间充质干细胞过滤分离器制备人羊膜间充质干细胞的研究

自然存在的间充质干细胞数量少,限制了其研究应用。依靠自主发明的间充质干细胞过滤分离器,分离制备了人羊膜间充质干细胞,并对制备的干细胞进行了三维培养扩增。结果表明,制备的干细胞形态长势良好,并能诱导分化为类胰岛样组织。与常规方法相比,干细胞收获率提高了8倍以上,且细胞活性状态良好。间充质干细胞过滤分离器可以批量制备高质量的各种间充质干细胞,有利于高效率地建设各种间充质干细胞库,以促进间充质干细胞的研究应用。     

Human Mesenchymal Stem Cells Signals Regulate Neural Stem Cell Fate

Neural stem cells (NSCs) differentiate into neurons, astrocytes and oligodendrocytes depending on their location within the central nervous system (CNS). The cellular and molecular cues mediating end-stage cell fate choices are not completely understood. The retention of multipotent NSCs in the adult CNS raises the possibility that selective recruitment of their progeny to specific lineages may facilitate repair in a spectrum of neuropathological conditions. Previous studies suggest that adult human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after a wide range of CNS insults, probably through their trophic influence. In the context of such trophic activity, here we demonstrate that hMSCs in culture provide humoral signals that selectively promote the genesis of neurons and oligodendrocytes from NSCs. Cell–cell contacts were less effective and the proportion of hMSCs that could be induced to express neural characteristics was very small. We propose that the selective promotion of neuronal and oligodendroglial fates in neural stem cell progeny is responsible for the ability of MSCs to enhance recovery after a wide range of CNS injuries. Special issue dedicated to Anthony Campagnoni.     

人骨髓间充质干细胞培养方法

骨髓间充质干细胞(BMMSCs)是一种多潜能的成体干细胞,在细胞治疗和组织工程上具有广阔的应用前景。对供体年龄、分离方法、培养密度、培养基和培养基质表面性质对细胞增殖的影响进行了比较,重点阐述了用人自体血清结合多种细胞因子,替代胎牛血清培养BMMSCs的效果,转染端粒酶基因的BMMSCs的增殖能力和分化潜能,以及灌注培养反应器用于大规模培养的技术进展。     

Secretome of Differentiated PC12 Cells Enhances Neuronal Differentiation in Human Mesenchymal Stem Cells Via NGF-Like Mechanism

The secretome-mediated responses over cellular physiology are well documented. Stem cells have been ruling the field of secretomics and its role in regenerative medicine since the past few years. However, the mechanistic aspects of secretome-mediated responses and the role of other cells in this area remain somewhat elusive. Here, we investigate the effects of secretome-enriched conditioned medium (CM) of neuronally differentiated PC12 cells on the neuronal differentiation of human mesenchymal stem cells (hMSCs). The exposure to CM at a ratio of 1:1 (CM: conditioned medium of PC12 cells) led to neuronal induction in hMSCs. This neuronal induction was compared with a parallel group of cells exposed to nerve growth factor (NGF). There was a marked increase in neurite length and expression of neuronal markers (β-III tubulin, neurofilament-M (NF-M), synaptophysin, NeuN in exposed hMSCs). Experimental group co-exposed to NGF and CM showed an additive response via MAPK signaling and directed the cells particularly towards cholinergic lineage. The ability of CM to enhance the neuronal properties of stem cells could aid in their rapid differentiation into neuronal subtypes in case of stem cell transplantation for neuronal injuries, thus broadening the scope of non-stem cell-based applications in the area of secretomics.     

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.     

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl₂ and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.     

间充质干细胞分泌组:创伤愈合的替代疗法

大量的证据表明,间充质干细胞(MSC)可以通过旁分泌或自分泌发挥治疗作用,其疗效与血管生成、免疫调节及细胞凋亡有关。MSC分泌组即MSC所分泌的生物活性分子的总和,包括细胞因子、趋化因子和生长因子等多种活性成分,对损伤组织的修复、再生及功能恢复等许多生理过程具有调控作用。在创伤治疗领域,基于MSC分泌组研制的制剂极有可能成为替代MSC治疗的新疗法。     

人羊膜间充质干细胞的生物学特性及临床前研究

近年来随着对间充质干细胞功能特性的深入研究,间充质干细胞已经成为细胞替代治疗的研究热点。而人羊膜间充质干细胞(human amniotic mesenchymal stem cell,hAMSC),除与时下研究热、应用广的骨髓间充质干细胞(Bone marrow mesenchymal stem cell,BM-MSC)一样具有多向分化潜能、免疫调节、造血支持等特性外,还具有来源广泛、取材方便、无伦理限制、细胞增殖能力强等优势。多项研究还证明,hAMSC移植免疫排斥少,成功率高,在各种组织器官中存活率高,存活时间长。以上诸多优势,使得其在细胞替代治疗和再生医学研究领域备受关注。     

人滑膜间充质干细胞与人脐带间充质干细胞的生物学性状比较

该文旨在比较人滑膜间充质干细胞(human synovial mesenchymal stem cells,hSMSCs)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状.流式细胞仪鉴定hSMSCs和hUC-MSCs.比较两种间...     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

胎盘间充质干细胞的体外培养及其生物学特性研究

目的：探讨胎盘间充质干细胞（PMSCs）的体外分离和培养方法,建立稳定的PMSCs体外培养扩增体系。方法：将胎盘组织经胶原酶消化、密度梯度离心、贴壁筛选法分离,获得并培养人PMSCs,观察细胞形态及其超微结构;应用流式细胞术测定细胞周期及CD14、CD29、CD34、CD44、CD45的表达,研究其增殖和生长特性。结果：在体外培养条件下,人PMSCs贴壁生长,为成纤维细胞样,与骨髓间充质干细胞相似;CD14/CD34/CD45阴性,CD29/CD44阳性,核浆比大,细胞周期检测G0/G1期约占95%,具有原始细胞的特征。结论：体外获得的PMSCs形态单一、生长稳定、增殖能力较强,具有与骨髓间充质干细胞相似的细胞形态、表面标志。由于其来源方便、丰富,无伦理学限制,因此可进一步用于细胞治疗的研究。     

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.     

Synthetic Surface for Expansion of Human Mesenchymal Stem Cells in Xeno-Free,Chemically Defined Culture Conditions

Human mesenchymal stem cells (hMSCs) possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM) coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT) plastic in fetal bovine serum (FBS) supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs.     

脂肪间充质干细胞的分离培养、多向分化及其应用前景

脂肪组织几乎遍布于动物体全身,在整个生命过程中有极强的可塑性. 近年研究表明,运用相似的分离方法,可从人、小鼠、大鼠、兔和猪等物种脂肪组织中分离获得脂肪间充质干细胞. 与骨髓来源的间充质干细胞相比,它具有相似的表面标记和分化潜能;在合适的诱导条件下,这种细胞能分别向3个胚层的细胞分化,如成肌细胞、心肌细胞、软骨细胞、成骨细胞、脂肪细胞、神经细胞、血管内皮细胞和肝细胞等;脂肪间充质干细胞具有来源丰富,取材安全方便和扩增速率高的特点,使其在细胞治疗和组织工程方面具有更广阔的应用前景.     

A Systems-level Characterization of the Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells,

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Highlights

• •Integrative multi-omics study characterizing the differentiation from hESCs into hMSCs.
• •Set of high confidence genes important in hESC to hMSC differentiation defined.
• •Two distinct expression waves of HOX genes and a AGO2-to-AGO3 switch in gene silencing identified.
• •AHNAK hypothesized as a defining factor in MSC biology.

     

人尿源性干细胞及人脐带间充质干细胞的生物学性状比较

该研究探讨人尿源性干细胞(human urine-derived stem cells,hUSCs)及人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状差异。分离培养hUSCs及hUC-MSCs,显微镜下观察细胞形态,流式细胞术检测干细胞表面标记物,锥虫蓝拒染实验及克隆形成实验检测细胞增殖能力,划痕实验及Transwell迁移实验检测细胞迁移能力,碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色、油红O染色及阿利新蓝染色评估多向分化潜能。hUSCs为米粒状贴壁生长细胞,hUC-MSCs为长梭形贴壁细胞,呈旋涡状排列生长,两种细胞表型分析相似,均表达多种间充质干细胞标志物,但CD24在hUC-MSCs表达阳性,而CD105在hUSCs表达阳性。hUC-MSCs的增殖及迁移能力优于hUSCs,但后者的克隆形成能力更强。hUSCs及hUCMSCs都具有成骨、成脂、成软骨分化能力,hUC-MSCs的成骨能力强而hUSCs的成脂能力强。该研究成功分离培养出增殖能力强并具有多向分化潜能的hUSCs,该细胞与hUC-MSCs相比具有相似的生物学性状,可作为再生医学自体移植的理想种子细胞来源。