小鼠脂肪来源干细胞体外培养、诱导分化及鉴定

目的:观察C57小鼠ASCs (Adipose-derived stem cells,ASCs)体外培养的生物学特性,探讨其成脂成骨诱导分化能力.方法:无菌条件下切取C57小鼠腹股沟处脂肪组织,0.25％Ⅰ型胶原酶消化,分离培养ASCs,37℃,5％CO2饱和湿度孵箱培养,细胞融合达80％时消化传代.观察其细胞形态;MTT比色法测细胞生长曲线;流式细胞仪检测细胞表面标志物;取第2代细胞行成骨及成脂诱导培养,3周后分别茜素红染色和油红O染色鉴定.结果:从C57小鼠脂肪组织中分离获取的ASCs呈长梭形,成纤维细胞样.细胞生长曲线呈“S”型,证明ASCs具有很强的增殖能力;流式细胞术分析结果:CD29、CD44、CD90阳性表达,CD31、CD34、CD45阴性表达;成骨诱导后茜素红染色阳性,成脂诱导后油红O染色阳性.结论:本试验分离培养的细胞为ASCs,具有确切分化能力.     

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。     

比格犬会厌黏膜来源干细胞的分离培养与生物学特性鉴定

目的:探讨建立喉部黏膜间充质干细胞的分离培养方法,并对其生物学特性进行鉴定,为进一步研究其在喉部瘢痕中形成的作用及喉部组织工程提供参考.方法:以比格犬喉部会厌背侧(舌面)黏膜为研究对象,采用消化培养的方法分离具有间充质干细胞样细胞.选取第3代细胞对其进行生物学特性鉴定,首先利用MTT法检测其增殖活性及克隆形成情况,然后通过流式细胞术检测细胞表面分子标记物CD29及CD34的表达情况,最后应用第3代细胞对其进行成脂肪细胞和成骨细胞分化培养,观察其分化能力.结果:分离培养细胞形态较为一致,绝大多数呈梭形,排列不规则.MTT增殖活性实验及克隆形成试验结果显示,所分离的细胞具有良好的增殖活性和克隆形成率;流式细胞术结果显示,该细胞表达CD29间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34;同时,该细胞诱导分化成脂肪细胞和成骨细胞实验表明,其具有多向分化潜能.结论:从比格犬会厌背侧黏膜分离的细胞具有间充质干细胞样特性,为进一步研究喉部瘢痕形成机制及喉部组织工程提供了技术基础.     

培养人皮下脂肪干细胞纵向分化的实验研究

目的:建立人脂肪干细胞(adipose-derived stem cells,ADSCs)分离、培养的方法,观察其生物学特性,探讨其纵向分化的能力.方法:自皮下脂肪组织获得梭形细胞,观察细胞生物学特征,免疫组化鉴定波形蛋白和CD44.以含有胰岛素、地塞米松、1.甲基-3-异丁基-黄嘌呤的无血清混合培养基诱导其向脂肪细胞纵向分化,以细胞形态学变化.油红O染色和测定甘油磷酸脱氢酶活性判定分化是否成功.结果:人皮下脂肪中能够分离培养出生长旺盛的脂肪干细胞,诱导培养7d后细胞由梭形逐渐变圆,胞质内出现脂滴后逐渐增多融合为脂泡,并与甘油磷酸脱氢酶活性变化相吻合.油红O染色显示约(78.6±2.2)%细胞转变为脂肪细胞.结论:人皮下脂肪中分离的脂肪干细胞在体外诱导条件下能纵向分化为脂肪细胞,可能成为修复软组织缺损及整形美容的又一干细胞来源.     

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.     

鼻黏膜间充质干细胞的培养、鉴定及诱导分化

目的建立Sprague Dawley(SD)大鼠鼻黏膜间充质干细胞(NM-MSCs)的获取、分离、培养方法,初步了解其生物学特性。方法通过SD大鼠鼻黏膜贴壁法体外分离、培养NM-MSCs,光镜下细胞形态学观察,用免疫荧光技术检测间充质干细胞和神经干细胞标记物,用流式细胞术检测细胞表面标记物,再诱导其向骨组织及脂肪组织方向分化,最后对其进行细胞周期检测及分析。结果分离培养的NM-MSCs光镜下以梭形与多角形细胞为主,呈放射状排列,且生长旺盛;免疫荧光染色显示NM-MSCs同时表达STRO-1和Nestin;第4代NM-MSCs不表达CD19、CD31、CD34、CD45及HLADR细胞表面标记物,但表达CD90、CD105基质细胞标记物;NM-MSCs经成骨、成脂诱导后,茜素红染色和油红O染色均呈阳性;细胞周期分析显示NM-MSCs符合干细胞生长的特性。结论 SD大鼠NM-MSCs组织块贴壁法获取容易,操作简单,且可大量扩增用于细胞实验研究,经体外诱导后具有多向分化潜能,具有间充质干细胞的一般生物学特性。SD大鼠鼻黏膜组织块贴壁法为组织细胞工程研究提供了充足的种子细胞来源。     

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。     

人体牙髓干细胞的分离与鉴定

目的探讨牙髓干细胞（DPSC）对牙周病,外伤及肿瘤等造成下颌骨缺损、口腔软组织与神经损伤的修复治疗作用。方法本研究利用组织块培养法分离出人体DPSC,用流式细胞仪进行了鉴定,并进行DPSC成骨、成脂、成神经的分化研究。结果分离出3株DPSC,流式细胞分析表明DPSC表达CD73和CD90标志物,但不表达生血干细胞标志物CD34。用茜素红染色表明DPSC能分化成骨细胞,油红O染色表明DPSC能分化成脂肪细胞,免疫免疫荧光染色表明DPSC分化的细胞表达神经细胞特异标志物TUJ1。结论组织块培养能够高效快速分离表达CD73和CD90的DPSC,在体外诱导条件下DPSC能分化为成骨细胞、脂肪细胞和神经细胞,此研究为DPSC在治疗和修复骨组织缺损和神经损伤中的临床应用提供了实验依据。     

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。     

Isolation,characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models

One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.     

Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose-derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non-cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+ CD45- cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood-derived cells (CD45+), ASCs (CD31- CD34+ CD45- CD90+ CD105- CD146-), endothelial (progenitor) cells (CD31+ CD34+ CD45- CD90+ CD105low CD146+), pericytes (CD31- CD34- CD45- CD90+ CD105- CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10-20 weeks. These results suggest that liposuction-derived cells may be useful and valuable for cell-based therapies.     

Myocardial regeneration potential of adipose tissue-derived stem cells

Various tissue resident stem cells are receiving attention from basic scientists and clinicians as they hold promise for myocardial regeneration. For practical reasons, adipose tissue-derived stem cells (ASCs) are attractive cells for clinical application in repairing damaged myocardium based on the following advantages: abundant adipose tissue in most patients and easy accessibility with minimally invasive lipoaspiration procedure. Several recent studies have demonstrated that both cultured and freshly isolated ASCs could improve cardiac function in animal model of myocardial infarction. The mechanisms underlying the beneficial effect of ASCs on myocardial regeneration are not fully understood. Growing evidence indicates that transplantation of ASCs improve cardiac function via the differentiation into cardiomyocytes and vascular cells, and through paracrine pathways. Paracrine factors secreted by injected ASCs enhance angiogenesis, reduce cell apoptosis rates, and promote neuron sprouts in damaged myocardium. In addition, Injection of ASCs increases electrical stability of the injured heart. Furthermore, there are no reported cases of arrhythmia or tumorigenesis in any studies regarding myocardial regeneration with ASCs. This review summarizes the characteristics of both cultured and freshly isolated stem cells obtained from adipose tissue, their myocardial regeneration potential, and the underlying mechanisms for beneficial effect on cardiac function, and safety issues.     

Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo

Mesenchymal stem-like cells identified in different tissues reside in a perivascular niche. In the present study, we investigated the putative niche of adipose-derived stromal/stem cells (ASCs) using markers, associated with mesenchymal and perivascular cells, including STRO-1, CD146, and 3G5. Immunofluorescence staining of human adipose tissue sections, revealed that STRO-1 and 3G5 co-localized with CD146 to the perivascular regions of blood vessels. FACS was used to determine the capacity of the CD146, 3G5, and STRO-1 specific monoclonal antibodies to isolate clonogenic ASCs from disassociated human adipose tissue. Clonogenic fibroblastic colonies (CFU-F) were found to be enriched in those cell fractions selected with either STRO-1, CD146, or 3G5. Flow cytometric analysis revealed that cultured ASCs exhibited similar phenotypic profiles in relation to their expression of cell surface markers associated with stromal cells (CD44, CD90, CD105, CD106, CD146, CD166, STRO-1, alkaline phosphatase), endothelial cells (CD31, CD105, CD106, CD146, CD166), haematopoietic cells (CD14, CD31, CD45), and perivascular cells (3G5, STRO-1, CD146). The immunoselected ASCs populations maintained their characteristic multipotential properties as shown by their capacity to form Alizarin Red positive mineralized deposits, Oil Red O positive lipid droplets, and Alcian Blue positive proteoglycan-rich matrix in vitro. Furthermore, ASCs cultures established from either STRO-1, 3G5, or CD146 selected cell populations, were all capable of forming ectopic bone when transplanted subcutaneously into NOD/SCID mice. The findings presented here, describe a multipotential stem cell population within adult human adipose tissue, which appear to be intimately associated with perivascular cells surrounding the blood vessels.     

Novel pathway of adipogenesis through cross-talk between adipose tissue macrophages, adipose stem cells and adipocytes: evidence of cell plasticity

Introduction

Previous studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes.

Research Design and Methods

Human adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs.

Results

Co-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes.

Conclusions

Cross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes.     

Adipose derived stem cells (ASCs) isolated from breast cancer tissue express IL-4, IL-10 and TGF-β1 and upregulate expression of regulatory molecules on T cells: do they protect breast cancer cells from the immune response?

Immunomodulatory function of bone marrow derived mesenchymal stem cells in cancer has recently been investigated. But the resident mesenchymal stem cells as whole in cancer and in the breast cancer tissue have not been studied well. In the present work we isolated adipose derived stem cells (ASCs) from breast cancer and normal breast tissues to investigate the expressions of IL-4, IL-10 and transforming growth factor (TGF)-β1 in ASCs and to see if ASCs isolated from patients can modulate the regulatory molecules on peripheral blood lymphocytes. Our results showed that IL-10 and TGF-β1 have significantly higher mRNA expressions in ASCs isolated from breast cancer patients than those from normal individuals (P value <0.05). The culture supernatant of ASCs isolated from breast cancer patients with pathological stage III induced upregulation of the mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4 and CD25 in PBLs. In addition, the percentage of CD4+CD25^highFoxp3⁺ T regulatory cells was increased in vitro. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of the mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF and SDF-1 in normal ASCs was also observed. These data collectively conclude that resident ASCs in breast cancer tissue may have crucial roles in breast tumor growth and progression by inducing regulatory molecules and promoting anti-inflammatory reaction within the tumor microenvironment. Further investigation is required to see if the immune suppression induced by ASCs is an independent property from tumor cells or ASCs gain their immunosuppressive potential from malignant cells.     

Characterization of hematopoietic potential of mesenchymal stem cells

Mesenchymal and hematopoietic tissues are important reservoirs of adult stem cells. The potential of tissue resident mesenchymal stem cells (MSCs) to differentiate into cells of mesodermal and ectodermal lineages has been reported previously. We examined the hypothesis that adherent adipose tissue resident mesenchymal stem cells (ASCs) are capable of generating cells with hematopoietic characteristics. When cultured in differentiation media, clonally isolated ASCs develop into cells with hematopoietic attributes. The hematopoietic differentiated cells (HD) express early hematopoietic (c‐kit, PROM1, CD4) as well as monocyte/macrophage markers (CCR5, CD68, MRC1, CD11b, CSF1R). Additionally, HD cells display functional characteristics of monocyte/macrophages such as phagocytosis and enzymatic activity of α‐Naphthyl Acetate Esterase. HD cells are also responsive to stimulation by IL‐4 and LPS as shown by increased CD14 and HLA‐DRB1 expressions and release of IL‐2, IL10, and TNF. Taken together, this study characterizes the potential of ASCs to generate functional macrophages in vitro, and therefore paves way for their possible use in cell therapy applications. J. Cell. Physiol. 225: 888–897, 2010. © 2010 Wiley‐Liss, Inc.