Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

Differentiation potential and GFP labeling of sheep bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an important cell population in the bone marrow microenvironment. MSCs have the capacity to differentiate in vitro into several mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma. This study was designed to isolate, expand, and characterize the differentiation ability of sheep bone marrow‐derived MSCs and to demonstrate the possibility to permanently express a reporter gene. Bone marrow was collected from the iliac crest and mononuclear cells were separated by density gradient centrifugation. Sheep MSCs cell lines were stable characterized as CD44+ and CD34? and then transfected with a green fluorescent protein (GFP) reporter gene. The GFP expression was maintained in about half (46.6%) of cloned blastocysts produced by nuclear transfer of GFP+ sheep MSCs, suggesting the possibility to establish multipotent embryonic cells' lines carrying the fluorescent tag for comparative studies on the differentiation capacity of adult stem cells (MSCs) versus embryonic stem cells. We found that sheep MSCs under appropriate culture conditions could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Our results confirm the plasticity of sheep MSCs and establish the foundation for the development of a pre‐clinical sheep model to test the efficiency and safety of cell replacement therapy. J. Cell. Biochem. 114: 134–143, 2012. © 2012 Wiley Periodicals, Inc.     

miRNA在人骨髓来源间充质干细胞软骨诱导分化过程中的表达

目的：研究miRNAs在人骨髓来源间充质干细胞软骨诱导分化过程中的表达情况。方法：以从骨髓中分离培养的MSCs及软骨诱导培养后的细胞为实验对象,利用基因芯片检测miRNAs的表达情况,由SAM分析得到MSCs较其诱导培养细胞中差异表达的miRNAs,再进行生物信息学分析。结果：①分离培养出的MSCs经软骨诱导培养21天后,已具有软骨细胞特性,经芯片检测并SAM分析,软骨诱导培养的细胞较MSCs高表达的miRNAs有6个：hsa-miR-572、hsa-miR-130b、hsa-miR-193b、hsa-miR-28、hsa-miR-152、hsa-miR-560;软骨诱导培养的细胞较MSCs低表达的miRNAs有2个：hsa-miR-424、hsa-miR-122a。②利用TargetScan预测其靶基因,并行生物信息学分析,其中hsa-miR-130b、hsa-miR-193b、hsa-miR-152及hsa-miR-424的预测靶基因中多为参与细胞分化、骨形成、软骨形成及干细胞表型相关的基因。结论：hsa-miR-130b、hsa-miR-193b、hsa-miR-152和hsa-miR-424等对人骨髓来源间充质干细胞的软骨分化起着重要调控作用。     

间充质干细胞体外调控骨髓造血前体细胞向单核系分化

研究间充质干细胞(MSC)能否在体外调控造血。体外分离培养人骨髓来源的MSC,RT-PCR检测其造血生长因子的表达,并以其为饲养层细胞,接种骨髓单个核细胞(MNC),观察生长情况,并通过形态学观察和流式细胞术分析,鉴定细胞来源和分化方向。结果显示,MSC构成性表达SCF、Flt3L和M-CSF,不表达C-CSF和GM-CSF,在骨髓MNC和MSC共培养体系中,大约2周左右可以看到大量的圆形细胞粘附在梭型MSC上生长,细胞胞体为圆形,胞浆较丰富,胞核为圆形、半月型或肾型,部分细胞呈典型的单核细胞形态,流式细胞术分析该类细胞表达CDl4,不表达CDl5、CD41、glycophorin A、CD5和CDl9。表明不需要添加外源性造血生长因子,间充质干细胞能在体外调控骨髓造血前体细胞向单核系分化,其定向分化可能与MSC分泌造血生长因子及MSC与造血细胞间相互作用有关。     

The role of catecholamines in mesenchymal stem cell fate

Mesenchymal stem cells (MSCs) are multipotent stem cells found in many adult tissues, especially bone marrow (BM) and are capable of differentiation into various lineage cells such as osteoblasts, adipocytes, chondrocytes and myocytes. Moreover, MSCs can be mobilized from connective tissue into circulation and from there to damaged sites to contribute to regeneration processes. MSCs commitment and differentiation are controlled by complex activities involving signal transduction through cytokines and catecholamines. There has been an increasing interest in recent years in the neural system, functioning in the support of stem cells like MSCs. Recent efforts have indicated that the catecholamine released from neural and not neural cells could be affected characteristics of MSCs. However, there have not been review studies of most aspects involved in catecholamines-mediated functions of MSCs. Thus, in this review paper, we will try to describe the current state of catecholamines in MSCs destination and discuss strategies being used for catecholamines for migration of these cells to damaged tissues. Then, the role of the nervous system in the induction of osteogenesis, adipogenesis, chondrogenesis and myogenesis from MSCs is discussed. Recent progress in studies of signaling transduction of catecholamines in determination of the final fate of MSCs is highlighted. Hence, the knowledge of interaction between MSCs with the neural system could be applied towards the development of new diagnostic and treatment alternatives for human diseases.     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

问充质干细胞体外调控骨髓造血前体细胞向单核系分化

研究间充质干细胞(MSC)能否在体外调控造血.体外分离培养人骨髓来源的MSC,RT-PCR检测其造血生长因子的表达,并以其为饲养层细胞,接种骨髓单个核细胞(MNC),观察生长情况,并通过形态学观察和流式细胞术分析,鉴定细胞来源和分化方向.结果显示,MSC构成性表达SCF、Flt3L和M-CSF,不表达G-CSF和GM-CSF,在骨髓MNC和MSC共培养体系中,大约2周左右可以看到大量的圆形细胞粘附在梭型MSC上生长,细胞胞体为圆形,胞浆较丰富,胞核为圆形、半月型或肾型,部分细胞呈典型的单核细胞形态,流式细胞术分析该类细胞表达CD14,不表达CD15、CD41、glycophorin A、CD5和CD19.表明不需要添加外源性造血生长因子,间充质干细胞能在体外调控骨髓造血前体细胞向单核系分化,其定向分化可能与MSC分泌造血生长因子及MSC与造血细胞间相互作用有关.     

间充质干细胞体外分化为多巴胺能神经元的研究进展

骨髓间充质干细胞（MSCs）有来源广泛、易于分离培养、不易引起免疫排斥等特点,使其成为细胞治疗和基因治疗的种子细胞,具有广泛的科研和临床应用价值。骨髓MSCs具有多向分化潜能,在特定条件下能诱导分化成神经元甚至是更为特异的多巴胺能神经元,为帕金森病进行细胞移植疗法提供了理想的细胞来源。本文就近年来体外诱导MSCs向多巴胺能神经元定向分化所涉及到的常用诱导因素和诱导方法及途径予以综述。     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

Neural stimulation on human bone marrow‐derived mesenchymal stem cells by extremely low frequency electromagnetic fields

Adult stem cells are considered multipotent. Especially, human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have the potential to differentiate into nerve type cells. Electromagnetic fields (EMFs) are widely distributed in the environment, and recently there have been many reports on the biological effects of EMFs. hBM‐MSCs are weak and sensitive pluripotent stem cells, therefore extremely low frequency‐electromagnetic fields (ELF‐EMFs) could be affect the changes of biological functions within the cells. In our experiments, ELF‐EMFs inhibited the growth of hBM‐MSCs in 12 days exposure. Their gene level was changed and expression of the neural stem cell marker like nestin was decreased but the neural cell markers like MAP2, NEUROD1, NF‐L, and Tau were induced. In immunofluorescence study, we confirmed the expression of each protein of neural cells. And also both oligodendrocyte and astrocyte related proteins like O4 and GFAP were expressed by ELF‐EMFs. We suggest that EMFs can induce neural differentiation in BM‐MSCs without any chemicals or differentiation factors. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type

Mesenchymal stem cells (MSCs) can differentiate not only into mesenchymal lineage cells but also into various other cell lineages. As MSCs can easily be isolated from bone marrow, they can be used in various tissue engineering strategies. In this study, we assessed whether MSCs can differentiate into multiple skin cell types including keratinocytes and contribute to wound repair. First, we found keratin 14-positive cells, presumed to be keratinocytes that transdifferentiated from MSCs in vitro. Next, we assessed whether MSCs can transdifferentiate into multiple skin cell types in vivo. At sites of mouse wounds that had been i.v. injected with MSCs derived from GFP transgenic mice, we detected GFP-positive cells associated with specific markers for keratinocytes, endothelial cells, and pericytes. Because MSCs are predominantly located in bone marrow, we investigated the main MSC recruitment mechanism. MSCs expressed several chemokine receptors; especially CCR7, which is a receptor of SLC/CCL21, that enhanced MSC migration. Finally, MSC-injected mice underwent rapid wound repaired. Furthermore, intradermal injection of SLC/CCL21 increased the migration of MSCs, which resulted in an even greater acceleration of wound repair. Taken together, we have demonstrated that MSCs contribute to wound repair via processes involving MSCs differentiation various cell components of the skin.     

Differentiation potential of bone marrow mesenchymal stem cells in duck

The bone marrow mesenchymal stem cells （MSCs） are multipotent stem cells which can differentiate into mesenchymal cells in vitro. In this study, MSCs in duck were isolated from bone marrow by density gradient centrifuge separation, purified and expanded in the me- dium. The primary MSCs were expanded for 11 passages. The different-passage MSCs were induced to differentiate into osteoblasts and neuron-like cells. Karyotype analysis indicated that MSCs kept diploid condition and the hereditary feature was stable. The different- passage MSCs expressed CD44, ICAM-1 and SSEA-4, but not CD34, CD45 and SSEA-1 when detected by immunofluorescence staining There was no significant difference among the positive rates of passages 2, 6 and 8 （P 〉 0.05）, but a significant difference existed among those of passages 2, 6, 8 and 11 （P 〈 0.05）. After the osteogenic inducement was added, the induced different-passage MSCs expressed high-level alkaline phosphatase （ALP）, and are positive for tetracycline staining, Alizarin Red staining and Von Kossa staining. After the neural inducement was added, about 70% cells exhibited typical neuron-like phenotype, the induced different-passage MSCs expressed Nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 3, 4 and 6 （P〉0.05）, but a significant difference existed among those of passages 3, 4, 6 and 8 （P〈0.05）. These results suggest that MSCs in duck were capable of differentiating into osteoblasts and neuron-like cells in vitro.     

Skin-derived multipotent stromal cells – an archrival for mesenchymal stem cells

Progenitor stem cells have been identified, isolated and characterized in numerous tissues and organs. However, their therapeutic potential and the use of these stem cells remain elusive except for a few progenitor cells from bone marrow, umbilical cord blood, eyes and dental pulp. The use of bone marrow-derived hematopoietic stem cells (HSC) or mesenchymal stem cells (MSCs) is restricted due to their extreme invasive procedures, low differentiation potential with age and rejection. Thus, we need a clinical grade alternative to progenitor stem cells with a high potential to differentiate, na?ve and is relatively easy in in vitro propagation. In this review, we summarize cell populations of adherent and floating spheres derived from different origins of skin, or correctly foreskin, by enzymatic digestion compared with established MSCs. The morphology, phenotype, differentiation capability and immunosuppressive property of the adherent cell populations are comparable with MSCs. Serum-free cultured floating spheres have limited mesodermal but higher neurogenic differentation potential, analogous to neural crest stem cells. Both the populations confirmed their plethora potential in in vitro. Together, it may be noted that the skin-derived adherent cell populations and floating cells can be good alternative sources of progenitor cells especially in cosmetic, plastic and sports regenerative medicine.     

再生障碍性贫血患者骨髓间充质干细胞生物学特性的初步研究

目的:研究再生障碍性贫血(aplasticanemia,从)患者骨髓间充质干细胞(mesenchymalstemcells,MSCs)的生物学特性和初步探讨其异常和AA发生的可能关系。方法:取AA患者骨髓间充质干细胞,测定其生长曲线和倍增时间;流式细胞仪检测其细胞周期和免疫表型;体外定向诱导其向脂肪、成骨、内皮和神经细胞分化;用real-timePCR及油红O染色法比较AA和正常对照组MSCs的成脂分化的不同。结果:AA患者和正常成人的MSCs均呈梭形贴壁生长;AA组细胞倍增时间长于对照组;CD105、CD44、CD29、CD106、FlK-1均阳性;96．51％的细胞处在G0／G1期;AA患者的MSCs保持了多向分化潜能,体外诱导形成脂滴较对照组早,诱导早期的脂蛋白脂酶表达增高。结论:再生障碍性贫血患者的骨髓间充质干细胞增殖能力较正常成人弱,骨髓间充质干细胞的易成脂性可能参与了再障的发病环节。     

从人胚胎干细胞畸胎瘤中有效获取间充质干细胞和神经前体细胞

通过人胚胎干细胞(human embryonic stem cells,hESC)体外分化方法和畸胎瘤形成可以分化获得多种成体细胞．但目前尚不清楚是否可以从hESCs畸胎瘤中分离某些特异性细胞．通过体外筛选方法,有效地从hESCs畸胎瘤中分离出神经前体细胞(neural progenitor cells,NPCs)和间充质干细胞(mesenchymal stem cells,MSCs)．这种hESCs畸胎瘤来源的NPCs和MSCs与体内神经前体细胞和间充质干细胞有着相似的分子标记和特性,并具有进一步的分化潜能——分别可以诱导成为神经元、神经胶质细胞、脂肪细胞和骨骼细胞等．根据人胚胎干细胞畸胎瘤中含有不同分化阶段的外胚层、中胚层和内胚层的组织或细胞,认为人胚胎干细胞畸胎瘤可以作为另一个细胞来源以获取多种(包括人胚胎干细胞体外分化难以得到的)各种前体/干细胞和终末分化细胞．     

Cellular retinol-binding protein 1 (CRBP-1) regulates osteogenenesis and adipogenesis of mesenchymal stem cells through inhibiting RXRα-induced β-catenin degradation

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can differentiate into osteoblasts, chondrocytes and adipocytes, providing a potential source for musculoskeletal tissue engineering. Retinoid signaling plays very important roles in skeletal development. CRBP1 (cellular retinol binding protein 1), a key component of retinoid signaling pathway, is known to take part in vitamin A metabolism and intracellular transporting of retinoids. However, the role of CRBP1 in MSCs remains still obscure. In this study, we investigated the cellular effects of CRBP1 on osteogenic and adipogenic differentiation of bone marrow derived MSCs in vitro and in vivo. Our results showed that CRBP1 overexpression promoted osteogenic differentiation of bone marrow derived MSCs, while inhibited their adipogenic differentiation. We also demonstrated that the possible underlying mechanism for CRBP1 promoting osteogenic differentiation of MSCs was by inhibiting RXRα-induced β-catenin degradation, maintaining β-catenin and pERK1/2 at higher levels. These findings reveal a potential role of CRBP1 in the regulation of β-catenin turnover which can greatly affect the process of osteogenesis and adipogenesis of MSCs.     

Comparison of the Neural Differentiation Potential of Human Mesenchymal Stem Cells from Amniotic Fluid and Adult Bone Marrow

Human mesenchymal stem cells (MSCs) are considered a promising tool for cell-based therapies of nervous system diseases. Bone marrow (BM) has been the traditional source of MSCs (BM-MSCs). However, there are some limitations for their clinical use, such as the decline in cell number and differentiation potential with age. Recently, amniotic fluid (AF)-derived MSCs (AF-MSCs) have been shown to express embryonic and adult stem cell markers, and can differentiate into cells of all three germ layers. In this study, we isolated AF-MSCs from second-trimester AF by limiting dilution and compared their proliferative capacity, multipotency, neural differentiation ability, and secretion of neurotrophins to those of BM-MSCs. AF-MSCs showed a higher proliferative capacity and more rapidly formed and expanded neurospheres compared to those of BM-MSCs. Both immunocytochemical and quantitative real-time PCR analyses demonstrated that AF-MSCs showed higher expression of neural stemness markers than those of BM-MSCs following neural stem cell (NSC) differentiation. Furthermore, the levels of brain-derived growth factor and nerve growth factor secreted by AF-MSCs in the culture medium were higher than those of BM-MSCs. In addition, AF-MSCs maintained a normal karyotype in long-term cultures after NSC differentiation and were not tumorigenic in vivo. Our findings suggest that AF-MSCs are a promising and safe alternative to BM-MSCs for therapy of nervous system diseases.