CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture

Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization andpurification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton’s jelly among others.     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

Tissue engineering for neurodegenerative diseases using human amniotic membrane and umbilical cord

Regenerative medicine, based on the use of stem cells, scaffolds and growth factors, has the potential to be a good approach for restoring damaged tissues of the central nervous system. This study investigated the use of human amniotic mesenchymal stem cells (hAMSC), human amniotic epithelial stem cells (hAESC), and human Wharton’s jelly mesenchymal stem cells (hWJMSC) derived from human umbilical cord as a source of stem cells, and the potential of the human amniotic membrane (HAM) as a scaffold and/or source of growth factors to promote nerve regeneration. The hAMSC and hAESC obtained from HAM and the hWJMSC from umbilical cords were cultured in induction medium to obtain neural-like cells. The morphological differentiation of hAMSC, hAESC and hWJMSC into neural-like cells was evident after 4–5 days, when they acquired an elongated and multipolar shape, and at 21 days, when they expressed neural and glial markers. On other way, the HAM was completely decellularized without affecting the components of the basement membrane or the matrix. Subsequently, hAMSC, hAESC and hWJMSC differentiated into neural-like cells were seeded onto the decellularized HAM, maintaining their morphology. Finally, conditioned media from the HAM allowed proliferation of hAMSC, hAESC and hWJMSC differentiated to neural-like cells. Both HAM and umbilical cord are biomaterials with great potential for use in regenerative medicine for the treatment of neurodegenerative diseases.     

Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO-PLEX technology

We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast-specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL-8, MCP-1 and VEGF, but did not express IL-2, IL-7, IL-17, eotaxin, G-CSF and IFN-gamma. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.     

Isolation and characterization of mesenchymal stem cells from caprine umbilical cord tissue matrix

Cord tissue fills the umbilical cord around the blood vessels and contains types of stem cells (mesenchymal stem cells or MSCs) that are not generally found in cord blood. MSCs are the stem cells that give rise to many of the “support tissues” in the body, including bone, cartilage, fat and muscle. Umbilical Cord Tissue cells (UCTs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes and adipocytes have been previously isolated from different species including human, canine, murine, avian species etc. The present study documents the existence of similar multipotential stem cells in caprine UCTs having similar growth and morphological characteristics. The cells were isolated from caprine umbilical cord and cultivated in DMEM (low glucose) supplemented with 15% FBS, L-glutamine and antibiotics. Primary culture achieved confluence in 5–7 days having spindle shaped morphology. The cells were morphologically homogeneous, showed robust proliferation ability with a population doubled time of 92.07 h as well as normal karyotype. In vitro self-renewal capacity was demonstrated by colony-forming unit assay (CFU). The cells expressed MSC specific markers and showed multi-differentiation capability into adipogenic and osteogeneic. The results indicated that caprine UCTs (cUCTs) were isolated and characterized from umbilical cord tissue which can be used for tissue regeneration.     

Long-term survival and characterisation of human umbilical cord-derived mesenchymal stem cells on dermal equivalents

During early embryogenesis, mesenchymal cells arise from the primitive epithelium and can revert to an epithelial phenotype by passing through mesenchymal-to-epithelial transition (MET). Mesenchymal stem cells (MSC) of the Wharton's Jelly of the umbilical cord (UC-MSC) express pluripotency markers underlining their primitive developmental state. As mesenchymal stem cells from bone marrow (BM-MSC) possess a strong propensity to ameliorate mesenchymal tissue damage, UC-MSC might also be able to differentiate into cells apart from the mesoderm, allowing replacement of ectodermal and mesodermal tissues.     

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.     

一种大量快速分离脐带间充质干细胞的新方法

目的:探讨体外快速大量分离脐带间充质干细胞的新方法。方法:采用复合胶原NB4、dispaseII、透明质酸酶三种酶消化3h,加入PBSA溶液稀释,离心获得脐带间充质干细胞,培养;用流式细胞仪对P3代细胞进行表面标记的鉴定,用化学诱导的方法使第3代细胞向脂肪、骨、软骨细胞分化,2~4周后,分别行oilred、Safranin'O和茜素红染色,倒置显微镜下观察诱导结果。结果:经3种酶消化和PBSA稀释,短时间内从脐带中获得了大量间充质干细胞;伴随着细胞的传代,形态逐渐均一,传至第3代,细胞的形态已基本相似;流式细胞仪鉴定,细胞强表达间充质细胞的特异性标记CD90,CD73,CD105,而不表达造血系或内皮系细胞的标记CD45、CD14、CD11、CD34、CD19,也不表达主要组织相容性抗原HLA-DR;向脂肪细胞诱导后第4周,oilred染色见细胞内大量红染的脂滴;向软骨细胞诱导后第4周,Safranin'O染色见多数切片呈阳性,细胞团块中存在大量软骨特异性的陷窝样结构;向骨细胞诱导后第4周,茜素红染色发现肉眼可见的广泛散在的红色阳性钙结节。结论:本研究所采用的3种酶消化结合PBSA稀释的方法可以快速获得脐带间...     

脐带间充质干细胞的生物学特性及旁分泌作用的研究进展

脐带是由胚胎外中胚层和/或胚胎中胚层发育而来的组织,脐带间充质干细胞是具有自我更新、多向分化以及高度增殖潜能的多功能干细胞。研究证明,脐带间充质干细胞具有以下功能：参与炎症反应,抑制炎症因子分泌并促进免疫调节;参与受损伤组织的治疗与修复使其再生并改善特定疾病症状;抑制肿瘤增殖和迁移以及促进其凋亡等。然而目前尚未明确以上功能是间充质干细胞本身发挥作用,还是其分泌的相关因子对机体修复产生作用。主要对脐带间充质干细胞的定义、来源、生物学特性、分泌功能等方面的研究进展进行了综述,旨在更好地利用间充质干细胞修复组织,以期为脐带间充质干细胞的后续研究提供参考依据。     

一种简单的人脐带间充质干细胞分离培养方法

目的 建立一种简单的人脐带间充质干细胞分离培养方法.方法 取新鲜脐带,剪成5 cm长的小段,直接剪碎为糊状,加入含10％胎牛血清的DMEM/F12在培养瓶中培养,光学显微镜下观察细胞的生长特征,运用流式细胞仪检测分析细胞的抗原标志表达,并检测其体外多向分化潜能.结果 运用不剥离血管组织、不用酶消化的组织贴块培养法可以从...     

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration.     

Human breast milk is a rich source of multipotent mesenchymal stem cells

Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be “reprogrammed” to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined.     

Human umbilical cord-derived MSC culture: the replacement of animal sera with human cord blood plasma

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great potential for their therapeutic use in various clinical diseases. Many publications have reported on human blood-derived alternatives to animal serum for culturing mesenchymal stem cells, such as human serum, allogenic umbilical cord blood serum, and human platelet derivatives. However, it is not clear whether human umbilical cord blood plasma (UCBP), as the surplusage of umbilical cord blood mesenchymal stem cell extraction, could be used. In this study, in order to make the best of umbilical cord blood, the human UCBP was dialyzed to replace fetal bovine serum (FBS) in the culture medium. hUC-MSCs were cultured in the new medium. Cell growth rate, specific biomarkers, and differentiation properties were detected to characterize the cell proliferation and MSC-specific properties. The hUC-MSCs cultured in such derived medium were verified with proliferation rate, cluster differentiation markers, cell cycle, as well as differentiation capabilities. Such dialyzed human UCBP is fully comparable with, if not superior to, FBS in deriving and culturing hUC-MSCs.     

三种不同转染剂介导钆喷酸葡胺标记人脐带间充质干细胞及体外MR成像的实验研究

人脐带组织富含间充质干细胞（human umbilical cord mesenchymal stem cells,hUCMSCs）,是干细胞研究理想的种子来源,如何从脐带组织中分离间充质干细胞及运用影像技术示踪干细胞生物学行为是当前研究的热点。该实验应用组织块贴壁法从足月孕妇脐带组织中分离纯化间充质干细胞并进行鉴定,结果为hUCMSCs。进一步应用磷酸钙、Effectene、脂质体2000三种转染试剂分别介导Gd-DTPA标记hUCMSCs,通过MRI（magnetic resonance imaging）检测钆喷酸葡胺（Gd-DTPA）标记细胞信号强度变化及细胞内钆离子浓度的测定评价三种转染试剂的转染能力,最终发现在三种转染试剂中,Effectene介导Gd-DTPA标记hUCMSCs效果最佳,为Gd-DTPA标记干细胞体外MR成像奠定了实验基础。     

人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。     

Human mesenchymal stem cells seeded on extracellular matrix-scaffold: viability and osteogenic potential

The development and the optimization of novel culture systems of mesenchymal osteoprogenitors are some of the most important challenges in the field of bone tissue engineering (TE). A new combination between cells and extracellular matrix (ECM)-scaffold, containing ECM has here been analyzed. As source for osteoprogenitors, mesenchymal stem cells obtained from human umbilical cord Wharton's Jelly (hWJMSCs), were used. As ECM-scaffold, a powder form of isolated and purified porcine urinary bladder matrix (pUBM), was employed. The goals of the current work were: (1) the characterization of the in vitro hWJMSCs behavior, in terms of viability, proliferation, and adhesion to ECM-scaffold; (2) the effectiveness of ECM-scaffold to induce/modulate the osteoblastic differentiation; and (3) the proposal for a possible application of cells/ECM-scaffold construct to the field of cell/TE. In this respect, the properties of the pUBM-scaffold in promoting and guiding the in vitro adhesion, proliferation, and three-dimensional colonization of hWJMSCs, without altering viability and morphological characteristics of the cells, are here described. Finally, we have also demonstrated that pUBM-scaffolds positively affect the expression of typical osteoblastic markers in hWJMSCs.     

Comparison of human mesenchymal stem cells isolated by explant culture method from entire umbilical cord and Wharton’s jelly matrix

Adult stem cells are of particular importance for applications in regenerative medicine. Umbilical cord was established recently as an alternative source of mesenchymal stem cell (MSC) instead of bone marrow (BM) and is superior to BM and other adult tissues according to several MSC properties. Additionally, for the purpose of cell therapy in clinical scale, steps of cell isolation, expansion and culture required to be precisely adjusted in order to obtain the most cost-effective, least time-consuming, and least labor-intensive method. Therefore, in this study, we are going to compare two simple and cost-effective explant culture methods for isolation of MSCs from human umbilical cord. One of the methods isolates cells from entire cord and the other from Wharton’s jelly matrix. Isolated cells then cultured in simple medium without addition of any growth factor. MSCs obtained via both methods display proper and similar characteristics according to morphology, population doubling time, post-thaw survival, surface antigenicity and differentiation into adipocytes, osteocytes, and chondrocytes. MSCs can easily be obtained from the entire cord and Wharton’s jelly, and it seems that both tissues are appropriate sources of stem cells for potential use in regenerative medicine. However, from technical largescale preview, MSC isolation from entire cord piece is less labor-intensive and time-consuming than from Wharton’s jelly part of the cord.