuroprotection and mediates anti-inflammatory effects following SE in mice, possibly through the secretion of soluble factors.     

自组装短肽用于SD大鼠头顶骨损伤的修复

使用SD大鼠头顶骨损伤模型,通过与骨蜡、空白作对照,研究了自组装短肽RADA16-I水凝胶修复骨缺损的能力.术后28天X-光片揭示在RADA16-I组中有新骨生成填充;而对照组中缺损部位清晰可见.其HE染色和Masson染色结果也表明RADA16-I能有效修复骨损伤.目前的研究表明RADA16-I引导成骨细胞迁移到受损部位,促进其修复.     

Bone Marrow Progenitor Cell Therapy-Mediated Paracrine Regulation of Cardiac miRNA-155 Modulates Fibrotic Response in Diabetic Hearts

Diabetes is associated with a higher incidence of myocardial infarction (MI) and increased risk for adverse vascular and fibrogenic events post-MI. Bone marrow-derived progenitor cell (BMPC) therapy has been shown to promote neovascularization, decrease infarct area and attenuate left ventricular (LV) dysfunction after MI. Unlike vascular effects, the anti-fibrosis mechanisms of BMPC, specifically under diabetic conditions, are poorly understood. We demonstrated that intramyocardial delivery of BMPCs in infarcted diabetic db/db mice significantly down-regulates profibrotic miRNA-155 in the myocardium and improves LV remodeling and function. Furthermore, inhibition of paracrine factor hepatocyte growth factor (HGF) signaling in vivo suppressed the BMPC-mediated inhibition of miR-155 expression and the associated protective effect on cardiac fibrosis and function. In vitro studies confirmed that the conditioned media of BMPC inhibited miR-155 expression and profibrotic signaling in mouse cardiac fibroblasts under diabetic conditions. However, neutralizing antibodies directed against HGF blocked these effects. Furthermore, miR-155 over-expression in mouse cardiac fibroblasts inhibited antifibrotic Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene, non-Alu-containing (SnoN) signaling and abrogated antifibrogenic response of HGF. Together, our data demonstrates that paracrine regulation of cardiac miRNAs by transplanted BMPCs contributes to the antifibrotic effects of BMPC therapy. BMPCs release HGF, which inhibits miR-155-mediated profibrosis signaling, thereby preventing cardiac fibrosis. These data suggest that targeting miR-155 might serve as a potential therapy against cardiac fibrosis in the diabetic heart.     

Loss of Sirt3 Limits Bone Marrow Cell-Mediated Angiogenesis and Cardiac Repair in Post-Myocardial Infarction

Sirtuin-3 (Sirt3) has a critical role in the regulation of human aging and reactive oxygen species (ROS) formation. A recent study has identified Sirt3 as an essential regulator of stem cell aging. This study investigated whether Sirt3 is necessary for bone marrow cell (BMC)-mediated cardiac repair in post-myocardial infarction (MI). In vitro, BMC-derived endothelial progenitor cells (EPCs) from wild type (WT) and Sirt3KO mice were cultured. EPC angiogenesis, ROS formation and apoptosis were assessed. In vivo, WT and Sirt3 KO mice were subjected to MI and BMCs from WT and Sirt3 KO mice were injected into ischemic area immediately. The expression of VEGF and VEGFR2 was reduced in Sirt3KO-EPCs. Angiogenic capacities and colony formation were significantly impaired in Sirt3KO-EPCs compared to WT-EPCs. Loss of Sirt3 further enhanced ROS formation and apoptosis in EPCs. Overexpression of Sirt3 or treatment with NADPH oxidase inhibitor apocynin (Apo, 200 and 400 microM) rescued these abnormalities. In post-MI mice, BMC treatment increased number of Sca1⁺/c-kit⁺ cells; enhanced VEGF expression and angiogenesis whereas Sirt3KO-BMC treatment had little effects. BMC treatment also attenuated NADPH oxidase subunits p47^phox and gp91^phox expression, and significantly reduced ROS formation, apoptosis, fibrosis and hypertrophy in post-MI mice. Sirt3KO-BMC treatment did not display these beneficial effects. In contrast, Sirt3KO mice treated with BMCs from WT mice attenuated myocardial apoptosis, fibrosis and improved cardiac function. Our data demonstrate that Sirt3 is essential for BMC therapy; and loss of Sirt3 limits BMC-mediated angiogenesis and cardiac repair in post-MI.     

Repair of Ear Cartilage Defects with Allogenic Bone Marrow Mesenchymal Stem Cells in Rabbits

The study aims to investigate the feasibility of repairing cartilaginous defects with chondrocytes induced from allogenic bone marrow mesenchymal stem cells (BMMSC) in rabbits’ ear. BMMSCs were isolated and purified from New Zealand rabbits, in vitro amplified, and cultured in chondrocyte induction medium in order to acquire chondrocytes. After 3 weeks of induction, their phenotypes were confirmed as chondrocytes, then they were implanted onto novel polymeric scaffolds made from Poly (dl-lactide-co-glycolide) (PLGA) embedded with chitosan nonwoven cloth. The experimental group was transplanted with tissue engineering cartilaginous grafts composed of chondrogenetic BMMSC/scaffolds; the scaffold group was treated with scaffolds without cells, while in the control group, nothing was implanted. Specimens were taken at 6, 12, and 18 weeks after implantation, and the healing condition was observed by hematoxylin-eosin staining and toluidine blue staining. The right and left ears with cartilage defects of eighteen rabbits were randomly divided into three groups. In the experimental group, after 18 weeks of transplantation, the gross observation indicated that the cartilaginous defects were completely repaired by chondrocytes with smooth surface and similar color with the surrounding tissue. Hematoxylin-eosin staining and toluidine blue staining suggested that the defective area was filled with mature cartilage cells with obvious lacunae but without obvious boundaries with the normal cartilage tissue, and that the new cartilage cells were evenly distributed with homogeneously dyed cytoplasm and smaller in size. The chondrocyte induced from allogenic BMMSC can be used to repair cartilage defects in rabbit’s ear.     

Bone Marrow Cell Recruitment to the Brain in the Absence of Irradiation or Parabiosis Bias

The engraftment of bone marrow-derived cells has been described not only during diseases of the central nervous system (CNS) but also under healthy conditions. However, previous studies pointing to an ample bone marrow cell engraftment used irradiation-induced bone marrow chimeras that evoked severe alterations of the CNS micromilieu including disturbances of the blood brain barrier (BBB), damage of endothelial cells and local induction of proinflammatory cytokines. On the other hand, parabiosis experiments using temporarily joined circulatory systems generally yielded low levels of myeloid cell chimerism thereby potentially underestimating bone marrow cell turnover with the CNS. To avoid these drawbacks we established a protocol using the alkylating agent busulfan prior to allogenic bone marrow transplantation from CX₃CR1^GFP/+ donors. This regimen resulted in a stable and high peripheral myeloid chimerism, significantly reduced cytokine induction and preserved BBB integrity. Importantly, bone marrow cell recruitment to the CNS was strongly diminished under these conditions and only weakly enhanced during local neurodegeneration induced by facial nerve axotomy. These results underscore the requirement of local CNS conditioning for efficient recruitment of bone marrow cells, establish busulfan as an alternative treatment for studying bone marrow chimeras and suggest a critical re-evaluation of earlier chimeric studies involving irradiation or parabiosis regimens.