Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells

Stem-cell-based therapies are regarded as promising treatments for neurological disorders, and adipose-derived stem cells (ASCs) are a feasible source of clinical application of stem cell. Recent studies have shown that stem cells have a therapeutic potential for use in the treatment of various illnesses through paracrine action. To examine the effects of cell components of ASCs on neural stem cells (NSCs), we treated cell-free extracts of ASCs (CFE-ASCs) containing various components with brain-derived NSCs. To elucidate the effects of CFE-ASCs in NSC proliferation, we treated mouse subventricular zone-derived cultured NSCs with various doses of CFE-ASCs. As a result, CFE-ASCs were found to induce the proliferation of NSCs under conditions of growth factor deprivation in a dose-dependent manner (p<0.01). CFE-ASCs increase the expression of neuron and astrocyte differentiation markers including Tuj-1 (p<0.05) and glial fibrillary acidic protein (p<0.01) without altering the cell’s fate in differentiating NSCs. In addition, treatment with CFE-ASCs induces an increase in neurite numbers (p<0.01) and lengths of NSCs (p<0.05). Furthermore, CFE-ASCs rescue the hydrogen peroxide-induced reduction of NSCs’ viability (p<0.05) and neurite branching (p<0.01). Findings from our study indicate that CFE-ASCs support the survival, proliferation and differentiation of NSCs accompanied with neurite outgrowth, suggesting that CFE-ASCs can modulate neurogenesis in the central nervous system.     

MicroRNA Regulation of Adipose Derived Stem Cells in Aging Rats

Background

Perturbations in abdominal fat secreted adipokines play a key role in metabolic syndrome. This process is further altered during the aging process, probably due to alterations in the preadipocytes (aka. stromal vascular fraction cells-SVF cells or adipose derived stem cells-ASCs) composition and/or function. Since microRNAs regulate genes involved both in development and aging processes, we hypothesized that the impaired adipose function with aging is due to altered microRNA regulation of adipogenic pathways in SVF cells.

Methodology and Principal Findings

Alterations in mRNA and proteins associated with adipogenic differentiation (ERK5 and PPARg) but not osteogenic (RUNX2) pathways were observed in SVF cells isolated from visceral adipose tissue with aging (6 to 30 mo) in female Fischer 344 x Brown Norway Hybrid (FBN) rats. The impaired differentiation capacity with aging correlated with altered levels of miRNAs involved in adipocyte differentiation (miRNA-143) and osteogenic pathways (miRNA-204). Gain and loss of function studies using premir or antagomir-143 validated the age associated adipocyte dysfunction.

Conclusions and Significance

Our studies for the first time indicate a role for miRNA mediated regulation of SVF cells with aging. This discovery is important in the light of the findings that dysfunctional adipose derived stem cells contribute to age related chronic diseases.     

脂肪干细胞的诱导分化及其来源的研究

目的:通过组织块培养法得到脂肪干细胞(adipose-derived stem cells,ADSCs),探讨其诱导分化潜能,并初步研究ADSCs的来源。方法:用脂肪组织块培养法培养原代人ADSCs。第三代ADSCs进行成脂和成骨诱导分化,分别用油红O和茜素红S染色进行鉴定。脂肪组织块培养七天后取脂肪组织进行Hematoxylin-eosin Staining(HE)染色观察ADSCs组织分布。结果:用脂肪组织块培养法成功培养出原代人ADSCs。ADSCs传代到第8代,依然保持着良好的增殖能力和细胞形态。ADSCs能成功诱导成脂肪细胞和骨细胞。通过对培养七天后的脂肪组织块进行HE染色,发现ADSCs主要分布在脂肪组织的间质血管和结缔组织周围。结论:用脂肪组织块培养出来的ADSCs具有成脂和成骨分化的潜能。ADSCs主要定位于间质血管和结缔组织周围。     

Transplantation of Induced Pluripotent Stem Cells Improves Functional Recovery in Huntington's Disease Rat Model

The purpose of this study was to determine the functional recovery of the transplanted induced pluripotent stem cells in a rat model of Huntington''s disease with use of ¹⁸F-FDG microPET/CT imaging.

Methods

In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle ten days after the quinolinic acid injection. The response to the treatment was evaluated by serial ¹⁸F-FDG PET/CT scans and Morris water maze test. Histological analyses and Western blotting were performed six weeks after stem cell transplantation.

Results

After induced pluripotent stem cells transplantation, higher ¹⁸F-FDG accumulation in the injured striatum was observed during the 4 to 6-weeks period compared with the quinolinic acid-injected group, suggesting the metabolic recovery of injured striatum. The induced pluripotent stem cells transplantation improved learning and memory function (and striatal atrophy) of the rat in six week in the comparison with the quinolinic acid-treated controls. In addition, immunohistochemical analysis demonstrated that transplanted stem cells survived and migrated into the lesioned area in striatum, and most of the stem cells expressed protein markers of neurons and glial cells.

Conclusion

Our findings show that induced pluripotent stem cells can survive, differentiate to functional neurons and improve partial striatal function and metabolism after implantation in a rat Huntington''s disease model.     

脂肪干细胞对神经创伤修复的研究进展

在医学上,神经系统对神经损伤的自我修复能力往往有限。因此,探索有效修复损伤神经的方法已经成为近年来的研究热点。研究发现脂肪干细胞对各类损伤神经都有一定的修复作用,可作为修复神经损伤的种子细胞。脂肪干细胞不仅具有干细胞的特性而且还具有自身的优点;首先它属于成体细胞,来源于中胚层,具有多种分化的潜能;其次免疫原性较低,易于取材;另外脂肪干细胞移植后的风险较低,因此被认为是修复神经损伤的优秀种子细胞。就脂肪干细胞的特性及脂肪干细胞在神经创伤修复方面的研究进展和存在的问题进行综述。     

骨质疏松大鼠脂肪基质细胞成骨能力的研究

目的:探讨骨质疏松大鼠的脂肪基质细胞有无成骨能力。方法:采用去势法构建SD大鼠骨质疏松模型,设置2组样本,分别为19月龄骨质疏松SD雌性大鼠,正常19月龄SD雌性大鼠。每组大鼠分别取腹股沟脂肪垫中脂肪,用胶原酶消化法培养获得脂肪间充质干细胞,绘制并分析细胞生长曲线;再用经典的骨向诱导液诱导2组ASCs,比较2组细胞骨向分化能力有无差别。结果:骨质疏松大鼠与正常老龄大鼠的ASCs骨向分化能力无明显差别。结论:可用去势法构建SD大鼠骨质疏松模型,并可取其细胞行进一步研究。老龄骨质疏松大鼠的ASCs也可以考虑作为种子细胞促进其自体骨愈合,从而为利用自体ASCs促进骨质疏松骨愈合打下一定的理论基础。     

Enhanced Osteogenesis of Adipose Derived Stem Cells with Noggin Suppression and Delivery of BMP-2

Bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.     

Comprehensive Behavioral Testing in the R6/2 Mouse Model of Huntington's Disease Shows No Benefit from CoQ10 or Minocycline

Previous studies of the effects of coenzyme Q10 and minocycline on mouse models of Huntington''s disease have produced conflicting results regarding their efficacy in behavioral tests. Using our recently published best practices for husbandry and testing for mouse models of Huntington''s disease, we report that neither coenzyme Q10 nor minocycline had significant beneficial effects on measures of motor function, general health (open field, rotarod, grip strength, rearing-climbing, body weight and survival) in the R6/2 mouse model. The higher doses of minocycline, on the contrary, reduced survival. We were thus unable to confirm the previously reported benefits for these two drugs, and we discuss potential reasons for these discrepancies, such as the effects of husbandry and nutrition.     

Ubiquilin-1 Overexpression Increases the Lifespan and Delays Accumulation of Huntingtin Aggregates in the R6/2 Mouse Model of Huntington's Disease

Huntington''s Disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in huntingtin (htt) protein. The expansion leads to increased htt aggregation and toxicity. Factors that aid in the clearance of mutant huntingtin proteins should relieve the toxicity. We previously demonstrated that overexpression of ubiqulin-1, which facilitates protein clearance through the proteasome and autophagy pathways, reduces huntingtin aggregates and toxicity in mammalian cell and invertebrate models of HD. Here we tested whether overexpression of ubiquilin-1 delays or prevents neurodegeneration in R6/2 mice, a well-established model of HD. We generated transgenic mice overexpressing human ubiquilin-1 driven by the neuron-specific Thy1.2 promoter. Immunoblotting and immunohistochemistry revealed robust and widespread overexpression of ubiquilin-1 in the brains of the transgenic mice. Similar analysis of R6/2 animals revealed that ubiquilin is localized in huntingtin aggregates and that ubiquilin levels decrease progressively to 30% during the end-stage of disease. We crossed our ubiquilin-1 transgenic line with R6/2 mice to assess whether restoration of ubiquilin levels would delay HD symptoms and pathology. In the double transgenic progeny, ubiquilin levels were fully restored, and this correlated with a 20% increase in lifespan and a reduction in htt inclusions in the hippocampus and cortex. Furthermore, immunoblots indicated that endoplasmic reticulum stress response that is elevated in the hippocampus of R6/2 animals was attenuated by ubiquilin-1 overexpression. However, ubiquilin-1 overexpression neither altered the load of htt aggregates in the striatum nor improved motor impairments in the mice.     

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.     

Efficient Generation of Neural Stem Cell-Like Cells from Rat Adipose Derived Stem Cells After Lentiviral Transduction with Green Fluorescent Protein

Neural stem cells (NSCs) can be isolated from nervous tissues or derived from embryonic stem cells. However, their procurement for clinical applications is limited, and there is a need for alternative types of cell that have NSCs properties. In the present study, the differentiation potential of rat adipose-derived stem cells (ADSCs) was evaluated by infecting these cells with a lentiviral vector-encoding green fluorescent protein (GFP). ADSCs transduced with lentivirus were able to generate NSC-like cells, without any effects on their growth, phenotype, and normal differentiation potential. NSC-like cells derived from ADSCs formed neurospheres and expressed high levels of the neural progenitor marker nestin. In the absence of selected growth factors, these neurospheres differentiated into neurons expressing NeuN and MAP2 and GFAP-expressing glia, as determined by immunocytochemistry, Western blotting, and quantitative real-time polymerase chain reaction. These results demonstrate that ADSCs can be induced to generate neurospheres that have NSC-like properties and may thus constitute a potential source of cells in stem cell therapy for neurological disorders.     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.     

小型猪ASCs生物学性状研究及其骨组织工程应用探讨

目的:观察小型猪脂肪来源干细胞(adipose derived stem/stromal cells,ASCs)的形态学特点,研究其多向分化潜能和生物学特性,探讨其在骨组织工程方面的应用。方法:切取雄性小型猪背部皮下脂肪,体外分离培养并鉴定ASCs,观察其生长、增殖特性和组织学形态;并诱导其向脂肪细胞和骨细胞多向分化,分别采用油红O染色和茜素红染色鉴定,细胞表面分子通过流式细胞术鉴定。结果:贴壁的ASCs为长梭形,生长增殖快,性状稳定,流式细胞术检测显示CD29、CD90阳性表达,CD14、CD31、CD34阴性表达。通过相应的诱导培养液诱导,ASCs可向脂肪细胞和骨细胞分化并表现出相应的生物学特征。结论:小型猪背部皮下脂肪取材方便,可获得大量脂肪用以分离ASCs,ASCs增殖分化能力较强,在条件培养基诱导下可成骨分化,可作为种子细胞应用于骨组织工程。     

Two-Point Magnitude MRI for Rapid Mapping of Brown Adipose Tissue and Its Application to the R6/2 Mouse Model of Huntington Disease

The recent discovery of active brown fat in human adults has led to renewed interest in the role of this key metabolic tissue. This is particularly true for neurodegenerative conditions like Huntington disease (HD), an adult-onset heritable disorder with a prominent energy deficit phenotype. Current methods for imaging brown adipose tissue (BAT) are in limited use because they are equipment-wise demanding and often prohibitively expensive. This prompted us to explore how a standard MRI set-up can be modified to visualize BAT in situ by taking advantage of its characteristic fat/water content ratio to differentiate it from surrounding white fat. We present a modified MRI protocol for use on an 11.7 T small animal MRI scanner to visualize and quantify BAT in wild-type and disease model laboratory mice. In this application study using the R6/2 transgenic mouse model of HD we demonstrate a significantly reduced BAT volume in HD mice vs. matched controls (n = 5 per group). This finding provides a plausible structural explanation for the previously described temperature phenotype of HD mice and underscores the significance of peripheral tissue pathology for the HD phenotype. On a more general level, the results demonstrate the feasibility of MR-based BAT imaging in rodents and open the path towards transferring this imaging approach to human patients. Future studies are needed to determine if this method can be used to track disease progression in HD and other disease entities associated with BAT abnormalities, including metabolic conditions such as obesity, cachexia, and diabetes.     

Inclusion formation in Huntington's disease R6/2 mouse muscle cultures

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion in the number of glutamine repeats in the N-terminal region of the huntingtin protein. Nuclear and cytoplasmic aggregates of the N-terminal portion of huntingtin have been found in the brains of HD patients and the brains and non-neuronal tissues of the R6/2 HD transgenic mouse. We have cultured myoblasts and myotubes from transgenic R6/2 mice and littermate controls to investigate the formation of these inclusions in post mitotic cells. Huntingtin immunoreactivity was intense in differentiating, desmin positive myoblasts and myotubes from both control and R6/2 mice suggesting that it may play a role in myotube differentiation. Following differentiation huntingtin and ubiquitin positive aggregates were observed in R6/2 but not control cultures. After 3 weeks in differentiation medium cytoplasmic huntingtin and ubiquitin immunoreactive aggregates were observed in non-myotube cells, while nuclear huntingtin aggregates were seen in a proportion of myotubes after 6 weeks. Growth in the absence of serum resulted in a marked increase in the number of R6/2 myotubes containing nuclear inclusions after 6 weeks demonstrating that environmental factors influenced huntingtin aggregate formation in these cells. Consequently, cultured myotubes from R6/2 mice may be a useful post mitotic cell culture model to study both the biochemical consequences of huntingtin aggregates and the factors that may influence aggregate formation.