Molecular markers distinguish bone marrow mesenchymal stem cells from fibroblasts

To characterize mesenchymal stem cells (MSC), we compared gene expression profiles in human bone marrow MSC (11 lines) and human fibroblasts (4 lines) by RT-PCR and real time PCR. Messenger RNA levels of MHC-DR-alpha, MHC-DR-beta, MHC-DR-associated protein CD74, tissue factor pathway inhibitor-2, and neuroserpin were much higher in MSC than in fibroblasts, even in the presence of large interindividual variations. Those of adrenomedullin, apolipoprotein D, C-type lectin superfamily member-2, collagen type XV alpha1, CUG triplet repeat RNA-binding protein, matrix metalloproteinase-1, protein tyrosine kinase-7, and Sam68-like phosphotyrosine protein/T-STAR were lower in MSC than in fibroblasts. FACS analysis showed that cell surface expression of MHC-DR was also higher in MSC than in fibroblasts. MHC-DR expression decreased after osteogenic differentiation, whereas the expression of adrenomedullin-a potent stimulator of osteoblast activity-along with collagen XV alpha1 and apolipoprotein D increased after osteogenic differentiation. The marker genes identified in this study should be useful for characterization of MSC both in basic and clinical studies.     

小型猪骨髓间充质干细胞的分离和培养

目的建立小型猪骨髓间充质干细胞（mesenchymal stem cells,MSCs）的体外分离和培养方法。方法穿刺小型猪髂后上嵴抽取骨髓,经密度梯度法离心得到骨髓单个核细胞,接种后形成单层贴壁细胞。用形态学方法鉴定培养的MSCs。结果经培养存活的MSCs原代和一代呈纺锤型、多边型或星型。至二代起呈均一纺锤型,似成纤维细胞样,长宽比例约为（2～3）？1。体外培养的原代MSCs8～10d达到融合,传代后仍具有较强的增殖能力。结论小型猪MSCs可在体外长期、稳定培养,其分离、培养体系的建立为基础研究和组织工程技术提供了一个有价值的动物模型。     

Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation

The first non-hematopoietic mesenchymal stem cells (MSCs) were discovered by Friedenstein in 1976, who described clonal, plastic adherent cells from bone marrow capable of differentiating into osteoblasts, adipocytes, and chondrocytes. More recently, investigators have now demonstrated that multi-potent MSCs can be recovered from a variety of other adult tissues and differentiate into numerous tissue lineages including myoblasts, hepatocytes and possibly even neural tissue. Because MSCs are multipotent and easily expanded in culture, there has been much interest in their clinical potential for tissue repair and gene therapy and as a result, numerous studies have been carried out demonstrating the migration and multi-organ engraftment potential of MSCs in animal models and in human clinical trials. This review describes the recent advances in the understanding of MSC biology.     

间充质干细胞基础研究与临床转化的中美比较

目的：从基础研究、专利申请、临床试验角度对比分析中美间充质干细胞领域发展现状和趋势,了解中美间充质干细胞领域研究的主要特征,为中国间充质干细胞领域的发展提供建议。方法：检索SCI论文数据、DII专利数据及Clinical Trials临床试验数据及新药审批情况,利用Excel、DDA工具对检索结果进行定量分析和讨论。结果：中国在间充质干细胞基础研究、专利申请及临床试验方面虽起步较晚,但近年来发展迅速,论文、专利和临床试验数量快速增长,2014年起发表论文数量及2016年申请专利数量均超过美国,临床试验注册已达200余项;在骨质疏松、脊髓损伤等研究领域,内分泌系统疾病、自体免疫疾病等临床试验方面形成一定优势,已具备坚实的团队与技术基础。但我国间充质干细胞研究仍面临激烈的竞争,存在进步与发展空间。结论：我国间充质干细胞研究应发挥已有优势,加强战略性布局;重视发展以企业为主导的新药开发路径;做精做细以增强国际竞争力与影响力;加大资金投入和产业政策的支持;健全监管机制及评价体系,抓住机遇的同时积极迎接挑战。     

脐静脉和骨髓来源的间充质干细胞的比较研究

间充质干细胞(MSCs)的来源有限,成人骨髓是MSCs的主要来源,这极大地限制了其在实验和临床中的应用。为拓宽MSCs来源,从细胞形态、生长特性、免疫表型和多向分化能力等四个方面对人脐静脉来源和成人骨髓来源的间充质干细胞进行了比较研究。结果表明,人脐静脉来源和成人骨髓来源的 MSCs具有相似的生物学特征,成纤维细胞样形态生长,并具有强大的体外扩增和多向分化能力。人脐静脉来源的MSCs可替代成人骨髓MSCs,作为满足实验和临床需要的重要来源。     

Mesenchymal cells emerge as primary contributors to fibrosis in multiple tissues

A longstanding controversy exists regarding the cellular origin of myofibroblasts in tissue fibrosis. A recent study by Hung and colleagues (Am J Respir Crit Care Med 188(7):820–830, 2013) used genetic fate mapping of FoxD1 embryonic progenitor cells to show a major and direct contribution of mesenchymal cells to fibrogenesis in the lung. Future studies using FoxD1-specific inducible knockout models of pro-fibrotic genes such as CCN2 will be valuable for determining anti-fibrotic drug targets. The emergence of pericyte-like myofibroblast precursors also raises the question of whether mesenchymal stem cells in various niches contribute to fibrotic responses throughout the body.     

Exposure to pro-inflammatory cytokines upregulates MMP-9 synthesis by mesenchymal stem cells-derived osteoprogenitors

An intimate interplay exists between the bone and the immune system, which has been recently termed osteoimmunology. The activity of immune cells affects the intrinsic balance of bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts. The aim of this study was to determine the possible interaction between inflammatory-induced conditions and matrix metalloproteinases-2,-9 (MMP-2,-9) synthesis and secretion by bone marrow-derived osteoprogenitor cells during advanced stages of osteogenesis. Rat bone marrow-derived mesenchymal stem cells (MSCs) were cultured in the presence of osteogenic supplements in order to direct the cells towards the osteogenic differentiation lineage. At the late stages of osteogenesis, assessed by histochemistry, immunohistochemistry and RT-PCR, cultures were exposed to pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-1alpha (IL-1α). Biochemical, histochemical and molecular biology techniques were used to discern the influence of pro-inflammatory cytokines on MMP-2,-9 synthesis and secretion. Results indicated that MMP-9 synthesis and secretion were significantly induced after exposure to the cytokines (TNF-α, IL-1α) treatment, while MMP-2 levels remained unchanged. These results indicate that in response to inflammatory processes, osteoblasts, in addition to osteoclasts, can also be involved and contribute to the process of active bone resorption by secretion and activation of MMPs.     

Immunological properties of embryonic and adult stem cells

The possibility of treating degenerative diseases by stem cell-based approaches is a promising therapeutical option. Among major concerns for the clinical application of stem cells, some derive from the possibility that stem cells may be rejected by the immune system as a consequence of histoincompatibility and that stem cells themselves may interfere with the normal functions of host immune response. Therefore, the immunogenicity and the immunomodulatory properties of stem cells must be carefully addressed. Although these properties are common features of different stem cell types, some peculiarities can be recognized and characterized for their proper clinical use.     

Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor γ agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-β1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.     

Galanin is highly expressed in bone marrow mesenchymal stem cells and facilitates migration of cells both in vitro and in vivo

Galanin peptide has recently been found to be highly abundant in early embryonic mouse mesenchyme, while galanin and its receptors are expressed in embryonic mouse stem cells. Bone marrow mesenchymal stem cells (BMMSCs) represent the primary source for adult stem cell therapy. In this study we examined the abundance of galanin and its receptors in BMMSCs and evaluated its possible function. Galanin mRNA and protein were highly expressed in BMMSCs cultures up to four passages, while among the three galanin receptor subtypes (GalR1, GalR2, and GalR3) only GalR2 and to a lesser extent GalR3 were expressed. Using chemotaxis and wound assays we found that galanin protein increased the migration of BMMSCs. Furthermore, increased serum galanin levels in a galanin transgenic model enhanced the mobilization (homing) of injected BMMSCs in vivo. These data suggest a role for galanin in BMMSC migration, probably through activation of the GalR2 receptor.     

Diet composition transiently modulates proliferative and potency features of human cord blood-derived mesenchymal stem cells

Mesenchymal stem cells (MSC) emerged in the last few years as a promise in regenerative medicine and have been actively tested in several clinical trials worldwide. However, the lack of common standards and a precise definition of MSC preparations remain a major obstacle in research and application. In this study, we compared the effects during culture of two different MSC commercial media (aMEM and SPE-IV) on the proliferative capacities, phenotypic and molecular features in human cord blood derived-MSC lines. Moreover, as miRNA are markers of stem cell multipotency and regulators of somatic cell reprogramming, we performed a miRNome analysis in both conditions. As a result, we observed that SPE-IV promoted a faster growth and modulated stemness and proliferation associated genes such as PDGFRB, p16 and p21. Notably, in aMEM miR-335 and miR-302b, both proposed as putative stemness markers, were upregulated together with miRNAs reported to decrease adipo- and osteogenesis confirming the observed reduced differentiation potential after growth in this condition. Intriguingly, phenotypic divergences were entirely due to culturing conditions and, most importantly, completely transitory since, after medium switch, the cells were able to revert their signatures. Thus, it emerges as crucial keeping constant the experimental settings, starting from culturing conditions, to avoid misleading characterization of stemness and/or potency markers when the eventual goal is unequivocal definition of such parameters for future clinical choice.