脐血来源间充质干细胞的分化潜能

背景：脐血源间充质干细胞是来源于发育早期中胚层的一类多能干细胞,因其具有多向分化潜能的特点而日益受到关注。 目的：综述脐血来源间充质干细胞的诱导分化潜能。 方法：应用计算机检索 Pubmed数据库中1999年1月至2012年12月关于脐血源间充质干细胞分化潜能的文章,在标题和摘要中以“umblical cord blood,mesenchymal stem cells,potential,differentiation”为检索词进行检索。最终选择关于脐血源间充质干细胞分化潜能的52篇文献进行综述。 结果与结论：虽然很多实验已经证实人脐血间充质干细胞可以成功向多种细胞系分化,但对其了解程度尚浅。如果能掌握脐血间充质干细胞分化潜能的特点,那么很可能用它来修复许多骨缺损、心肌缺损等。目前研究正处于起步阶段,在分离纯化技术、分化方向的调控、体外扩增、免疫原性等方面尚有待进一步深入研究。     

脐血间充质干细胞移植与免疫调节

背景：近年研究显示,脐血间充质干细胞的自我更新和多向分化潜能为细胞移植治疗提供了基础条件,而其免疫调节功能也极大地拓展了细胞治疗的方向和范围。 目的：就近期脐血间充质干细胞的免疫调节和细胞移植研究进行回顾分析。 方法：应用计算机以“umbilical cord blood-derived mesenchymal stem cells”为关键词检索Pubmed数据库,以“脐血间充质干细胞”为关键词检索知网数据库,时间限定为2008-01/2011-06,语言种类为“English”和汉语。通过阅读标题和摘要进行初筛,排除研究内容与此文无关的文献、重复性研究及Meta分析,选择近期发表或发表在权威杂志的30篇文献进行综述。 结果与结论：脐血间充质干细胞具有与骨髓间充质干细胞相似的自我更新和多向分化潜能。通过细胞移植技术,脐血间充质干细胞在糖尿病、神经退化性疾病如阿尔茨海默病和帕金森病等以及神经损伤后的修复治疗方面显示出很强的潜力。同时,脐血间充质干细胞又具有免疫调节作用,其可通过下调T细胞的增殖,降低免疫反应。利用该特点,脐血间充质干细胞在一些免疫性疾病,如移植物抗宿主病和狼疮性肾炎等疾病的细胞治疗方面也取得了积极的进展。     

脐血单个核细胞与骨髓间充质干细胞滋养层的共培养

背景：骨髓间充质干细胞具有维持骨髓正常造血功能,在造血调控中发挥重要的作用。 目的：观察骨髓间充质干细胞的生物学性状和多向分化能力,并检测其在体外支持造血的能力。 方法：利用密度梯度培养法分离骨髓单个核细胞进行培养,流式细胞仪检测骨髓间充质干细胞表型;接种脐血单个核细胞于骨髓间充质干细胞滋养层培养板上共培养,观察粒-单系祖细胞集落变化。 结果与结论：骨髓间充质干细胞呈典型的成纤维样细胞形态,强表达CD44,CD29,不表达CD34和CD106。可促进脐血单个核细胞扩增并形成造血祖细胞集落。提示骨髓间充质干细具有造血支持作用。     

In vitro cultivation of islet-like cell clusters from human umbilical cord blood-derived mesenchymal stem cells

A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. In vitro transdifferentiation of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into insulin-producing cells could provide an abundant source of cells for this procedure. For this study, we isolated and characterized human UCB-MSCs and induced them in vitro to differentiate into islet-like cell clusters using a 15-day protocol based on a combination of high-glucose, retinoic acid, nicotinamide, epidermal growth factor, and exendin-4. These clusters appeared about 9 days after pancreatic differentiation; expressed pancreatic beta-cell markers, including insulin, glucagon, Glut-2, PDX1, Pax4, and Ngn3; and could synthesize and secrete functional islet proteins at the end of the inducing protocol. The insulin-positive cells accounted for (25.2-3.36)% of whole induced cells. Although insulin secretion of those insulin-producing cells did not respond to glucose challenge very well, human UCB-MSCs have the ability to differentiate into islet-like cells in vitro and may be a potential new source for islet transplantation.     

Human umbilical cord blood-derived stromal cells are superior to human umbilical cord blood-derived mesenchymal stem cells in inducing myeloid lineage differentiation in vitro

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.     

The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro

Bone marrow-derived mesenchymal stem cells (BM-MSC) are currently being investigated in preclinical and clinical settings because of their self-renewal and multipotent differentiative capacity or their immunosuppressive function. However, BM may be detrimental because of the highly invasive donation procedure and BM-MSC decline with age. Therefore, MSC derived from other sources have been considered as an alternative. However, there is only limited knowledge on their immunomodulatory properties. Human umbilical cord blood (UCB) cells are good substitutes for BM-MSC because of the immaturity of newborn cells. In this study, we successfully isolated MSC from UCB. The morphological phenotypes, cell cycle status, surface markers and differentiation potential of these clonally expanded cells are consistent with BM-MSC. Furthermore, UCB-MSC expanded in vitro retain low immunogenicity and an immunomodulatory effect. Flow cytometry analysis showed that UCB-MSC did not express CD40, CD40 ligand, CD80, CD86 and major histocompatibility complex class II molecules. We have demonstrated that UCB-MSC are incapable of inducing allogeneic peripheral blood mononuclear cell (PBMC) proliferation and have a dose-dependent inhibition of PBMC immune responses in mixed lymphocyte reactions (MLR) and phytohaemagglutinin activation assays, even after interferon-gamma treatment. Additionally, we have found that UCB-MSC can suppress the function of mature dendritic cells. Using transwell systems, we have demonstrated an inhibition mechanism that depends on both cell contact and soluble factors. Based on the findings we conclude that banked UCB could serve as a potential alternative source of MSC for allogeneic application in the future.     

不同培养基培养人脐血间充质干细胞的差异

背景：脐血间充质干细胞为干细胞领域的研究热点,但目前传代及扩增此类细胞尚无简单、有效、完美培养方法。 目的：采用不同的培养基分离培养融合状态的间充质干细胞,以筛选一种较好的体外培养人脐血间充质干细胞的方法。 方法：无菌条件下取正常足月剖宫产新生儿的脐血,随机分为5组：低糖DMEM(Dulbecco改良的Eagle培养基)组、高糖DMEM组、α-DMEM组、低糖DMEM+干细胞因子组、低糖DMEM +骨髓间充质干细胞培养上清组。用淋巴细胞分离液分离脐血的单个核细胞。将脐血单个核细胞接种于含体积分数为10%胎牛血清的上述培养基中,放置于37 ℃、体积分数为5%的CO2培养箱内培养,倒置显微镜观察细胞数量和形态的变化并用流式细胞技术分析细胞的表面抗原。 结果与结论：①各组间充质干细胞培养48 h后贴壁细胞数和细胞存活率的比较：低糖DMEM+干细胞因子组、低糖DMEM +骨髓间充质干细胞培养上清组的贴壁细胞数明显多于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05),细胞存活率亦明显高于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05)。②各组间充质干细胞在不同培养时间下生长状态的比较：培养第3,6,9,12,15,18,21天低糖DMEM+干细胞因子组、低糖DMEM+骨髓间充质干细胞培养上清组细胞增殖的速度均快于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05)。低糖DMEM+干细胞因子组与低糖DMEM +骨髓间充质干细胞培养上清组比较差异无显著性意义。结果可见人脐血间充质干细胞与骨髓间充质干细胞培养上清或干细胞因子共孵育,对脐血间充质干细胞体外分离培养及扩增有支持作用。     

The i blood group antigen as a marker for umbilical cord blood-derived mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. However, there is a lack of methods to quickly and efficiently isolate, characterize, and ex vivo expand desired cell populations for therapeutic purposes. Single markers to identify cell populations have not been characterized; instead, all characterizations rely on panels of functional and phenotypical properties. Glycan epitopes can be used for identifying and isolating specific cell types from heterogeneous populations, on the basis of their cell-type specific expression and prominent cell surface localization. We have now studied in detail the cell surface expression of the blood group i epitope (linear poly-N-acetyllactosamine chain) in umbilical cord blood (UCB)-derived MSCs. We used flow cytometry and mass spectrometric glycan analysis and discovered that linear poly-N-acetyllactosamine structures are expressed in UCB-derived MSCs, but not in cells differentiated from them. We further verified the findings by mass spectrometric glycan analysis. Gene expression analysis indicated that the stem-cell specific expression of the i antigen is determined by β3-N-acetylglucosaminyltransferase 5. The i antigen is a ligand for the galectin family of soluble lectins. We found concomitant cell surface expression of galectin-3, which has been reported to mediate the immunosuppressive effects exerted by MSCs. The i antigen may serve as an endogenous ligand for this immunosuppressive agent in the MSC microenvironment. Based on these findings, we suggest that linear poly-N-acetyllactosamine could be used as a novel UCB-MSC marker either alone or within an array of MSC markers.     

The significant cardiomyogenic potential of human umbilical cord blood-derived mesenchymal stem cells in vitro

We tested the cardiomyogenic potential of the human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs). Both the number and function of stem cells may be depressed in senile patients with severe coronary risk factors. Therefore, stem cells obtained from such patients may not function well. For this reason, UCBMSCs are potentially a new cell source for stem cell-based therapy, since such cells can be obtained from younger populations and are being routinely utilized for clinical patients. The human UCBMSCs (5 x 10(3) per cm(2)) were cocultured with fetal murine cardiomyocytes ([CM] 1 x 10(5) per cm(2)). On day 5 of cocultivation, approximately half of the green fluorescent protein (GFP)-labeled UCBMSCs contracted rhythmically and synchronously, suggesting the presence of electrical communication between the UCBMSCs. The fractional shortening of the contracted UCBMSCs was 6.5% +/- 0.7% (n = 20). The UCBMSC-derived cardiomyocytes stained positive for cardiac troponin-I (clear striation +) and connexin 43 (diffuse dot-like staining at the margin of the cell) by the immunocytochemical method. Cardiac troponin-I positive cardiomyocytes accounted for 45% +/- 3% of GFP-labeled UCBMSCs. The cardiomyocyte-specific long action potential duration (186 +/- 12 milliseconds) was recorded with a glass microelectrode from the GFP-labeled UCBMSCs. CM were observed in UCBMSCs, which were cocultivated in the same dish with mouse cardiomyocytes separated by a collagen membrane. Cell fusion, therefore, was not a major cause of CM in the UCBMSCs. Approximately half of the human UCBMSCs were successfully transdifferentiated into cardiomyocytes in vitro. UCBMSCs can be a promising cellular source for cardiac stem cell-based therapy. Disclosure of potential conflicts of interest is found at the end of this article.     

The therapeutic potential of human umbilical cord blood-derived mesenchymal stem cells in Alzheimer's disease

The neuropathological hallmarks of Alzheimer's disease (AD) include the presence of extracellular amyloid-β peptide (Aβ) in the form of amyloid plaques in the brain parenchyma and neuronal loss. The mechanism associated with neuronal death by amyloid plaques is unclear but oxidative stress and glial activation has been implicated. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are being scrutinized as a potential therapeutic tool to prevent various neurodegenerative diseases including AD. However, the therapeutic impact of hUCB-MSCs in AD has not yet been reported. Here we undertook in vitro work to examine the potential impact of hUCB-MSCs treatment on neuronal loss using a paradigm of cultured hippocampal neurons treated with Aβ. We confirmed that hUCB-MSCs co-culture reduced the hippocampal apoptosis induced by Aβ treatment. Moreover, in an acute AD mouse model to directly test the efficacy of hUCB-MSCs treatment on AD-related cognitive and neuropathological outcomes, we demonstrated that markers of glial activation, oxidative stress and apoptosis levels were decreased in AD mouse brain. Interestingly, hUCB-MSCs treated AD mice demonstrated cognitive rescue with restoration of learning/memory function. These data suggest that hUCB-MSCs warrant further investigation as a potential therapeutic agent in AD.     

脐血浆分离培养人脐带间充质干细胞

目的:利用脐带血血浆分离、培养人脐带间充质干细胞(HUCMSCs).方法:将脐血浆经盐析、透析等处理后,按10％体积成分分离、培养扩增HUCMSCs,观察细胞的形态,流式细胞仪检测细胞表面标志,MTT法检测细胞生长曲线、增殖能力,ELISA检测培养液中碱性成纤维细胞生长因子(bFGF)、noggin的分泌浓度.结果:利用含10％脐带血血浆的培养液能分离、扩增HUCMSCs,分离纯化的HUCMSCs成梭形、增殖能力强、具有分化为成骨细胞、脂肪细胞及神经干细胞的能力,高表达CD29、CD44,低表达或不表达CD34、CD45及HLA-DR.而且在含10％脐带血血浆的培养液中生长的HUCMSCs还能分泌人胚胎干细胞(hESC)生长所需的的bFGF及noggin.结论:脐带血血浆经简单处理后能分离培养HUCMSCs,而且其分泌的细胞因子更适合hESC培养需要.     

脐带间充质干细胞治疗代谢综合征的作用机制

背景：研究表明,干细胞的主要功能有直接参与损伤修复、分泌生长因子促进损伤修复、促血管再生改善血液循环、调节免疫和炎症、抗氧化应激等,可用于治疗多种慢性疾病。 目的：综述脐带间充质干细胞治疗代谢综合征的作用机制。 方法：以“干细胞,脐带间充质干细胞,代谢综合征,胰岛素抵抗,高血糖,高血脂”为中文捡索词,以“stem cells,mbilical cord-derived mesenchymal stem cells,stem cells transplantation, metabolic syndrome,insulin resistance,diabetes, hyperlipidemia”为英文检索词,在维普、中国知网(CNKI)期刊全文数据库和PubMed数据库检索2004年1月至2014年10月有关干细胞移植治疗代谢综合征的文献。排除陈旧性和重复性研究,最终纳入42篇文献进行综述。 结果与结论：脐带间充质干细胞具有自我复制能力和多向分化潜能,是细胞替代治疗的理想种子细胞,同时它还具有旁分泌和免疫调节功能。目前脐带间充质干细胞的分离、培养、鉴定以及体外扩增等技术已相当成熟,且有动物实验研究证实脐带间充质干细胞治疗代谢综合征安全有效,可从根本上治疗靶器官损伤,改善胰岛素抵抗和糖、脂代谢紊乱,但应用于临床仍然有很多问题尚待解决。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Human umbilical cord blood-derived mesenchymal stem cells undergo cellular senescence in response to oxidative stress

Since human mesenchymal stem cells (MSCs) are therapeutically attractive for tissue regeneration and repair, we examined the physiological responses of human umbilical cord blood-derived MSCs (hUCB-MSCs) to genotoxic stress. We found that that sublethal doses of reactive oxygen species (ROS) and ionizing radiation cause DNA damage and reduce DNA synthesis and cell proliferation in hUCB-MSCs, resulting in cellular senescence. In contrast, these physiological changes were limited in human fibroblast and cancer cells. Our data show that reduced activities of antioxidant enzymes, which may occur due to low gene expression levels, cause hUCB-MSCs to undergo cellular senescence in response to oxidative stress and ionizing radiation. Resistance of hUCB-MSCs to oxidative stresses was restored by increasing the intracellular antioxidant activity in hUCB-MSCs via exogenous addition of antioxidants. Therefore, the proliferation and fate of hUCB-MSCs can be controlled by exposure to oxidative stresses.     

甲状腺素诱导人脐血间充质干细胞向软骨细胞分化中的作用

背景：研究发现共培养可诱导人源干细胞向特定细胞分化,但如何获得更多的组织工程所需的种子细胞是研究中的难题。 目的：探讨不同浓度甲状腺素在人脐血间充质干细胞向兔软骨细胞分化中的作用。 方法：将人脐血间充质干细胞与兔软骨细胞按照2∶1比例共培养,并用含有不同浓度甲状腺素(0,0.01,0.1,1,10 μmol/L)的培养基分组刺激,共培养14 d后分别观察各组细胞形态,并提取细胞RNA和蛋白质,Real-time PCR检测聚集蛋白多糖mRNA及Ⅱ型胶原mRNA表达量,Western blotting技术检测Ⅱ型胶原及聚集蛋白多糖蛋白表达水平。 结果与结论：加入0.01,0.1,1,10 μmol/L的甲状腺素干预后,聚集蛋白多糖、Ⅱ型胶原mRNA和蛋白的表达水平均随甲状腺素浓度增加而增强,与单纯共培养组比较差异有显著性意义(P < 0.05)。实验证明高浓度甲状腺素可增强人脐血间充质干细胞与兔软骨细胞共培养后向软骨细胞分化的能力。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

脐带血间充质干细胞的成骨诱导及与β-磷酸3钙的生物相容性

目的探讨脐带血间充质干细胞(umbilical cord blood-derived mesenchymal stem cells,UCB-MSCs)的成骨诱导及与β-磷酸3钙(β-tricalcium phosphate,β-TCP)的生物相容性。方法在无菌条件下抽取人脐血,用密度梯度离心的方法获得脐血单个核细胞,接种到含10%胎牛血清的DMEM/F12培养基中。单个核细胞行贴壁培养后,进行细胞形态学观察,绘制细胞生长曲线,分析细胞周期,检测细胞表面抗原;应用条件培养基进行成骨诱导后,应用免疫组织化学方法检测碱性磷酸酶(alkaline phosphatase,AKP)的表达,应用放免方法分析骨钙素(osteocalcin,OCN)的含量、荧光法检测细胞内钙离子的含量;与β-TCP复合培养后,用扫描电镜检测成骨诱导后的UCB-MSCs与β-TCP的生物相容性。结果采用Percoll(1.073g/ml)分离的脐血间充质干细胞大小较为均匀,梭形或星形的成纤维细胞样细胞。细胞生长曲线测定表明接种后第5d细胞进入指数增生期,至第9d后数量减少;流式细胞检测表明50%～70%细胞为CD29、CD45和CD105阳性;成骨诱导后,UCB-MSCs内AKP、培养基中的OCN含量和细胞内钙离子的含量均明显高于对照组(p0.01);成骨诱导的细胞在β-TCP表面生长良好,细胞周围有白色的钙盐沉积。结论 UCB-MSCs成骨诱导后具有骨细胞特性,与β-TCP具有良好的生物相容性,可作为骨组织工程的种子细胞。     

胎儿脐血间充质干细胞治疗大鼠急性心肌梗死

背景：胎儿脐血间充质干细胞对病变心肌有修复和再生能力,胎儿脐血间充质干细胞移植是治疗心肌梗死的一种新途径。 目的：探讨胎儿脐血间充质干细胞移植治疗大鼠心肌梗死的效果。 方法：选取32只大鼠结扎左冠状动脉前降支制作心肌梗死动物模型,随机等分为移植组和梗死组,从胎儿脐血中分离培养脐血间充质干细胞,制备脐血间充质干细胞悬液,对移植组大鼠进行脐血间充质干细胞移植。 结果与结论：胎儿脐血间充质干细胞在体外可以被成功分离培养;与梗死组相比,移植组大鼠心肌梗死边缘区的微血管密度、左室收缩末压、左室内压最大上升和下降速率显著增加(P < 0.05),左室舒张末压显著下降(P < 0.05),心电图情况稍有好转。表明胎儿脐血间充质干细胞治疗心肌梗死大鼠可以促进心肌血管再生,改善心脏功能。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

人脐带间充质干细胞研究进展及应用

人脐带间充质干细胞是一类具有自我更新和多向分化潜能的成体干细胞。随着细胞技术的提高,人们对于脐带间充干细胞的研究越发深入,在医学的各个领域都取得了长足的进步,如脐带间充质干细胞分离技术及脐带间充质干细胞的诱导分化技术。本文就脐带间充质干细胞的生物学特性、诱导分化潜能、免疫原性进行综述,重点讨论其最新的临床应用。     

血小板裂解液对脐带间充质干细胞增殖与分化的影响

背景：脐带间充质干细胞是近年来干细胞生物学研究中发现的重要细胞之一,在细胞治疗领域有着良好的应用前景。血小板裂解液对于干细胞的增殖及诱导分化都存在不同程度的有利作用,在一定程度上甚至可以代替甚至优于血清在培养基中的作用。 目的：对血小板裂解液结合脐带间充质干细胞的研究作一综述。 方法：于万方等中文数据库和PubMed等英文数据库中,以“脐带,间充质干细胞,血小板裂解液,分化,增殖”为关键词检索文献,总结脐带间充质干细胞及血小板裂解液的生物学特性和相关临床应用,并汇总有关血小板裂解液对脐带间充质干细胞增殖活性和诱导分化能力影响的研究结果。 结果与结论：血小板裂解液对脐带间充质干细胞的增殖活性及诱导分化有正面影响,这使血小板裂解液在干细胞移植治疗中将会起到无可比拟的作用。