人脐静脉间充质干细胞的分离培养及生物学特性鉴定

为了对人脐静脉间充质干细胞(MSC)进行分离培养及其生物学特性鉴定。采用胶原酶分步消化法获得人脐静脉间充质干细胞(hUVMSC2)并对其进行体外培养、形态学观察及绘制生长曲线;利用条件培养基诱导法分析该细胞分别向成骨细胞和脂肪细胞分化能力;流式细胞术检测细胞表面标志物CD54、CD105、CD29、CD166、CD44、CD31、CD34、CD49、CD106等表达情况。结果该细胞形态为梭形或成纤维样,不表达内皮来源的vWF因子。在不同诱导条件下,该细胞可分别向成骨细胞和脂肪细胞分化。hUVMSC2细胞表面表达CD54、CD105、CD29、CD166、CD44等间质细胞黏附分子,不表达CD31、CD34、CD49、CD106等内皮或造血细胞相关标志物。该细胞指数生长期倍增时间约为26h,在添加bFGF条件下可迅速增殖,指数生长期倍增时间缩短为16h。研究证实人脐静脉内皮层下存在间充质干细胞,采用分步酶消化法可同时分别获得单根脐静脉的内皮细胞和间充质干细胞。hUVMSC2间充质干细胞具有向脂肪细胞和成骨细胞分化潜能并表达多种黏附分子。     

小鼠脐带间充质干细胞的体外诱导分化

目的分离扩增小鼠脐带间充质干细胞(mouse umbilical cord mesenchymal stem cells,m UCMSCs)探讨其是否可诱导成软骨、脂肪和成骨细胞。方法通过贴壁培养法将m UCMSCs体外分离、扩增、纯化,倒置显微镜下观察细胞的形态特征,运用流式细胞仪检测分析细胞的抗原标志表达进行鉴定。运用诱导培养液对分离的m UCMSCs分别定向诱导培养为软骨、脂肪和成骨细胞。结果运用组织贴块培养法可从新鲜脐带中分离到贴壁生长的成纤维样细胞,这些细胞高表达CD29、CD90和CD105,低表达CD34。成软骨诱导后阿新兰染色呈蓝色;成脂诱导后油红O染色,出现红色脂滴;茜红素染色成骨诱导的m UCMSC,可见红色结节。结论贴壁培养法分离培养所获得的m UCMSCs在体外可诱导分化为软骨、脂肪和成骨细胞。     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的：研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法：采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果：纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论：分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的:研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法:采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果:纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论:分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

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。     

阿尔巴斯绒山羊骨髓间充质干细胞体外分离培养及鉴定

探讨阿尔巴斯绒山羊骨髓间充质干细胞(Arbas cashmere goat bone marrow-derived mesenchymal stem cells,g BMMSCs)体外分离培养,并对其生物学特性进行系统的鉴定,进一步深入分析体外诱导时多向分化相关基因动态表达情况。结果显示,原代培养的g BM-MSCs呈梭形或纺锤形、放射状排列的细胞集落,能够连续稳定传代至15代以上;免疫荧光及流式细胞术检测显示,g BM-MSCs表达间质系特异性表面标志物,如:CD13、CD29、CD44、CD106、CD90及CD166,不表达造血干细胞表面标志物CD45及CD34;体外诱导实验证实该细胞具有多向分化潜能,能够向脂肪细胞、成骨细胞和软骨细胞分化;荧光定量PCR实验结果显示,g BM-MSCs多向分化相关基因的表达水平随着诱导天数的增加呈上升趋势。实验结果证实阿尔巴斯绒山羊骨髓中分离培养的细胞具备g BM-MSCs的生物学特性。     

雪貂脂肪间充质干细胞的分离、分化与鉴定

目的分离、多向诱导分化并进行鉴定雪貂脂肪间充质干细胞。方法无菌采取雪貂腹部皮下脂肪,机械剪碎为0.5 mm~2,采用含0.075%的胶原酶I消化,应用高糖-DMEM培养基(添加4 ng/mLβ-FGF)培养,观察细胞形态特征,流式细胞术分析细胞表面抗原标志的表达,并检测其体外成脂、成骨、成软骨分化能力,还进一步尝试了诱导神经分化。结果运用组织块贴壁法可以从新鲜脂肪组织中分离到贴壁生长的类似成纤维细胞样细胞,流式分析结果显示高表达CD29(53.1%),CD90(99.6%),CD105(93.7%),低表达CD11b(36.3%),CD45(28.3%)。这些细胞在体外经诱导可以分化成为脂肪、骨、软骨和神经元。结论从雪貂脂肪组织中可以分离得到脂肪间充质干细胞,这些细胞具有多向分化能力。     

人尿源干细胞的提取和鉴定

目的从正常人尿液中分离得到具有增殖和分化能力的人尿源干细胞(h USC)。方法收集16例健康成人新鲜尿液,分离培养得到贴壁细胞,显微镜观察拍照。WST-1检测并绘制细胞生长曲线。流式细胞术分析细胞表面分子表达率。成骨和成脂诱导培养基诱导h USC分化。结果采用优化的细胞培养基得到12株具有较快增殖能力的h USC。分离的细胞中表达CD13、CD29、CD73、CD105的细胞数目均大于97﹪,表达CD90的细胞数目不足50﹪,表达造血系表面抗原CD14、CD19、CD34、CD45,内皮细胞表面抗原CD31以及HLA-DR的细胞数目均小于1﹪。h USC经诱导可分化成为骨和脂肪细胞。结论采用本研究自建培养体系可培养得到形态单一、具有较强增殖分化能力的人尿源干细胞。     

脐带血来源间充质干细胞体外分离培养条件的优化

脐带血间充质干细胞（umbilical cord blood mesenchymal stem cells,UCB-MSCs）不仅可以作为滋养层细胞支持造血干细胞在体外的大规模扩增,在造血移植过程中还能够降低并发症的发生率以及加速造血重建功能的恢复．但是,目前UCB-MSCs的原代分离培养成功率一般只有30％左右,为进一步提高该成功率,利用正交实验方法对UCB-MSCs体外培养的主要影响因素：细胞的接种密度、细胞因子的组合及用量、是否添加血清和滋养层细胞,进行逐层筛选,并对培养出的间充质干细胞进行了流式细胞分析和向成骨、软骨及脂肪方向的诱导分化检测,以期获得UCB-MSCs培养的最佳方法．实验结果表明,细胞的接种密度是UCB-MSCs培养最显著的影响因素（P〈0．1）,接种密度越大,MSCs越容易生长,能够培养出MSCs的几率就越大,其次为细胞因子,添加细胞因子能有效地刺激MSCs的生长．在高接种密度的基础上,添加细胞因子IL-3（15μg／L）和GM-CSF（5μg/L）,可大大提高UCB-MSCs体外原代培养的成功率,从30％左右提高到90％以上．流式细胞检测结果显示,所分离培养的细胞表达间充质干细胞的抗原（CD13^＋、CD29^＋、CD44^＋、CD105^＋、CD166^＋）,不表达造血细胞的抗原（CD34^-、CD45^-、HLA-DR^-）,并能够向成骨、软骨及脂肪方向分化,这与源于骨髓的间充质干细胞相一致．所建立的培养方法能够为UCB-MSCs的临床应用提供大量优质的种子细胞．     

体外诱导小鼠脂肪来源干细胞向内皮细胞分化的实验研究

目的：探索体外小鼠脂肪来源干细胞（Adipose—derivedstemcells,ASCs）诱导分化为内皮细胞（endothelialprogenitorcells,EPCs）的可行性。方法：利用I型胶原酶消化法和传代培养纯化法从小鼠脂肪组织中分离、培养及扩增ASCs,通过流式细胞仪检测ASCs特异性表面抗原CD29和CD44、CD105的表达;取第2代ASCs进行内皮诱导：即BD基质胶包被＋内皮细胞生长诱导培养基（M199＋10％血清＋10ng／mL血管内皮细胞生长因子VEGF＋10ng／ml碱性生长因子bFGF）。诱导两周左右,倒置显微镜观察诱导前后细胞的一般形态学特征;同时通过成血管实验观察成管腔能力;通过免疫荧光鉴定CD31表型分子的表达。结果：成功培养小鼠ASCs;CD29、CD44、CD90、呈阳性表达,而CD31、CD34、CD45呈阴性表达;诱导后的细胞形态呈三角形或多边形;HE染色观察可见明显的管腔样结构;免疫荧光法CD31表达阳性;MTT法成功记录EPCs增殖生长曲线。结论：成功的诱导小鼠脂肪来源干细胞分化为血管内皮细胞,为后续体内移植实验提供理论基础,同时也为临床受损组织或器官的重建再生提供实验基础。     

Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure

BACKGROUND: Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. METHODS: Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. RESULTS: The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34+ CD31- CD105+ CD166+ CD45- CD90+ ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. DISCUSSION: We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.     

Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles

Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.     

A rapid and efficient method for primary culture of human adipose-derived stem cells

Adipose tissue contains some populations, adipose-derived stem cells (ADSCs) which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, and endothelial cells. Furthermore, adipose tissue can be easily obtained in large quantities through a simple liposuction. ADSCs are thought to be an alternate source of autologous adult stem cells for cell-based therapy. However, it is time-consuming and inefficient to harvest ADSCs by using a traditional collagenase-digestion method. To meet the demand of large quantities of ADSCs in the basic and applied research of regenerative medicine, we developed a rapid and efficient method for isolation and culture of primary ADSCs. The results indicated that the ADSCs obtained with our method possessed strong abilities of proliferation and colony formation in vitro, and could keep low level of cell senescence with stable population doubling during long-term culture in vitro. Furthermore, these harvested ADSCs were capable to differentiate into osteogenic and adipogenic lineages in the specific induction medium. In addition, the results of flow cytometry analysis indicated that these ADSCs could positively express multiple CD markers, such as CD44, CD105, CD29, CD90, and CD13, and hardly expressed CD31, CD34, CD45, and CD106, which was homologous to the mesenchymal stem cells. Therefore, the ADSCs isolated with our method are consistent with previously reported characteristics of the ADSCs. This new method that we established in this study is an efficient tool to isolate and culture the stem cells from adipose tissue.     

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

Human placenta-derived mononuclear cells （MNC） were isolated by a Percoll density gradient and cultured in mesenchymal stem cell （MSC） maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and m-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class Ⅰ major histocompatibility complex （MHC-I）, but they did not express MHC-Ⅱ molecules. Additionally these cells could suppress umbilical cord blood （UCB） lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells （HSC） from UCB to reduce the potential graft-versus-host disease （GVHD） in recipients.     

Human bone marrow mesenchymal cells express NG2: possible increase in discriminative ability of flow cytometry during mesenchymal stromal cell identification

Background aimsMesenchymal stromal cells (MSC) exhibit non-specific hematopoietic cell and/or stromal cell markers (e.g. CD73, CD105 and CD166) that have been used to identify MSC by flow cytometry. Because a neural glial antigen, NG2 (a progenitor cell marker in the central nervous system), is expressed by several tissue cells originating in the mesenchyme but not hematopoietic cells, it might be useful for isolating and identifying MSC. We investigated NG2 expression on culture-expanded MSC by flow cytometry.MethodsHuman bone marrow (BM) samples taken from 12 donors were cultured for MSC to be used in up to nine serial passages. Using flow cytometry, the neural glial antigen NG2 and commonly used MSC markers CD73, CD105 and CD166, were analyzed on the surface of culture-expanded MSC. The multipotential differentiation of the MSC was examined by adipogenic and osteogenic induction.ResultsThe percentage of cells positive for NG2 was similar to the percentages of cells positive for CD73, CD105 and CD166 in all passages of BM samples. The mean fluorescent intensities of NG2 did not change with culture passage. The MSC was successfully differentiated into adipogenic and osteogenic lines. The cells showed no karyotypic abnormalities.ConclusionsNG2 seems to be a promising marker for investigating the biology of MSC.     

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies

Background: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem‐cell exhibits tri‐lineage differentiation potential and is able to maintain its proliferation potential and cell‐surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad‐derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. Materials and methods: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell‐surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. Results: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell‐surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad‐derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator‐activated receptor γ2 and lipoprotein lipase, and oil red O staining. Discussion: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem‐cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.     

Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers

We first identified and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). Cells from osteoarthritic cartilage were enzymatically isolated and analyzed directly or after culture expansion over several passages by FACS using various combinations of surface markers that have been identified on human MPCs (CD9, CD44, CD54, CD90, CD166). Culture expanded cells combined and the subpopulation derived from initially sorted CD9+, CD90+, CD166+ cells were tested for their osteogenic, adipogenic and chondrogenic potential using established differentiation protocols. The differentiation was analyzed by immunohistochemistry and by RT-PCR for the expression of lineage related marker genes. Using FACS analysis we found that various triple combinations of CD9, CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2-12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints.