A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.     

体外扩增过程中人骨髓间充质干细胞的增殖与分化规律

目的：系统考察体外扩增过程中人骨髓间充质干细胞（MSC）的增殖与分化规律，为MSC任组织修复以及细胞治疗中的应用提供参考、方法：以全骨髓贴壁法分离成人肋骨骨髓MSC，在相同条件下分别考察各代细胞形态、生长、表面标记、细胞周期、成骨、成软骨及成脂肪能力的变化情况。结果：随代次增加，MSC增殖能力、成骨、成脂肪能力均有所下降，而成软骨能力无明显降低；成骨、成软骨及成脂肪能乃均保持到细胞衰老。存扩增过程中，MSC始终保持较高的纯度，CD29、CD44、CD105的阳性率均在90％以上，CD14、CD34和CD45的阳性率均在4％以下、结论：在体外培养过程中MSC干细胞特性逐渐丢失，其中向骨、脂肪方向的分化潜能较软骨方向更易失去；而多向分化能力的保持较之自我更新能力更为持久。MSC在7代以前可作为基础研究及临床应用的良好对象。     

Differences in Surface Marker Expression and Chondrogenic Potential among Various Tissue-Derived Mesenchymal Cells from Elderly Patients with Osteoarthritis

Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells that could potentially be used to repair injured cartilage in diseases such as osteoarthritis (OA). In this study we used bone marrow, adipose tissue from articular and subcutaneous locations, and synovial fluid samples from 18 patients with knee OA to find a suitable alternative source for the isolation of MSCs with high chondrogenic potential. MSCs from all tissues analysed had a fibroblastic morphology, but their rates of proliferation varied. Subcutaneous fat-derived MSCs proliferated faster than bone marrow- and Hoffa's fat pad-derived MSCs, while synovial fluid-derived MSCs grew more slowly. CD36 and CD54 expression was similar across all groups of MSCs with several minor differences. High expression of these surface markers in subcutaneous fat-derived MSCs was correlated with poor differentiation into hyaline cartilage. Synovial fluid-derived MSCs presented a relatively small chondrogenic differentiation capacity while Hoffa's fat pad-derived MSCs had strong chondrogenic potential. In conclusion, MSCs from elderly patients with OA may still display significant chondrogenic potential, depending on their origin.     

Comparison of potentials of stem cells isolated from tendon and bone marrow for musculoskeletal tissue engineering

The use of tendon-derived stem cells (TDSCs) as a cell source for musculoskeletal tissue engineering has not been compared with that of bone marrow stromal cells (BMSC). This study compared the mesenchymal stem cell (MSC) and embryonic stem cells (ESC) markers, clonogenicity, proliferative capacity, and multilineage differentiation potential of rat TDSC and BMSC in vitro. The MSC and ESC marker profiles of paired TDSC and BMSC were compared using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Their clonogenicity and proliferative capacity were compared using colony-forming and 5-bromo-2'-deoxyuridine assays, respectively. The expression of tenogenic, osteogenic, and chondrogenic markers at basal state were examined using qRT-PCR. Their osteogenic, chondrogenic, and adipogenic differentiation potentials were compared using standard assays. TDSC and BMSC showed similar expression of CD90 and CD73. TDSC expressed higher levels of Oct4 than BMSC. TDSC exhibited higher clonogenicity, proliferated faster, and expressed higher tenomodulin, scleraxis, collagen 1 α 1 (Col1A1), decorin, alkaline phosphatase, Col2A1, and biglycan messenger RNA levels than BMSC. There was higher calcium nodule formation and osteogenic marker expression in TDSC than BMSC upon osteogenic induction. More chondrocyte-like cells and higher glycosaminoglycan deposition and chondrogenic marker expression were observed in TDSC than BMSC upon chondrogenic induction. There were more oil droplets and expression of an adipogenic marker in TDSC than BMSC upon adipogenic induction. TDSC expressed higher Oct4 levels, which was reported to positively regulate mesendodermal lineage differentiation, showed higher clonogenicity and proliferative capacity, and had greater tenogenic, osteogenic, chondrogenic, and adipogenic markers and differentiation potential than BMSC. TDSC might be a better cell source than BMSC for musculoskeletal tissue regeneration.     

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin,and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.     

A Novel Semisynthetic Molecule Icaritin Stimulates Osteogenic Differentiation and Inhibits Adipogenesis of Mesenchymal Stem Cells

Background We previously reported that the constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common nuclear stem) exerted beneficial effects on the bone, including promoting bone formation and inhibiting bone marrow fat deposition. Recent in vivo study showed that Icaritin was a common metabolite of these constitutional flavonoid glycosides, indicating that Icaritin is a bioactive compound. The present study was designed to investigate whether Icaritin could promote osteogenic differentiation and suppress adipogenic differentiation of marrow mesenchymal stem cells (MSCs).Methods Primary MSCs were harvested from adult mice and exposed to Icaritin to evaluate whether it could promote osteogenesis and suppress adipogenesis using the following assays: determination of alkaline phosphatase (ALP) activity and mineralization; mRNA expression of osteogenic differentiation marker Runx2; osteocalcin and bone sialoprotein (BSP) by RT-PCR; quantification of adipocyte-like cells by Oil Red O staining assay and mRNA expression for adipogenic differentiation markers peroxisome proliferator-activated receptor gamma (PPARγ); adipocyte fatty acid binding protein (aP2) and lipoprotein lipase (LPL) by RT-PCR. For the underlying mechanism, glycogen synthase kinase-3beta (GSK3β) and β-catenin were also explored by western blotting.Results Icaritin promoted osteogenic differentiation and maturation of MSCs as indicated by increased mRNA expression for Runx2, osteocalcin and BSP, and enhanced ALP activity and mineralization; Icaritin inhibited adipogenic differentiation, as indicated by decreased mRNA expression for PPARγ, LPL, aP2, and suppressed formation of adipocyte-like cells; Icaritin inactivated GSK3β and suppressed PPARγ expression when promoting osteogenesis and suppressing adipogenesis of MSCs.Conclusion This was the first study demonstrating that the novel semisynthetic molecule Icaritin could stimulate osteogenic differentiation and inhibit adipogenesis of MSCs, which was associated with the suppression of GSK3β and PPARγ.     

microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.     

Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.     

单轴、双轴循环拉力对小鼠肌腱源性干细胞肌腱样分化的影响

目的 探讨单轴、双轴循环拉力对小鼠肌腱源性干细胞(TDSCs)分化的影响,为临床肌腱损伤后的康复治疗提供理论基础。方法 取6~8周龄C57BL/6小鼠10只,无菌条件下暴露双侧后腿至脚掌,显微镜下解剖收集小鼠髌腱和跟腱组织块,体外分离培养细胞,观察第3代细胞的形态特点。(1)取传代至第3代的细胞,采用流式细胞术检测间充质干细胞标志物(CD44、CD90、Sca-1)、内皮细胞标志物(CD34、Flk-1)、造血细胞标志物(CD45),鉴定细胞是否符合TDSCs特点。(2)取传代至第3代的TDSCs进行成骨细胞、软骨细胞和脂肪细胞分化培养,分别采用茜素红、油红和阿尔新蓝染料对培养的三系细胞进行染色,鉴定细胞是否具有多向分化潜能。(3)取传代至第3代的TDSCs接种到硅胶底培养皿上,分为双轴循环拉力组、单轴循环拉力组、对照组3组。双轴循环拉力组细胞使用Flexcell^􀆿 FX-4000TM柔性基底拉伸加载系统,单轴循环拉力组细胞使用自制拉伸力生物反应器,对照组细胞无拉力。双轴循环拉力组、单轴循环拉力组施加机械负荷组的参数均设置为0.25 Hz、6%的循环拉力,在培养期间进行机械负荷加载,每天加载8 h,共加载6 d。第6天机械负荷刺激结束后,收集3组细胞进行实时荧光定量PCR (qPCR),检测肌腱、成骨、脂肪和软骨相关转录因子的表达。结果 显微镜下观察第3代TDSCs形态一致,呈梭形纤维状。(1)流式细胞技术检测结果显示,间充质干细胞标志物CD44、CD90和Sca-1表达阳性、内皮细胞标志物CD34和Flk-1表达阴性、造血细胞标志物CD45表达阴性,符合TDSCs标记鉴定特点。(2)三系分化细胞检测结果显示,提取的细胞成功分化为成骨细胞、脂肪细胞和软骨细胞,验证了提取的细胞具有向成骨细胞、软骨细胞和脂肪细胞分化的潜能。(3)对照组、单轴循环拉力组、双轴循环拉力组3组间比较,肌腱、成骨、软骨、脂肪相关转录因子的相对表达量差异均有统计学意义(P值均＜0.05)。组间两两比较:单轴循环拉力组与对照组比较,肌腱、成骨相关转录因子以及脂肪相关转录因子PPARγ的相对表达均增高,软骨相关转录因子的相对表达均降低,差异均有统计学意义(P值均＜0.05),而脂肪相关转录因子CEB/P的相对表达差异无统计学意义(P＞0.05);双轴循环拉力组与对照组比较,肌腱相关转录因子Scx、Mohawk的相对表达降低,成骨相关转录因子Runx2的相对表达增高、碱性磷酸酶(ALP)的相对表达降低,软骨相关转录因子Sox9相对表达增高、Col2a1的相对表达降低,脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均＜0.05);单轴循环拉力组与双轴循环拉力组比较,双轴循环拉力组肌腱相关转录因子Scx、Mohawk、Col1a1的相对表达均降低,成骨相关转录因子ALP的相对表达降低,软骨、脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均＜0.05)。结论 单轴循环拉力诱导TDSCs向肌腱细胞、成骨细胞分化,而双轴循环拉力诱导TDSCs向成骨细胞、脂肪细胞、软骨细胞分化。单轴循环拉力的作用下可以促进体外TDSCs向肌腱细胞分化,有利于肌腱组织的再生和损伤后的修复,为临床肌腱损伤后的康复治疗提供了理论依据。     

Hydrostatic Pressure Enhances Chondrogenic Differentiation of Human Bone Marrow Stromal Cells in Osteochondrogenic Medium

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-β1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-β1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.     

Distinct Stem Cells Subpopulations Isolated from Human Adipose Tissue Exhibit Different Chondrogenic and Osteogenic Differentiation Potential

Recently adipose tissue has become a research topic also for the searching for an alternative stem cells source to use in cell based therapies such as tissue engineer. In fact Adipose Stem Cells (ASCs) exhibit an important differentiation potential for several cell lineages such as chondrogenic, osteogenic, myogenic, adipogenic and endothelial cells. ASCs populations isolated using standard methodologies (i.e., based on their adherence ability) are very heterogeneous but very few studies have analysed this aspect. Consequently, several questions are still pending, as for example, on what regard the existence/ or not of distinct ASCs subpopulations. The present study is originally aimed at isolating selected ASCs subpopulations, and to analyse their behaviour towards the heterogeneous population regarding the expression of stem cell markers and also regarding their osteogenic and chondrogenic differentiation potential. Human Adipose derived Stem Cells (hASCs) subpopulations were isolated using immunomagnetic beads coated with several different antibodies (CD29, CD44, CD49d, CD73, CD90, CD 105, Stro-1 and p75) and were characterized by Real Time RT-PCR in order to assess the expression of mesenchymal stem cells markers (CD44, CD73, Stro-1, CD105 and CD90) as well as known markers of the chondrogenic (Sox 9, Collagen II) and osteogenic lineage (Osteopontin, Osteocalcin). The obtained results underline the complexity of the ASCs population demonstrating that it is composed of several subpopulations, which express different levels of ASCs markers and exhibit distinctive differentiation potentials. Furthermore, the results obtained clearly evidence of the advantages of using selected populations in cell-based therapies, such as bone and cartilage regenerative medicine approaches.     

小鼠骨髓间充质干细胞生物学特性和体外诱导分化

目的研究小鼠骨髓间充质干细胞的生物学性状和多系分化潜能。方法取Balb／c小鼠骨髓单个核细胞在低糖的培养液中培养出贴壁生长的细胞,进行形态学观察、细胞周期和免疫表型分析;在不同的因子作用下诱导向成骨细胞、软骨细胞,脂肪细胞分化,并检测诱导后细胞相应的基因表达。结果小鼠骨髓间充质干细胞贴壁生长后形态较均一,增殖能力随着传代逐渐增强,但从第8代后增殖能力明显减退。细胞表达CD29,CD38,CD44,CD106等标记,但CD34和H-2k表达阴性。在不同的诱导培养体系里间充质干细胞能分化为成骨细胞、软骨细胞和脂肪细胞,相应的骨钙蛋白基因,Ⅱ型胶原基因,脂蛋白脂酶基因表达都明显增强。结论从小鼠骨髓可以分离培养出间充质干细胞,在体外有效扩增和诱导分化。表明可以以小鼠为模型研究间充质干细胞在组织工程、细胞移植、基因治疗等领域的运用。     

Adipose tissue-derived stem cells for cell therapy of airway allergic diseases in mouse

The purpose of this study was to compare murine mesenchymal stem cells (MSCs) isolated from bone marrow (BM) and adipose tissue (AT) for the selection of suitable MSCs in cell therapy of an airway allergic animal model. We compared MSCs of BALB/c mice derived from BM and AT with respect to proliferation potential, immunophenotype, and multilineage differentiation capacity. In proliferation potential, MSCs from AT (ASCs) showed higher fibroblastoid colony-forming units frequencies and colony-forming efficiency than MSCs from BM (BMSCs). The flow cytometry analysis showed that both ASCs and BMSCs expressed MSCs-related antigens (CD90 and CD105), whereas they did not express hematopoiesis-related antigens (CD45 and CD11b). There was no significant difference in adipogenic, osteogenic, and chondrogenic differentiation between the murine ASCs and BMSCs. In conclusion, the present study has shown that ASCs had higher CFU-F frequencies and colony-forming efficiency than BMSCs. ASCs and BMSCs presented a similar surface immunophenotype and multilineage differentiation capacity. Therefore, ASCs in BALB/c mice might be a more useful material for cell therapy of the airway allergic experiment due to the abundance, relatively easy harvesting and high proliferation potential.     

Multiple differentiation capacity of STRO-1+/CD146+ PDL mesenchymal progenitor cells

Although mesenchymal progenitor cells can be isolated from periodontal ligament (PDL) tissues using stem cell markers STRO-1 and CD146, the proportion of these cells that have the capacity to differentiate into multiple cell lineages remains to be determined. This study was designed to quantify the proportions of primary human PDL cells that can undergo multilineage differentiation and to compare the magnitude of these capabilities relative to bone marrow-derived mesenchymal stem cells (MSCs) and parental PDL (PPDL) cells. PDL mesenchymal progenitor (PMP) cells were isolated from PPDL cells using the markers STRO-1 and CD146. The colony-forming efficiency and multilineage differentiation potential of PMP, PPDL, and MSCs under chondrogenic, osteogenic, and adipogenic conditions were determined. Flow cytometry revealed that on average 2.6% of PPDL cells were STRO-1(+)/CD146(+), whereas more than 63% were STRO-1(-)/CD146(-). Colony-forming efficiency of STRO-1(+)/CD146(+) PMP cells (19.3%) and MSCs (16.7%) was significantly higher than that of PPDL cells (6.8%). Cartilage-specific genes, early markers of osteoblastic differentiation, and adipogenic markers were significantly upregulated under appropriate conditions in PMP cells and MSCs compared to either their noninduced counterparts or induced PPDL cells. Consistent with these findings, immunohistochemistry revealed substantial accumulation of cartilaginous macromolecules, mineralized calcium nodules, and lipid vacuoles under chondrogenic, osteogenic, or adipogenic conditions in PMP and MSC cultures, respectively, compared to noninduced controls or induced PPDL cells. Thus STRO-1(+)/CD146(+) PMP cells demonstrate multilineage differentiation capacity comparable in magnitude to MSCs and could potentially be utilized for regeneration of the periodontium and other tissues.     

Breast and abdominal adipose multipotent mesenchymal stromal cells and stage-specific embryonic antigen 4 expression

We assessed human mesenchymal stem cells (MSCs) harvested from breast and abdominal adipose tissues enriched in embryonic stage-specific antigen (SSEA-4) expression for osteogenic and adipogenic differentiation in comparison to a mixed cell population. Human adipose was obtained from abdominal and breast tissues of females undergoing gastric bypass and breast reduction, respectively. SSEA-4-expressing cells were enriched from the mixed cell population by magnetic cell sorting and expanded in culture. The results showed that freshly isolated cells from breast and abdominal tissues based on adipose from 3 patients comprised 12 and 10% SSEA-4+ cells, respectively. At passage 0, 48% of the cells from breast adipose tissue were positive for SSEA-4 while 12% of the cells from abdominal adipose tissue were positive for this antigen. The level of SSEA-4-expressing cells remained relatively constant with passaging; SSEA-4-expressing cells from breast tissue comprised 45% of the total while 27% of the cells from abdominal adipose tissue expressed SSEA-4 at passage 5. Cells sorted for SSEA-4 expression exhibited a higher potential for differentiation toward osteogenic and adipogenc cell lineages in vitro when compared to a mixed population. Interestingly, SSEA-4 expression was lost upon differentiation, suggesting that the antigen marks a subpopulation of MSCs. Taken together, the data demonstrate that breast adipose tissue is highly enriched in a subpopulation of MSCs expressing SSEA-4 and suggest that SSEA-4 may be a marker of a subpopulation of MSCs with high potential for osteogenic and adipogenic differentiation.