Effect of cooling to low temperatures on viability of human skin keratinocytes at different stages of differentiation

The goal of this study was to conduct a comparative analysis of the degree of resistance to low temperatures of human epidermal cells found at different stages of differentiation. The action of liquid-nitrogen vapors was analyzed in experiments in vitro at various temperature regimes on fragments of the human integral skin and on isolated from them and cultivated keratinocytes. The degree of resistance of keratinocytes to the action of cooling to low temperatures was evaluated by their ability to form a multilayer stratum in culture, which indicates the preservation of the viability of the cells treated with cold. This approach allowed one to reveal the diapason of optimal regimes of the action of low temperatures on cells in the composition of tissue and after their conversion into culture. The quantitative ratios of the epidermal stem, transitory, and differentiated cells in a population of viable cells before and after exposure to low temperatures were determined with antibodies that correspond to their different stages of differentiation. The degree of resistance of keratinocytes to action of cooling to low temperatures was evaluated by their ability to form a multilayer stratum in culture, which indicates preservation of viability of the cells treated with cold. The results of this study show that the resistance of human epidermal cells to low temperature differs depending on their stage of differentiation both in situ and in vitro. The epidermal stem and transitory cells are more stable than the differentiated cells.     

The effect of low temperatures on the viability of human epidermal keratinocytes found at different stages of differentiation

The aim of this study was a comparative analysis to the degree of stability of human epidermal cells found at different stages of differentiation to low temperatures. The effect of different subzero temperatures of liquid nitrogen vapor on keratinocytes found both in human skin fragments and as isolated cells extracted from skin fragments has been studied. The degree of stability of epidermal cells low temperatures was evaluated by their ability to form a multilayer stratum in culture; hence this phenomenon explains the survival of a sufficient amount of proliferative cells after exposure to subzero temperatures. Quantitative analysis of the ratio of epidermal stem, transitory and differentiated cells in a population of viable cells before and after exposure to low temperatures were determined using antibodies corresponding to their different stages of differentiation. The results of this research show that the stability of human epidermal cells to low temperature differs depending on their stage of differentiation both in situ and in vitro. Epidermal stem cells and transitory cells are more stable than differentiated cells.     

Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation

Metallothionein (MT), a cysteine-rich, metal-binding protein, is involved in homeostatic regulation of essential metals and protection of cells against oxidative injury. It has been shown that oxidative stress is associated with pathogenesis of osteoporosis and is capable of inhibiting osteoblastic differentiation of bone cells by nuclear factor-kappaB (NF-kappaB). In this study, the effect of MT on oxidative stress-induced inhibition of osteoblast differentiation was examined. 50-200 microM hydrogen peroxide-induced oxidative stress suppressed the osteoblastic differentiation process of primary mouse bone marrow stromal cells (BMSCs), manifested by a reduction in the differentiation marker alkaline phosphatase (ALP). The presence of exogenous MT (20-500 microM) or induction of endogenous MT by ZnCl2 (50-200 microM) could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by a resumption of H2O2-inhibited ALP activity and ALP positive cells. Furthermore, adding exogenous MT or inducing endogenous MT expression impaired H2O2-stimulated NF-kappaB signaling. These data indicate the ability of MT to protect BMSCs against oxidative stress-induced inhibition of osteoblastic differentiation.     

DDR2基因缺失对小鼠骨髓间充质干细胞生物学特性的影响

目的:骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs)是骨再生工程中重要的种子细胞,它对骨组织缺损的修复有着良好的效果。但是BMSCs向成骨细胞分化并修复骨组织缺损是是由细胞外因子共同作用产生的结果。DDR2(Discoidin Domain Receptor 2)作为I型胶原的特异性受体在成骨细胞的分化中发挥重要的调节作用。而对于其在BMSCs向成骨细胞的分化过程中的所起到的作用还鲜有研究,对其作用机理尚不明确。因此我们希望通过分离、培养并鉴定比较DDR2基因缺失小鼠与野生型小鼠来源的骨髓间充质干细胞了解其生物学特性,为后续的实验奠定理论基础。方法:采用改良型的全骨髓贴壁细胞分离方法分离培养两种小鼠来源的骨髓间充质干细胞,采用流式细胞技术鉴定其表面标记物的表达,并利用诱导培养液诱导骨髓间充质干细胞向成骨细胞和成脂肪细胞分化。结果:分离培养的两种骨髓间充质干细胞形态一致,增殖能力和自我更新能力强,流式细胞术检测其表面标记物CD29,Sca-1均表达阳性,CD105,CD45表达为阴性,分离得到的两种细胞均有向成骨细胞和成脂肪细胞分化的能力,但可以明显观察到DDR2基因缺失小鼠的骨髓间充质干细胞的成骨分化能力减弱。结论:本实验通过对于DDR2基因缺失小鼠BMSCs分离、培养和鉴定,初步探索DDR2基因缺失在在成骨过程中的作用结果,为进一步研究提高BMSCs的成骨分化能力奠定理论基础。经实验证明,DDR2基因缺失小鼠来源的骨髓间充质干细胞虽然仍具备干细胞的生物学特性,但其向成骨细胞的分化能力明显减弱,说明DDR2基因缺失对其骨髓间充质干细胞的成骨分化等有着重要的影响。     

The Time-Dependent Manner of Sinusoidal Electromagnetic Fields on Rat Bone Marrow Mesenchymal Stem Cells Proliferation,Differentiation, and Mineralization

Electromagnetic fields (EMFs) are used clinically to promote fracture healing and slow down osteoporosis without knowledge of optimal parameters and underlying principles. In the present study, we investigate the effects of irritation for different durations with 15 Hz 1 mT sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs) proliferation, differentiation, and mineralization potentials. Our results show that SEMFs irritation promote rat BMSCs proliferation in a time-dependent manner, and the expression of osteogenic gen [Cbfa 1/RUNX2, bone sialoprotein (BSP), osteopontin (OPN)], alkaline phosphatase activity, and calcium deposition were enhanced after SEMFs treatment depending on the time duration of treatment. To determine the role of MEK/ERK signaling pathway, U0126, a MEK/ERK inhibitor was used. It can suppress rat BMSCs’ proliferation with or without SEMF exposure, and partly attenuate the expression of osteogenesis related proteins (RUNX2, BSP, OPN) which were improved by SEMF. This finding suggests that the effects of SEMF on rat BMSCs’ proliferation differentiation and mineralization are time duration dependent and MEK/ERK signaling pathway plays important role.     

In vitro study of the differentiation of bone marrow stromal cells into cardiomyocyte-like cells

Bone marrow multipotent stromal cells (BMSCs) have the ability to transdifferentiate into various cell types, including: osteoblasts, chondrocytes, adipocytes, neurons, and cardiomyocytes. This study aimed to differentiate the BMSCs into cardiomyocyte. BMSCs were exposed to 5-azacytidine for 24 h. Seven days after the induction of cell differentiation by 5-azacytidine, the cardiomyogenic cells were stained by fushin and binucleated cells were counted and compared with the neonate cardiomyocyte as positive control. In addition, immunofluorescence analysis and western blot were performed using the antibodies against α-actinin, desmin, troponin T, and β-myosin heavy chain. Our results showed that there was no significant difference between the number of binucleated cells within the cardiomyogenic cell group and positive control group; however, a statistically significant difference was observed between both of these groups and undifferentiated cell group (P < 0.005). In addition, after 5-azacytidine treatment, BMSCs had a higher expression of cardiac-specific markers such as desmin, α-actinin, troponin T and β-myosin heavy chain compared with the untreated groups (P < 0.005). We concluded that 5-azacytidine is an effective inducer for the differentiation of BMSCs into cardiomyocytes and could produce a population of binucleated cells, which express α-actinin, desmin, troponin T, and β-myosin heavy chain, four markers of cardiomyocytes.     

Fibromatosis stem cells rather than bone-marrow mesenchymal stem cells recapitulate a murine model of fibromatosis

Palmar fibromatosis is a benign fibroproliferative tumor of unknown etiology, with a high rate of recurrence after excision. The offending cells of palmar fibromatosis are myofibroblasts and the cellular origin of other myofibroblasts has previously been reported to be the bone marrow. However, further clarification of the relationship between bone marrow precursors and palmar fibromatosis is required. Stem cells (SCs) are known to exist in various tissues, but whether SCs can be isolated from fibromatosis tissue is still unclear. The purpose of this study was to isolate and identify stem cells from human palmar fibromatosis, and to evaluate the differences in the differentiation and fibrogenic capacities of bone marrow stem cells (BMSCs) and fibromatosis-derived stem cells (FSCs). We found that FSCs had better fibrogenic differentiation potential than BMSCs, whereas BMSCs had better adipogenic and chondrogenic differentiation capacities. Treatment with transforming growth factor-β1 increased the expression of α-smooth muscle actin, and types III and I collagen significantly more in FSCs than in BMSCs. An in vivo study further confirmed the results of fibrogenesis and suggested that FSCs can recapitulate the fibromatosis nodule. In summary, their myofibroblastic differentiation both in vivo and in vitro makes FSCs a potential cell source for future applications in murine models of fibromatosis or fibrogenesis.     

Polydatin promotes the neuronal differentiation of bone marrow mesenchymal stem cells in vitro and in vivo: Involvement of Nrf2 signalling pathway

Bone marrow mesenchymal stem cell (BMSC) transplantation represents a promising repair strategy following spinal cord injury (SCI), although the therapeutic effects are minimal due to their limited neural differentiation potential. Polydatin (PD), a key component of the Chinese herb Polygonum cuspidatum, exerts significant neuroprotective effects in various central nervous system disorders and protects BMSCs against oxidative injury. However, the effect of PD on the neuronal differentiation of BMSCs, and the underlying mechanisms remain inadequately understood. In this study, we induced neuronal differentiation of BMSCs in the presence of PD, and analysed the Nrf2 signalling and neuronal differentiation markers using routine molecular assays. We also established an in vivo model of SCI and assessed the locomotor function of the mice through hindlimb movements and electrophysiological measurements. Finally, tissue regeneration was evaluated by H&E staining, Nissl staining and transmission electron microscopy. PD (30 μmol/L) markedly facilitated BMSC differentiation into neuron‐like cells by activating the Nrf2 pathway and increased the expression of neuronal markers in the transplanted BMSCs at the injured spinal cord sites. Furthermore, compared with either monotherapy, the combination of PD and BMSC transplantation promoted axonal rehabilitation, attenuated glial scar formation and promoted axonal generation across the glial scar, thereby enhancing recovery of hindlimb locomotor function. Taken together, PD augments the neuronal differentiation of BMSCs via Nrf2 activation and improves functional recovery, indicating a promising new therapeutic approach against SCI.     

Protection of bone marrow-derived CD45+/CD34-/lin- stromal cells with immunosuppressant activity against ischemia/reperfusion injury in rats

Non-hematopoietic CD45+ precursor cells are not known to differentiate into cardiomyocytes. We found that CD45+/CD34-/lin- stromal cells isolated from mouse bone marrow (BMSCs) potentially differentiated into cardiomyocyte-like cells in vitro. Therefore, we hypothesized that the CD45+/CD34-/ lin- BMSCs might protect rat hearts against ischemia/reperfusion (IR) injury following xeno-transplantation. In the present study, BMSCs were isolated by immunoselection and their cellular phenotype and biochemical properties were characterized. The immunological inertness of BMSCs was examined by the allogeneic and xenogeneic mixed lymphocyte reaction (MLR). The potential role of BMSCs for cardioprotection was evaluated by intravenous introduction of 1 x 10(6) cells into rat IR hearts, induced by left coronary ligation for 45 min and released for 72 h. Changes in cardiac contractility and the degree of myocardial injury were assessed. Our findings indicated that BMSCs expressed the muscle-cell marker alpha-actinin after 5-azacytidine treatment. CD45+/CD34-/lin- stromal cells were characterized as mesenchymal progenitor cells based on the expression of Sca-1 and Rex-1. The MLR assay revealed an immunosuppression of BMSCs on mouse and rat lymphocytes. After xeno-transplantation, the BMSCs engrafted into the infarct area and attenuated IR injury. However, increases in intracardial TGF-beta and IFN-gamma contents of IR hearts were not affected by BMSC treatment. Interestingly, ex vivo evidence indicated that CXCR4, SDF-1 and TGFbeta-1 receptors were up-regulated after the cells were exposed to tissue extracts prepared from rat post-IR hearts. In addition, IFN-gamma treatment also markedly increased Sca-1 expression in BMSCs. Mechanistically, these results indicated that CXCR4/SDF-1 and TGF-beta signals potentially enhanced the interaction of BMSCs with the damaged myocardium, and increased IFN-gamma in post-ischemic hearts might cause BMSC to behave more like stem cells in cardioprotection. These data show that CD45+/CD34-/lin- BMSCs possess cardioprotective capacity. Evidently, the accurate production of soluble factors TGF-beta and IFN-gamma in parallel with increased expression of both TGF-beta and Sca-1 receptors may favor BMSCs to achieve a more efficient protective capacity.     

Reorganization of the tubulin and actin cytoskeleton under acclimation and abscisic acid treatment of Triticum aestivum L. plants

Only scanty and contradictory data are available concerning effects of low temperatures and ABA on the structural organization of microtubules (MTs) and microfilaments (MFs), and no information exists on the interaction of these parameters at cold acclimation of plants. Therefore, in cold acclimate and ABA-treated winter wheat plants, a comparative study was made of the state (localization, orientation, structure) and stability of actin and tubulin cytoskeleton in root cells taken from different zones, using indirect immunofluorescent microscope. The plant cold acclimation caused MT aggregation, the rise of MT and MF fluorescence, and the increase of their stability (a decrease of oryzalin effect) mainly in the root differentiation zone, that may testify to the strengthening of contacts between MTs and MFs. Like the cold acclimation, ABA induced the formation of MT bunches only in meristem and elongation zone cells. However in the zone of differentiation, the hormone stimulated the increase of tubulin structure stability, well correlating with a decrease in MT content, aggregation degree, and immunofluorescence, and, in addition with a complete depolymerization of MFs. Low temperatures removed the hormone effect on the structural organization of tubulin and actin cytoskeleton in the zone of differentiation. It is suggested that MT destruction, the decrease of instable MT populations, and the increase of stable MT populations may slow down growth processes in ABA-treated plants, similarly as in seedlings being on the initial stages of cold acclimation. By the end of this process, the induction of plant growth is determined evidently by the increase in the number of instable, highly labile MT populations, and in the status of MF polymerization.     

The effect of combined regulation of the expression of peroxisome proliferator-activated receptor-γ and calcitonin gene-related peptide on alcohol-induced adipogenic differentiation of bone marrow mesenchymal stem cells

Studies have shown that alcohol can upregulate the expression of peroxisome proliferator-activated receptor-γ (PPARγ) gene in bone marrow mesenchymal stem cells (BMSCs). High expression of PPARγ can promote adipogenic differentiation of BMSCs, and reduce their osteogenic differentiation. Abnormal proliferation of adipocytes and fatty accumulation in osteocytes can result in high intraosseous pressure and disturbance of blood circulation in the femoral head, which induces osteonecrosis of the femoral head (ONFH). Downregulation of PPARγ is efficient in inhibiting adipogenesis and maintaining osteogenesis of BMSCs, which might potentially reduce the incidence of ONFH. Calcitonin gene-related peptide (CGRP) is a neuropeptide gene which has been closely associated with bone regeneration. In this study, we aimed to observe the effect of combined regulation of the expression of PPARγ and CGRP genes on alcohol-induced adipogenic differentiation of BMSCs. Our results demonstrated that simultaneous downregulation of PPARγ and upregulation of CGRP was efficient in suppressing adipogenic differentiation of BMSCs and promoting their osteogenic differentiation. These findings might enlighten a novel approach for the prevention of ONFH.     

Self-assembled extracellular macromolecular matrices and their different osteogenic potential with preosteoblasts and rat bone marrow mesenchymal stromal cells

Extracellular environment is a physical support that is critical to cell adhesion, migration, and differentiation. In this work, cell-derived matrices (CDMs) were obtained by separately culturing fibroblasts, preosteoblasts, and chondrocytes. The cells were grown on a coverslip and subjected to decellularization using detergents and enzymes. The resulting matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). We hypothesize that the unique compositional and structural feature of each CDM provides cells with a distinct microenvironment capable of functioning as a different signaling cue in the regulation of preosteoblast and rat bone marrow mesenchymal stromal cell (BMSC) osteogenic differentiation. SEM images show that each cell type creates its unique surface texture in a fibrillar structure. Three major macromolecules, fibronectin, type I collagen, and laminin, were clearly identified using both immunofluorescence and Western blot, in which FDM exhibited a much stronger signal of each ECM component than that of PDM or CHDM. For early cell morphology, BMSCs on the CDMs were highly elongated in a spindle-like shape. Both preosteoblasts and BMSCs proliferated well on CDMs comparable to the control. Once preosteoblasts were cultured for 2 weeks, their osteogenic activity was significantly different depending on the type of CDM. Using Alizarin red and von Kossa staining, we found that the cells on the FDM were much more osteogenic than the other groups. Furthermore, FDM was the most effective in upregulating the osteogenic markers, such as alkaline phosphatase (ALP), osteopontin, osteocalcin, and type I collagen. In particular, we observed a 2.5-fold increase in ALP activity with FDM compared to that of control and CHDM. In stark contrast, CHDM was very poor in stimulating osteogenic differentiation of preosteoblasts. Interestingly, these results were reproducible with the use of BMSCs, which are much more heterogeneous in cell populations than preosteoblasts. CHDM was still very weak in triggering the osteogenesis of BMSCs, whereas both FDM and PDM were equally competitive. This study demonstrates that a combination of factors (surface texture and composition) shape a unique cellular microenvironment, which serves as a physical cue toward the osteogenic differentiation of preosteoblasts and BMSCs.     

Expression of Pdx-1 in bone marrow mesenchymal stem cells promotes differentiation of islet-like cells in vitro

Bone marrow mesenchymal stem cells (BMSCs) have the ability of self-renewal and multi-directional differentiation. Recent reports showed that BMSCs could differentiate into endocrine cells of pancreas. However, the differentiation is not efficient enough to produce insulin-producing cells for the future therapeutic use. Pdx-1 is a crucial regulator for pancreatic development. Therefore we constructed a eukaryotic expression vector containing Pdx-1 to determine the effect of Pdx-1 expression on differentiation of BMSCs in vitro. The results showed that BMSCs could self-assemble to form functional pancreatic islet-like structures after differentiation in vitro. The proportion of insulin-producing cells differentiated from Pdx-1⁺BMSCs was 28.23%±2.56%, higher than that from BMSCs transfected with vacant vector and Pdx-1 ⁻ BMSCs (7.23%±1.56% and 4.08%±2.69% respectively) by flow cytometry. Immunocytochemical examination also testified the expression of multiple β-cells-specific genes such as insulin, glucagons, somatostatin in differentiated BMSCs. The results also revealed that the expressions of genes mentioned above in Pdx-1⁺BMSCs were higher than that in Pdx-1⁻BMSCs, which was confirmed by Western blotting analysis and RT-PCR. Glucose-induced insulin secretion from Pdx-1⁺BMSCs in 5mmol/L and 25mmol/L glocuse was (56.61±4.82) μU/mL and (115.29±2.56) μU/mL respectively, which were much higher than those from Pdx-1⁻BMSCs((25.53±6.49) μU/mL and (53.26±7.56) μU/mL respectively). Grafted animals were able to maintain their body weight and survive for relatively longer periods of time than hyperglycemic sham-grafted controls, which demonstrated an overall beneficial effect of the grafted cells on the health of the animals. These findings thus suggested that exogenous expression of Pdx-1 should provide a promising approach for efficiently producing islet-like cells from BMSCs for the future therapeutic use in diabetic patients.     

The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells

Multi-differentiation capability is an essential characteristic of bone marrow mesenchymal stem cells (BMSCs). Method on obtaining higher-quality stem cells with an improved differentiation potential has gained significant attention for the treatment of clinical diseases and developmental biology. In our study, we investigated the multipotential differentiation capacity of BMSCs under simulated microgravity (SMG) condition. F-actin staining found that cytoskeleton took on a time-dependent change under SMG condition, which caused spindle to round morphological change of the cultured cells. Quantitative PCR and Western Blotting showed the pluripotency marker OCT4 was up-regulated in the SMG condition especially after SMG of 72 h, which we observed would be the most appropriate SMG duration for enhancing pluripotency of BMSCs. After dividing BMSCs into normal gravity (NG) group and SMG group, we induced them respectively in endothelium oriented, adipogenic and neuronal induction media. Immunostaining and Western Blotting found that endothelium oriented differentiated BMSCs expressed higher VWF and CD31 in the SMG group than in the NG group. The neuron-like cells derived from BMSCs in the SMG group also expressed higher level of MAP2 and NF-H. Furthermore, the quantity of induced adipocytes increased in the SMG group compared to the NG group shown by Oil Red O staining, The expression of PPARγ2 increased significantly under SMG condition. Therefore, we demonstrated that SMG could promote BMSCs to differentiate into many kinds of cells and predicted that enhanced multi-potential differentiation capacity response in BMSCs following SMG might be relevant to the changes of cytoskeleton and the stem cell marker OCT4.     

The Methods and Mechanisms to Differentiate Endothelial-Like Cells and Smooth Muscle Cells from Mesenchymal Stem Cells for Vascularization in Vaginal Reconstruction

Endothelial cells and smooth muscle cells (SMCs) are important aspects of vascularization in vaginal reconstruction. Research has confirmed that mesenchymal stem cells could differentiate into endothelial-like cells and SMCs. But the methods were more complicated and the mechanism was unknown. In the current study, we induced the bone mesenchymal stem cells (BMSCs) to differentiate into endothelial-like cells and SMCs in vitro by differentiation medium and investigated the effect of Wnt/β-catenin signaling on the differentiation process of BMSCs. Results showed that the hypoxic environment combined with VEGF and bFGF could induce increased expression of endothelial-like cells markers VEGFR1, VEGFR2, and vWF. The SMCs derived from BMSCs induced by TGF-β1 and PDGF-AB significantly expressed SMC markers SMMHC11 and α-SMA. The data also showed that activation of Wnt/β-catenin signaling could promote the differentiation of BMSCs into endothelial-like cells and SMCs. Thus, we established endothelial-like cells and SMCs in vitro by more simple methods, presented the important role of hypoxic environment on the differentiation of BMSCs into endothelial-like cells, and confirmed that the Wnt/β-catenin signaling pathway has a positive impact on the differentiation of BMSCs into endothelial-like cells and SMCs. This is important for vascular reconstruction.     

TGF-beta expression by allogeneic bone marrow stromal cells ameliorates diabetes in NOD mice through modulating the distribution of CD4+ T cell subsets

BMSCs could promote the regeneration of islet beta-cell, but the status of BMSCs under diabetes is still unknown. Our study verified the effect of allogeneic BMSCs (ICR) transferred into NOD mice on blood glucose and CD4+ T cells subsets function. In vivo experiment, BMSCs could decrease blood glucose, weaken lymphocytes proliferation. In vitro experiment, the distribution of CD4+ T cell subsets was changed after co-culture with BMSCs, resulting in a greater frequency of Treg cells and reduced representation of Th17 cells. After TGF-beta blockade, CD4+ T cells differentiated along a route favoring development of Th17, but not Treg cells. Thus, NOD can be treated by BMSCs which changes the distribution of CD4+ T cells, increases the number of Treg cells, and inhibits the differentiation of Th17 cells. And the positive effects of allogeneic BMSCs in the treatment of NOD mice depend on the regulation of TGF-beta secreted by BMSCs.     

Human bone marrow stroma cells display certain neural characteristics and integrate in the subventricular compartment after injection into the liquor system

Because the neural differentiation capacity of bone marrow stromal cells (BMSCs) is still a matter of controversial debate, we performed a thorough investigation into the differentiation capacity of human BMSCs and examined their therapeutic potency. BMSCs were isolated from the femur and kept in cell cultures with various cultivation protocols being applied. In standard culture conditions using a fetal calf serum-enriched medium, while not exhibiting a neural phenotype, the majority of cells expressed a variety of neuronal marker proteins as well as the astrocyte marker GFAP. Only a minority of stem cells expressed nestin, a marker for neural precursor cells. Cultivation in serum-free medium supplemented with specific growth factors resulted in a markedly higher percentage of nestin-positive cells. To establish the therapeutic potency of bone marrow-derived cells, the synthesis of neurotrophic factors such as NGF, BDNF and GDNF was analyzed under non-stimulating standard culture conditions as well as after a neural selection procedure. The therapeutic potency of BMSCs was further examined with regard to their migratory potential in vitro and after transplantation in vivo. After stereotactic engraftment into the lateral ventricle of adult rats, mesenchymal stem cells were seen to adhere to the ependymocytes and cells of the choroids plexus. Afterwards grafted cells passed through the ependymal barrier, locating in the subventricular space. Their BMSCs took up a close host graft interaction without any degenerative influence on the host cells. Furthermore, there was morphological as well as immunohistochemical evidence for a transdifferentiation within the host tissue. In addition, BMSCs could be efficiently transduced using a third-generation adenoviral vector, indicating their potential feasibility for a gene-therapeutic option.     

骨髓间充质干细胞移植对急性脊髓损伤脱髓鞘病变的保护作用

骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)已被广泛应用于治疗脊髓损伤,但目前对其治疗机制了解甚少。BMSCs被移植至脊髓钳夹损伤模型大鼠,以研究其保护作用。通过LFB(Luxol fast blue)染色、锇酸染色、TUNEL(Td T-mediated d UTP nick-end labeling)染色和透射电镜对白质有髓神经纤维进行观察。免疫印迹检测BMSCs移植对脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)和caspase 3蛋白表达的影响。通过脊髓损伤后1、7、14 d三个时间点移植BMSCs并进行后肢运动评分(Basso,beattie and bresnahan;BBB评分)和CNPase(2′,3′-cyclic-nucleotide 3′-phosphodiesterase)、髓鞘碱性蛋白(Myelin basic protein,MBP)、caspase 3蛋白水平的检测。免疫荧光观察BMSCs移植到受损脊髓后分化情况及CNPase-caspase 3~+共表达情况。骨髓间充质干细胞移植7 d后,部分移植的BMSCs可表达神经元和少突胶质细胞标记物,大鼠后肢运动能力和髓鞘超微结构特征均明显改善。骨髓间充质干细胞移植后BDNF蛋白表达水平增加,caspase 3蛋白表达水平则降低。相对于脊髓损伤后1 d和14 d,7 d移植BMSCs后MBP和CNPase蛋白表达水平最高;caspase 3蛋白表达水平则最低。骨髓间充质干细胞移植后CNPase-caspase 3~+细胞散在分布于脊髓白质。结果表明,急性脊髓损伤后,BMSCs移植到受损脊髓有分化为神经元和少突胶质细胞的倾向,并促进BDNF的分泌介导抗少突胶质细胞凋亡而对神经脱髓鞘病变有保护作用,且最佳移植时间为脊髓损伤后7 d。     

Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles

Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.     

The frequency, growth kinetics, and osteogenic/adipogenic differentiation properties of canine bone marrow stromal cells

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38 × 10⁴ bone marrow mononucleated cells (0.0042 ± 0.0019%, n = 5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.