Nuclear heterogeneity and proliferative capacity of human adipose derived MSC-like cells

Adipose derived stem cells (ADSCs) are MSC-like cells which could be easily used for regenerative medicine. Here, the morphology and proliferative capacity of human ADSCs is discribed. ADSCs were analyzed after one month of cultivation at a density of 10 cells/cm2. 21 colonies were counted. Few atypical cells (huge nuclei and cytoplasm) were found in 9 out of 17 colonies analyzed. ANOVA demonstrated that colonies also differed (P = 0.0025) in nuclei dimensions and scatter in the dimensions in each colony. Nuclei dimensions and cell density logarithms correlated in reverse proportion (-0.7; P = 0.002). Thus, ADSCs were heterogeneous and represented two types of cells: small highly proliferative and large low proliferative cells. Cell heterogeneity observed in some colonies might be due to cells registered at different cell cycle phases. Stable and typical morphology, colony-formation capability and high proliferative capacity of cells indicate visceral adipose tissue as a rich source of ADSCs.     

In Vitro Evaluation of Endothelial Progenitor Cells from Adipose Tissue as Potential Angiogenic Cell Sources for Bladder Angiogenesis

Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.     

Expression pattern of embryonic stem cell markers in DFAT cells and ADSCs

Mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability under the condition of ceiling method, named dedifferentiated fat cells (DFAT cells). These cells exhibit multilineage potential as adipose tissue-derived stromal cells (ADSCs). However, the stem molecular signature of DFAT cells and the difference distinct from ADSCs are still not sure. To study the molecular signature of DFAT cells better, highly purified mature adipocytes were obtained from rats and the purity was more than 98%, and about 98.6% were monocytes. These mature adipocytes dedifferentiated into fibroblast-like cells spontaneously by the ceiling culture method, these cells proliferated rapidly in vitro, grew in the same direction and formed vertex, and expressed extensively embryonic stem cell markers such as Oct4, Sox2, c-Myc, and Nanog, surface antigen SSEA-1, CD105, and CD31, moreover, these cells possessed ALP and telomerase activity. The expression level was Oct4 1.3%, Sox2 1.3%, c-Myc 1.2%, Nanog 1.2%, CD105 0.6%, CD31 0.6% and SSEA-1 0.4%, respectively, which was lower than that in ADSCs, but the purity of DFAT cells was much higher than that of ADSCs. In conclusion, DFAT cells is a highly purified stem cell population, and expressed some embryonic stem cell markers like ADSCs, which seems to be a good candidate source of adult stem cells for the future cell replacement therapy.     

Therapeutic Potential of Human Adipose-Derived Stem Cells (ADSCs) from Cancer Patients: A Pilot Study

Mesenchymal stem cells from adipose tissue (ADSCs) are an important source of cells for regenerative medicine. The therapeutic effect of culture-expanded adipose derived stem cells has been shown; however, optimal xeno-free culture conditions remain to be determined. Cancer patients, specifically those undergoing invasive surgery, constitute a subgroup of patients who could benefit from autologous stem cell transplantation. Although regenerative potential of their ADSCs could be affected by the disease and/or treatment, we are not aware of any study that has evaluated the therapeutic potential of ADSCs isolated from cancer patients in reference to that of ADSCs derived from healthy subjects. Here we report that ADSCs isolated from subabdominal adipose tissue of patients with urological neoplasms yielded similar growth kinetics, presented equivalent mesenchymal surface markers and showed similar differentiation potential into distinct mesodermal cell lineages: adipocytes, chondroblasts and osteoblasts than ADSCs isolated from adipose tissue of age-matched non-oncogenic participants, all under xeno-free growth culture conditions. Molecular karyotyping of patient expanded ADSCs genomes showed no disease-related alterations indicating their safety. In addition, vesicles <100 nm identified as exosomes (EXOs) which may be at least partly responsible for the attributed therapeutic paracrine effects of the ADSCs were effectively isolated from ADSCs and showed equivalent miRNA content regardless they were derived from cancer patients or non-oncogenic participants indicating that the repair capabilities of xeno-free expanded ADSCs are not compromised by patient condition and therefore their xeno-free culture expanded ADSCs should be suitable for autologous stem cell transplantation in a clinical setting.     

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.     

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.     

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.     

Nuclear heterogeneity and proliferation activity of human adipose derived MSC-like cells

Adipose tissue (AT) is an easily available source of mesenchymal-like stem cells (MSCs) that are appropriate for applications in regenerative medicine. There is conflicting evidence on the morphology of AT-derived stem cells (ADSCs). Here, we described the morphology and proliferation activity of human ADSCs. The cells were plated at a density of 10 cells/sm² and cultivated for 1 month. Twenty-one colonies were grown. In nine out of 17 analyzed colonies, few atypical cells (large nuclei and cytoplasm) were found. ANOVA demonstrated that colonies also differed (p = 0.0025) in the size and diameter of cell nuclei. The size of nuclei and logarithm of cell density were correlated in the reverse proportion (−0.7; p = 0.002). Thus, a culture obtained from the stromal vascular fraction (SVF) is heterogeneous and composed of two types of cells, i.e., highly proliferative and large, low proliferative cells. These cells are typical of the MSC and ADSC cultures described in literature. The cell heterogeneity observed in some colonies probably resulted from variations in the cell-cycle phases.     

Exercise ameliorates high-fat diet-induced impairment of differentiation of adipose-derived stem cells into neuron-like cells in rats

Adipose-derived stem cells (ADSCs) can differentiate into neurons under particular conditions. It remains largely unknown whether this differentiation potential is affected by physical conditions such as obesity, which modulates the functions of adipose tissue. In this study, we determined the impact of either a 9-week high-fat diet (60% fat; HFD) or 9-week exercise training on the differentiation potential of ADSCs into neuron-like cells in male Wistar rats. Rats were randomly assigned to a normal diet-fed (ND-SED) group, HFD-fed (HFD-SED) group, or exercise-trained HFD-fed group (HFD-EX). After a 9-week intervention, ADSCs from all groups differentiated into neuron-like cells. Expression of neuronal marker proteins (nestin, βIII-tubulin, and microtubule-associated protein 2 [MAP2]) and the average length of cell neurites were lower in cells from HFD-SED rats than in other groups. Instead, protein expression of COX IV and Cyt-c, the Bax/Bcl-2 and LC3-II/I ratio, and the malondialdehyde level in culture medium were higher in cells from HFD-SED rats. No significant difference between ND-SED and HFD-EX rats was observed, except for the average length of cell neurites in MAP2. Thus, HFD impaired the differentiation potential of ADSCs into neuron-like cells, which was accompanied by increases in apoptotic activity and oxidative stress. Importantly, exercise training ameliorated the HFD-induced impairment of neurogenesis in ADSCs. The adipose tissue microenvironment could influence the differentiation potential of ADSCs, a source of autologous stem cell therapy.     

Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

Adipose tissue-derived stem cells (ADSCs), which are easily harvested and show excellent pluripotency potential, have generated considerable interest in regenerative medicine. In this study, the differentiation of ADSCs was assessed after treatment with dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs). ADSCs exhibited a fibroblast-like morphology and high proliferative capacity. However, after treatment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and in vitro mineralization behaviour of ADSCs were significantly enhanced. Mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype. These results demonstrate that ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment and could be a potential source of cementogenic cells for periodontal regeneration.     

Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle

Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P < 0.0001). After 28-day myogenic induction, higher expression levels of skeletal muscle-specific genes were observed in MDSCs than FMSCs (P < 0.01), and the lowest expression levels were demonstrated in ADSCs among three cells (P < 0.01). Besides, M-Cad and MyHC expressions in ADSCs were not detected by immunofluorescence or real-time quantitative PCR. Furthermore, after 14 days adipogenic induction, PPARγ2, LPL and aP2 mRNA expressions were higher in ADSCs vs. MDSCs (P < 0.01). Besides, MSCs from adult or fetal muscle expressed higher OCN and OPN than ADSCs after 28 days osteogenic induction (P < 0.01). Taken together, our results suggested that cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.     

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.     

Aldehyde dehydrogenase activity helps identify a subpopulation of murine adipose-derived stem cells with enhanced adipogenic and osteogenic differentiation potential

AIM To identify and characterize functionally distinct subpopulation of adipose-derived stem cells(ADSCs).METHODS ADSCs cultured from mouse subcutaneous adipose tissue were sorted fluorescence-activated cell sorter based on aldehyde dehydrogenase(ALDH) activity, a widely used stem cell marker. Differentiation potentials were analyzed by utilizing immunocytofluorescece and its quantitative analysis.RESULTS Approximately 15% of bulk ADSCs showed high ALDH activity in flow cytometric analysis. Although significant difference was not seen in proliferation capacity, the adipogenic and osteogenic differentiation capacity was higher in ALDHHi subpopulations than in ALDHLo. Gene set enrichment analysis revealed that ribosome-related gene sets were enriched in the ALDHHi subpopulation. CONCLUSION High ALDH activity is a useful marker for identifying functionally different subpopulations in murine ADSCs. Additionally, we suggested the importance of ribosome for differentiation of ADSCs by gene set enrichment analysis.     

Neural Differentiation of Rat Adipose-Derived Stem Cells in Vitro

It is reported that adipose-derived stem cells (ADSCs) had multilineage differentiation potential, and could differentiate into neuron-like cells induced by special induction media, which may provide a new idea for restoration of erectile dysfunction (ED) after cavernous nerve injury. The aim of this research was to explore the neuronal differentiation potential of ADSCs in vitro. ADSCs isolated from inguinal adipose tissue of rat were characterized by flow cytometry, and results showed that ADSCs were positive for mesenchymal stem cell markers CD90 and CD44, but negative for hematopoietic stem cell markers. ADSCs maintained self-renewing capacity and could differentiate into adipocytes and neurocytes under special culture condition. In this research, two methods were used to induce ADSCs. In method 1, ADSCs were treated with the preinduction medium including epithelium growth factor, basic fibroblast growth factor, and brain derived neurotrophic factor (BDNF) for 3?days, then with the neurogenic induction medium containing isobutylmethylxanthine, indomethacin, and insulin. While in method 2, BDNF was not used to treat ADSCs. After induction, neuronal differentiation of ADSCs was evaluated. Neuronal markers, glial fibrillary acidic protein (GFAP), and ??-tubulin III (Tuj-1) were detected by immunofluorescence and Western Blot analyses. The expressions of GFAP and Tuj-1 in method 1 were obviously higher then those in method 2. In addition, the positive rate of the neuron-like cells was higher in method 1. It suggested that ADSCs are able to differentiate into neural-like cells in vitro, and the administration of BDNF in the preinduction medium may provide a new way to modify the culture method for getting more neuron-like cells in vitro.     

Isolation, characterization, and differentiation of stem cells derived from the rat amniotic membrane

Abstract Stem-cell-based therapies may offer treatments for a variety of intractable diseases. A fundamental goal in stem-cell biology concerns the characterization of diverse populations that exhibit different potentials, growth capabilities, and therapeutic utilities. We report the characterization of a stem-cell population isolated from tissue explants of rat amniotic membrane. Similar to mesenchymal stem cells, these amnion-derived stem cells (ADSCs) express the surface markers CD29 and CD90, but were negative for the lymphohematopoietic markers CD45 and CD11b. ADSCs exist in culture in a multidifferentiated state, expressing neuroectodermal (neurofilament-M), mesodermal (fibronectin), and endodermal (α-1-antitrypsin) genes. To assess plasticity, ADSCs were subjected to a number of culture conditions intended to encourage differentiation into neuroectodermal, mesodermal, and endodermal cell types. ADSCs cultured in a defined neural induction media assumed neuronal morphologies and up-regulated neural-specific genes. Under different conditions, ADSCs were capable of differentiating into presumptive bone and fat cells, indicated by the deposition of mineralized matrix and accumulated lipid droplets, respectively. Moreover, ADSCs cultured in media that promotes liver cell differentiation up-regulated liver-specific genes (albumin) and internalized low-density lipoprotein (LDL), consistent with a hepatocyte phenotype. To determine whether this observed plasticity reflects the presence of true stem cells within the population, we have derived individual clones from single cells. Clonal lines recapitulate the expression pattern of parental ADSC cultures and are multipotent. ADSCs have been cultured for 20 passages without losing their plasticity, suggesting long-term self-renewal. In sum, our data suggest that ADSCs and derived clonal lines are capable of long-term self-renewal and multidifferentiation, fulfilling all the criteria of a stem-cell population.     

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.     

Effect of Calcitonin Gene-Related Peptide on the Neurogenesis of Rat Adipose-Derived Stem Cells In Vitro

Calcitonin gene-related peptide (CGRP) promotes neuron recruitment and neurogenic activity. However, no evidence suggests that CGRP affects the ability of stem cells to differentiate toward neurogenesis. In this study, we genetically modified rat adipose-derived stem cells (ADSCs) with the CGRP gene (CGRP-ADSCs) and subsequently cultured in complete neural-induced medium. The formation of neurospheres, cellular morphology, and proliferative capacity of ADSCs were observed. In addition, the expression of the anti-apoptotic protein Bcl-2 and special markers of neural cells, such as Nestin, MAP2, RIP and GFAP, were evaluated using Western blot and immunocytochemistry analysis. The CGRP-ADSCs displayed a greater proliferation than un-transduced (ADSCs) and Vector-transduced (Vector-ADSCs) ADSCs (p<0.05), and lower rates of apoptosis, associated with the incremental expression of Bcl-2, were also observed for CGRP-ADSCs. Moreover, upon neural induction, CGRP-ADSCs formed markedly more and larger neurospheres and showed round cell bodies with more branching extensions contacted with neighboring cells widely. Furthermore, the expression levels of Nestin, MAP2, and RIP in CGRP-ADSCs were markedly increased, resulting in higher levels than the other groups (p<0.05); however, GFAP was distinctly undetectable until day 7, when slight GFAP expression was detected among all groups. Wnt signals, primarily Wnt 3a, Wnt 5a and β-catenin, regulate the neural differentiation of ADSCs, and CGRP gene expression apparently depends on canonical Wnt signals to promote the neurogenesis of ADSCs. Consequently, ADSCs genetically modified with CGRP exhibit stronger potential for differentiation and neurogenesis in vitro, potentially reflecting the usefulness of ADSCs as seed cells in therapeutic strategies for spinal cord injury.     

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。