Differentiation potential of rabbit CD90-positive cells sorted from adipose-derived stem cells in vitro

To investigate the differentiation potential of purified CD90⁺ cells sorted from adipose-derived stem cells (ADSCs), CD90⁺ cells were sorted from rabbit ADSCs using flow cytometry. Then, cell expansion of CD90⁺ cells and unsorted ADSCs was observed using an inverted microscope. Furthermore, cell surface markers including CD40, CD105, and CD90 on CD90⁺ cells and unsorted ADSCs were quantified using flow cytometry. Additionally, multi-lineage differentiation ability between CD90⁺ cells and unsorted ADSCs was compared, and expression of adipocyte-related genes PPAR-r and CEBPA as well as stem cell-related gene SOX2 in CD90⁺ cells and unsorted ADSCs was determined using real-time quantitative PCR. We found that CD90⁺ cells had a stronger cell proliferation ability than unsorted ADSCs. CD90⁺ cells showed a stronger ability of osteoblast and chondrocyte differentiation than unsorted ADSCs and CD90^? cells, whereas the adipose differentiation ability of CD90⁺ cells was similar to that of ADSCs and CD90^? cells. CD14, CD105, and CD90 on CD90⁺ cells were expressed more highly than those on ADSCs. Additionally, the mRNA expression level of SOX2 in CD90⁺ cells was significantly higher than that in ADSCs, whereas the expression of PPAR-r and CEBPA was markedly lower than that in ADSCs. These results suggested that the purified CD90⁺ cells sorted from ADSCs exhibit a stronger differentiation potential than the unsorted ADSCs.     

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.     

胰岛素样生长因子-1对脂肪间充质干细胞增殖的影响

目的：探讨胰岛素样生长因子-1（insulin-like growth factor-1,IGF-1）对脂肪间充质干细胞（adipose-derived stem cells,ADSCs）增殖的影响。方法：采用密度梯度离心法结合贴壁法分离脂肪间充质干细胞,接种于含体积分数为10%的胎牛血清的DMEM培养基中行贴壁培养。流式细胞仪检测ADSCs表面标志物（CD90、CD29、CD31、CD34、CD45）的表达情况,利用成骨、成脂诱导液诱导ADSCs向成骨细胞、成脂细胞分化,用碱性磷酸酶、油红O染色观察。采用终浓度为0、5、10、15、20、30 ng/mL IGF的培养基培养ADSCs,利用Edu染色标记ADSCs,分析不同浓度的IGF-1对ADSCs增殖的影响。结果：流式细胞术显示ADSCs的表型分子CD90、CD29呈阳性,CD31、CD45呈阴性,成骨诱导后碱性磷酸酶染色阳性,成脂诱导后油红O染色可见大量脂滴,表明培养的ADSCs具有成骨、成脂分化的能力。IGF-1促进ADSCs增殖的作用随IGF-1的作用浓度的增加而增加,并逐渐趋于饱和,在趋于15μg/mL的浓度时达到最大促增殖作用,且随着IGF-1作用时间的延长其促ADSCs增殖的作用逐渐增强。结论：本实验成功分离培养ADSCs,IGF-1对体外培养的ADSCs有促进增殖的作用。     

脂肪来源细胞体外增殖规律及定向诱导分化研究

脂肪组织由整形外科吸脂术获得(19例,31．5±5．8岁)。酶消化法分离抽吸物中细胞,体外扩增至第10代．测定细胞生长曲线、累计倍增数目,明确其体外生长规律和增殖能力;通过对表面抗原CD29、CD105、CD106、CD166、CD49d、CD34、CD31、3G5等的检测分析脂肪来源细胞的群体组成：分别向软骨、骨、脂肪定向诱导,进一步明确该细胞群体定向分化能力。实验表明,每300ml脂肪抽吸物平均可获得5×10~7个有核细胞,体外扩增10代,平均每代倍增数目为1．59±0．224．累计倍增数目为15．53。流式细胞学及免疫细胞化学检测显示,干细胞相关抗原CD29、CD105、CD106、CD166等表达率均＞60%,但与造血系相关的CD34、CD31表达率也分别达到7．3%、29．2%。ADC向软骨诱导可检测到Ⅱ型胶原表达;向成骨诱导可见矿化结节形成,并可检测到AKP、Osteonectin基因表达;向脂肪诱导可检测到PPARr2、GLU-4、Leptin基因表达,细胞内有脂滴形成。脂肪来源的细胞获得量大,体外增殖能力强,并含有具有多向分化潜能细胞,有可能作为组织构建的种子细胞。     

Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells

In this study, adipose-derived mesenchymal stem cells (ADSCs) were isolated from adipose tissues of rats. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by the results of oil red O staining, alkaline phosphatase (ALP), and alcian blue staining, respectively. ADSCs at a density of 1 × 10⁶/cm² were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 d. The cell sheets were positive with von kossa staining. The transmission electron microscopy (TEM) result showed that needle-like calcium salt crystals were deposited on the ECM. These results suggested that the osteogenic cell sheets may have potential osteogenesis ability. ADSCs at a density of 1 × 10⁶/cm² were cultured in the endothelial cell growth medium-2 and the endothelial cell sheets can be formed after 16 d of culture. The TEM image confirmed that the Weibel-Palade corpuscle was seen in the cells. The expression of CD31 was positive, suggesting that the endothelial cell sheets may have a strong ability to form blood vessels. In this study, two types of cell sheets with the potential abilities of osteogenesis and blood vessels formation were obtained by induced culture of ADSCs in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defects.     

Evaluation of CD49f as a novel surface marker to identify functional adipose‐derived mesenchymal stem cell subset

ObjectivesCD49f is expressed on a variety of stem cells and has certain effects on their cytological functions, such as proliferation and differentiation potential. However, whether CD49f is expressed on the surface of adipose tissue‐derived mesenchymal stem cells (ADSCs) and its effect on ADSCs has not been clarified.Materials and methodsThe effects of in vitro culture passage and inflammatory factor treatment on CD49f expression and the adhesion ability of ADSCs from mice and rats were investigated. CD49f⁺ cells were selected from rat ADSCs (rADSCs) by magnetic‐activated cell sorting (MACS), and the cellular functions of CD49f⁺ ADSCs and unsorted ADSCs, including their clonogenic, proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities, were compared.ResultsCD49f expression and the adhesion ability of ADSCs decreased with increasing in vitro culture passage number. TNF‐α and IFN‐γ treatment decreased CD49f expression but increased the adhesion ability of ADSCs. After CD49f was blocked with an anti‐CD49f antibody, the adhesion ability of ADSCs was decreased. No significant difference in clonogenic activity was observed between unsorted ADSCs and CD49f⁺ ADSCs. CD49f⁺ ADSCs had greater proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities than unsorted ADSCs.ConclusionIn the current study, the expression of CD49f on ADSCs was identified for the first time. The expression of CD49f on ADSCs was influenced by in vitro culture passage number and inflammatory factor treatment. Compared with unsorted ADSCs, CD49f ⁺ ADSCs exhibited superior cellular functions, thus may have great application value in mesenchymal stem cell (MSC)‐based therapies.     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

胰岛素样生长因子-1 对脂肪间充质干细胞增殖的影响

目的：探讨胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)对脂肪间充质干细胞(adipose-derived stem cells,ADSCs) 增殖的影响。方法：采用密度梯度离心法结合贴壁法分离脂肪间充质干细胞,接种于含体积分数为10%的胎牛血清的DMEM 培 养基中行贴壁培养。流式细胞仪检测ADSCs表面标志物(CD90、CD29、CD31、CD34、CD45)的表达情况,利用成骨、成脂诱导液诱 导ADSCs 向成骨细胞、成脂细胞分化,用碱性磷酸酶、油红O 染色观察。采用终浓度为0、5、10、15、20、30 ng/mL IGF的培养基培 养ADSCs,利用Edu 染色标记ADSCs,分析不同浓度的IGF-1 对ADSCs增殖的影响。结果：流式细胞术显示ADSCs的表型分子 CD90、CD29 呈阳性,CD31、CD45 呈阴性,成骨诱导后碱性磷酸酶染色阳性,成脂诱导后油红O染色可见大量脂滴,表明培养的 ADSCs具有成骨、成脂分化的能力。IGF-1 促进ADSCs 增殖的作用随IGF-1 的作用浓度的增加而增加,并逐渐趋于饱和,在趋于 15 滋g/mL的浓度时达到最大促增殖作用,且随着IGF-1 作用时间的延长其促ADSCs 增殖的作用逐渐增强。结论：本实验成功分 离培养ADSCs,IGF-1 对体外培养的ADSCs 有促进增殖的作用。     

一种体外快速分离脂肪来源干细胞的方法

目的:以小鼠为模型,建立一种基于流式细胞仪为检测手段的快速分离脂肪来源干细胞的方法,解决间充质干细胞进入实际应用过程中遇到的难题。方法:取BALB/c小鼠腹股沟内侧的皮下脂肪组织,采用Ⅰ型胶原酶消化等系列措施,获取脂肪干细胞(adipose-derived stem cells,ADSCs)。所得细胞分离培养4代后,流式细胞仪分选出CD73+CD45-ADSCs后成骨诱导分化。碱性磷酸酶染色和实时荧光定量PCR检测其分化情况。结果:刚分离培养的ADSCs细胞普遍呈圆形或椭圆形,传至第三代的细胞,非MSCs细胞逐渐被淘汰,剩余的细胞形态逐渐变得一致,细胞形态呈梭形。流式细胞仪检测发现ADSCs细胞表面抗原标记CD73+CD45-为20.7%。所得的ADSCs成骨诱导分化后碱性磷酸酶(alkaline phosphatase,ALP)染色呈阳性,实时荧光定量PCR检测成骨标志基因发现它们表达上调,其中ALP的表达高达22倍。结论:本方法可以获得纯度较高的ADSCs,且耗时少成本低;且提示可采用该方法来获得大量的人源ADSCs用于组织工程修复。     

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。     

人脐带间充质干细胞的分离培养与鉴定简

目的：建立并优化人脐带间充质干细胞分离纯化方法,并对其表面标志与多向分化潜能进行鉴定。方法：收集健康足月产胎儿脐带组织,采用组织块贴壁法进行原代培养,流式细胞仪对其表面标志进行检测,通过向成骨成脂分化对其多向分化潜能进行鉴定,RT-PCR对其干细胞特性基因Oct4、Nanog、Sox2、Nestin进行检测。结果：采用组织块贴壁法可在2周左右获得大量间充质干细胞,培养的细胞经流式细胞仪检测,高表达CD29、CD44、CD105、CD106,低表达CD34、CD45;经成骨成脂诱导2周后可分化为成骨细胞和成脂细胞,RT-PCR检测发现原代细胞表达Oct4、Nanog、Sox2、Nestin基因。结论：人脐带间充质干细胞可在体外扩增培养,具有多向分化潜能,可作为组织工程种子细胞来源。     

Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy.     

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.     

Differentiation of human adipose derived stem cells into Leydig‐like cells with molecular compounds

Leydig cells (LCs) are the primary source of testosterone in the testis, and testosterone deficiency caused by LC functional degeneration can lead to male reproductive dysfunction. LC replacement transplantation is a very promising approach for this disease therapy. Here, we report that human adipose derived stem cells (ADSCs) can be differentiated into Leydig‐like cells using a novel differentiation method based on molecular compounds. The isolated human ADSCs expressed positive CD29, CD44, CD59 and CD105, negative CD34, CD45 and HLA‐DR using flow cytometry, and had the capacity of adipogenic and osteogenic differentiation. ADSCs derived Leydig‐like cells (ADSC‐LCs) acquired testosterone synthesis capabilities, and positively expressed LC lineage‐specific markers LHCGR, STAR, SCARB1, SF‐1, CYP11A1, CYP17A1, HSD3B1 and HSD17B3 as well as negatively expressed ADSC specific markers CD29, CD44, CD59 and CD105. When ADSC‐LCs labelled with lipophilic red dye (PKH26) were injected into rat testes which were selectively eliminated endogenous LCs using ethylene dimethanesulfonate (EDS, 75 mg/kg), the transplanted ADSC‐LCs could survive and function in the interstitium of testes, and accelerate the recovery of blood testosterone levels and testis weights. These results demonstrated that ADSCs could be differentiated into Leydig‐like cells by few defined molecular compounds, which might lay the foundation for further clinical application of ADSC‐LC transplantation therapy.     

Xenotransplantation of Human Adipose-Derived Stem Cells in Zebrafish Embryos

Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3–4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.     

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44⁺CD24^-/lowEpCAM⁺ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.     

Nurr-1基因转染促进脂肪干细胞的神经分化

目的:研究孤儿核受体相关基因1(Nurr-1)对脂肪干细胞(adipose tissue-derived stem cells,ADSC)向神经元方向分化的潜在作用。方法:流式细胞术与成骨、成脂诱导技术鉴定脂肪干细胞;Nurrr-1基因转染脂肪干细胞后,应用神经特异性标志物MAP-2,β-tubulin的免疫荧光染色评估其向神经方向分化的能力。结果:流式细胞术结果表明培养的细胞CD29,CD44表达90%以上,CD45,CD90表达均低于1.5%,经过诱导后,油红O、茜素红S染色均呈阳性,表明所培养的细胞为脂肪干细胞;慢病毒转染Nurr-1基因后,免疫荧光染色检测MAP-2,β-tubulin的免疫荧光强度显著增加;RT-PCR结果显示Nurr-1转染的脂肪干细胞的MAP-2、β-tubulin、NF200的表达量显著提高。结论:Nurr-1基因转染能促进脂肪干细胞向神经方向分化,为神经损伤和神经退行性病变的治疗提供了新途径。     

Stimulatory effect of 17β-estradiol on osteogenic differentiation potential of rat adipose tissue-derived stem cells

Adipose tissue-derived stem cells (ADSCs) are considered as a potential cell source for regenerative medicine and tissue engineering. Although ADSCs have greater proliferation capacity than bone marrow stem cells (BMSCs), lower differentiation ability of these cells limits their utility in experimental and clinical studies. The purpose of this study was to investigate whether 17β-estradiol (E(2)) has a stimulatory effect on osteogenic differentiation potential of ADSCs in vitro. ADSCs were isolated from visceral adipose tissues of rats and treated with different concentrations of E(2) in osteogenic medium (OM) for 21 days. The differences in osteogenic differentiation potential of the cultures were assessed by von Kossa staining, measurement of alkaline phosphatase (ALP) activity and calcium levels. ADSCs cultured in OM supplemented with E(2) showed greater bone-like nodule formation and mineral deposition in comparing with the cells grown in OM. In addition, ALP activity and calcium levels also were significantly higher in the cultures exposed to E(2) than the cells treated only with OM (p < 0.005, n = 5). Our results suggest that E(2) may stimulate the osteogenic differentiation of ADSCs and therefore, can be used as an inducing agent to improve the efficiency of these cells in in vitro and in vivo studies.