Chondrogenesis of adipose stem cells in a porous PLGA scaffold impregnated with plasmid DNA containing SOX trio (SOX-5,-6 and -9) genes

We developed a chondrogenic scaffold system in which plasmid DNA (pDNA) containing SOX trio (SOX-5, -6, and -9) genes was incorporated into a PLGA scaffold and slowly released to transfect adipose stem cells (ASCs) seeded in the scaffold. The purpose of this study was to test the in vitro and in vivo efficacy of the system to induce chondrogenic differentiation of ASCs. The pDNA/PEI-PEG complex-incorporated PLGA/Pluronic F127 porous scaffolds were fabricated by a precipitation/particulate leaching method. The following five kinds of pDNA were incorporated into the scaffolds: 1) pECFP-C1 vector without an interposed gene (control group); 2) SOX-5 plasmids; 3) SOX-6 plasmids; 4) SOX-9 plasmids; and 5) one-third doses of each plasmid (SOX-5, -6, and -9). ASCs were seeded on pDNA-incorporated PLGA scaffolds and cultured in chondrogenic media for 21 days. ASCs were also isolated from rabbits, seeded in pDNA-incorporated PLGA scaffolds, and then implanted in the osteochondral defect created on the patellar groove. The rabbits were sacrificed and analyzed grossly and microscopically 8 weeks after implantation. The percentage of transfected cells was highest on day 14, around 70%. After 21 days, PLGA scaffolds incorporated with each gene showed markedly increased expression of the corresponding gene and protein. Glycosaminoglycan (GAG) assay and Safranin-O staining showed an increased proteoglycan production in SOX trio pDNA-incorporated scaffolds. The COL2A1 gene and protein were notably increased in SOX trio pDNA-incorporated scaffolds than in the control, while COL10A1 protein expression decreased. Gross and histological findings from the in vivo study showed enhanced cartilage regeneration in ASCs/SOX trio pDNA-incorporated PLGA scaffolds.     

Electroporation-mediated transfer of SOX trio genes (SOX-5, SOX-6, and SOX-9) to enhance the chondrogenesis of mesenchymal stem cells

The purpose of this study was to test the hypothesis that the SOX trio genes (SOX-5, SOX-6, and SOX-9) have a lower level of expression during the chondrogenic differentiation of mesenchymal stem cells (MSCs) compared with chondrocytes and that the electroporation-mediated gene transfer of SOX trio promotes chondrogenesis from human MSCs. An in vitro pellet culture was carried out using MSCs or chondrocytes at passage 3 and analyzed after 7 and 21 days. Then, MSCs were transfected with SOX trio genes and analyzed for the expression of chondrogenic markers after 21 days of in vitro culture. Without transforming growth factor-β1, the untransfected MSCs had a lower level of SOX trio gene and protein expression than chondrocytes. However, the level of SOX-9 gene expression increased in MSCs when treated with transforming growth factor-β1. GAG level significantly increased 7-fold in MSCs co-transfected with SOX trio, which was corroborated by Safranin-O staining. SOX trio co-transfection significantly increased COL2A1 gene and protein and decreased COL10A1 protein in MSCs. It is concluded that the SOX trio have a significantly lower expression in human MSCs than in chondrocytes and that the electroporation-mediated co-transfection of SOX trio enhances chondrogenesis and suppresses hypertrophy of human MSCs.     

Co-delivery of SOX9 genes and anti-Cbfa-1 siRNA coated onto PLGA nanoparticles for chondrogenesis of human MSCs

Some genes expressed in stem cells interrupt and/or enhance differentiation. Therefore, the aim of this study was to inhibit the expression of unnecessary genes and enhance the expression of specific genes involved in stem cell differentiation by using small interfering RNA (siRNA) and plasmid DNA (pDNA) incorporated into cationic polymers as co-delivery factors. To achieve co-delivery of siRNA and pDNA to human mesenchymal stem cells (hMSCs), two different genes were complexed with poly(ethyleneimine) (PEI) and then coated onto poly(lactide-co-glycolic acid) (PLGA) nanoparticles (NP). To evaluate co-delivery of siRNA and pDNA into hMSCs, cells were transfected with green fluorescence protein (GFP) pDNA (GFP pDNA) and GFP siRNA (GFP siRNA). The percentage of GFP-expressing hMSCs decreased from 25.35 to 3.7% after transfection with GFP-DNA/PLGA NP (NPs) or GFP siRNA/PLGA NPs, whereas GFP-DNA/PLGA NPs and scramble siRNA (MOCK)/PLGA NPs had no effect on GFP expression. hMSCs cotransfected with coSOX9-pDNA/NPs and Cbfa-1-siRNA/NPs were tested both in vitro and in vivo using gel retardation, dynamic light scattering (DLS), and scanning electron microscope (SEM). The expression of genes and proteins associated with chondrogenesis was evaluated by FACS, RT-PCR, real time-qPCR, Western blotting, immunohistochemistry, and immunofluorescence imaging.     

Long-term durable repaired cartilage induced by SOX9 in situ with bone marrow-derived mesenchymal stem cells

Background: Microfracture is a common procedure for cartilage repair, but it often produces inferior fibrocartilage. We previously reported that a super positively charged SOX9 (scSOX9) promoted hyaline-like cartilage regeneration by inducing bone marrow derived mesenchymal stem cell differentiation into chondrocytes in vivo. Here we examined the long-term efficacy of cartilage repair induced by microfracture with scSOX9 by assessing the biomechanical property of the repaired cartilage.Methods: A cartilage defect was created at the right femoral trochlear groove in New Zealand female rabbits and microfracture was performed. The scSOX9 protein was administered at the site of microfracture incorporated in a collagen membrane.Results: At 12 and 24 weeks, scSOX9 treatment induced hyaline-like cartilage while collagen-membrane alone induced fibrocartilage and mutant scSOX9-A76E poorly induced cartilage repair. The cartilage matrix in scSOX9-treated group showed highly enriched proteoglycan content. Consistent with the histological feature and the thickness of the repaired cartilage, the mechanical property of scSOX9-induced cartilage was also similar to that of normal cartilage.Conclusion: This long-term in vivo study demonstrated that in combination with microfracture, scSOX9 was able to induce reparative tissue with features of hyaline cartilage which was durable in long-term. This technology has the potential to translate into clinical use for cartilage repair to prevent progression to osteoarthritis.     

滑膜间充质干细胞与软骨细胞三维条件下混合培养向软骨细胞的分化

背景：软骨细胞通过自分泌及旁分泌的作用可以为滑膜间充质干细胞向软骨细胞分化提供所需的生长因子及微环境,三维条件下更有利于细胞的黏附增殖与分化。目的：观察滑膜间充质干细胞与软骨细胞混合培养于壳聚糖/Ⅰ型胶原复合支架材料中向成软骨细胞分化的能力。方法：取SD大鼠滑膜组织及软骨组织,用酶消化法获得滑膜间充质干细胞及软骨细胞分别进行培养。取第3代滑膜间充质干细胞及第2代软骨细胞,将二者以1∶2的比例混合培养负载于壳聚糖/Ⅰ型胶原复合支架材料21 d,进行激光共聚焦扫描及免疫组织化学检测。结果与结论：培养72 h后,扫描电镜观察细胞黏附于支架材料表面,并可见细胞分泌大量基质成分。培养     21 d后,激光共聚焦扫描可见细胞在支架表面分布均匀,逐层扫描后细胞逐渐减少。免疫组织化学检测可见基质能被Ⅱ型胶原染色,细胞染色呈现棕黄色。结果表明壳聚糖/Ⅰ型胶原复合支架材料提供三维生长空间,利用软骨细胞分泌生长因子及细胞间的相互作用可以诱导滑膜间充质干细胞向软骨细胞分化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

microRNA-130b downregulation potentiates chondrogenic differentiation of bone marrow mesenchymal stem cells by targeting SOX9

Osteoarthritis (OA) is a chronic health condition. MicroRNAs (miRs) are critical in chondrocyte apoptosis in OA. We aimed to investigate the mechanism of miR-130b in OA progression. Bone marrow mesenchymal stem cells (BMSCs) and chondrocytes were first extracted. Chondrogenic differentiation of BMSCs was carried out and verified. Chondrocytes were stimulated with interleukin (IL)-1β to imitate OA condition in vitro. The effect of miR-130b on the viability, inflammation, apoptosis, and extracellular matrix of OA chondrocytes was studied. The target gene of miR-130b was predicted and verified. Rescue experiments were performed to further study the underlying downstream mechanism of miR-130b in OA. miR-130b first increased and drastically reduced during chondrogenic differentiation of BMSCs and in OA chondrocytes, respectively, while IL-1β stimulation resulted in increased miR-130b expression in chondrocytes. miR-130b inhibitor promoted chondrogenic differentiation of BMSCs and chondrocyte growth and inhibited the levels of inflammatory factors. miR-130b targeted SOX9. Overexpression of SOX9 facilitated BMSC chondrogenic differentiation and chondrocyte growth, while siRNA-SOX9 contributed to the opposite trends. Silencing of SOX9 significantly attenuated the pro-chondrogenic effects of miR-130b inhibitor on BMSCs. Overall, miR-130b inhibitor induced chondrogenic differentiation of BMSCs and chondrocyte growth by targeting SOX9.     

炎症因子干预影响间充质干细胞的免疫调控特性

文题释义： 免疫调节的可塑性：患者免疫系统对炎症反应的应答程度,局部微环境炎症递质的种类、浓度及参与的免疫细胞都会发生不同程度的改变,此时输注间充质干细胞既可以促进免疫应答也可以抑制免疫反应。 炎症因子影响间充质干细胞免疫调节特性研究的意义：间充质干细胞的免疫调节特性是其发挥疾病治疗作用的重要机制之一,在自身免疫性疾病和多种炎症损伤性疾病的治疗中显示了良好疗效。不同炎症环境对间充质干细胞功能、特性的改变也直接影响间充质干细胞的免疫调控作用,阐明间充质干细胞、免疫细胞和炎症环境3者之间的相互作用和变化,不但有利于明确间充质干细胞的作用机制,更有利于针对不同疾病病理机制精确提供疾病治疗的新策略,尤其是有的放矢地研发免疫增强型间充质干细胞制剂,进一步提高疗效,恢复免疫平衡,提供治疗学理论基础和实验依据。 背景：间充质干细胞拥有强大的免疫调节能力,主要表现在调控免疫细胞的增殖、分化和功能状态,调节炎症因子的分泌等。间充质干细胞的免疫调节能力受炎症因子调控,依据微环境中炎症因子的种类、水平,间充质干细胞的免疫调节功能也会发生变化。 目的：综述炎症因子影响间充质干细胞免疫调控特性的研究进展。 方法：应用计算机检索PubMed数据库、Elsevier数据库、CNKI数据库,中文检索关键词或主题词为“间充质干细胞,免疫调节,可塑性,干扰素γ,转化生长因子β,白细胞介素17,白细胞介素35,前列腺素E2”,英文检索关键词为“mesenchymal stem cells,immune regulation,plasticity,interferon gamma,transforming growth factor beta,interleukin17,interleukin 35,prostaglandin E2”。纳入炎症因子干预影响间充质干细胞免疫调控特性的相关文献。 结果与结论：采用干扰素γ、转化生长因子β、白细胞介素17、白细胞介素35和前列腺素E2干预或预处理后,间充质干细胞的生物学特性随之改变,不但可重塑组织微环境、影响炎症反应,而且可重建免疫平衡,进而治疗疾病或缓解疾病进展。针对炎症、免疫反应的不同,在疾病的不同阶段进行合理的个体化、差异化治疗,都有望进一步提高间充质干细胞对免疫炎症性疾病的治疗效果。 ORCID: 0000-0003-4724-0848(王婕) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Different roles of PD-L1 and FasL in immunomodulation mediated by human placenta-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from either bone marrow (BMSCs) or placenta (PMSCs) have the capacity to suppress immune responses to mitogenic and allogeneic stimulations. Both cell contact and soluble factor dependent mechanisms have been proposed to explain this immunosuppression. This study explored the roles of some of cell surface molecules expressed on human PMSCs (hPMSCs) in hPMSC mediated immunomodulation. hPMSCs strongly suppressed mitogen and allogeneic peripheral mononuclear cells (PBMCs) induced T cell activation and proliferation. hPMSCs constituently expressed programmed death-ligand 1 (PD-L1) and Fas ligand (FasL) molecules. Neutralising antibodies to-PD-L1 and FasL significantly reduced the suppressive effect of hPMSCs on T cell proliferation. However, only anti-PD-L1 antibody partially restored early T cell activation suppressed by hPMSCs. Anti-FasL antibody but not anti-PD-L1 antibody reduced apoptosis of activated T cell indicating that FasL molecule plays a role in inducing apoptosis of activated T cells, although overall hPMSCs diminished T cell apoptosis. Different effects of PD-L1 and FasL molecules on T cell activation and activated T cell apoptosis suggest that these two molecules influence T cell response at different stages. hPMSCs significantly prevented activated T cells from going into S phase. Both antibodies to PD-L1 and FasL had significant effect on reversing the effect of hPMSCs on cell cycles. hPMSCs reduced INF-γ but increased IL-10 production by mitogen activated T cells. Both antibodies partially abolished the effect of hPMSCs on INF-γ and IL-10 production. These data demonstrated that PD-L1 and FasL molecules play significant roles in immunomodulation mediated by hPMSCs. This study provides a rational basis for modulation of negative costimulators on hPMSCs to increase their immunosuppressive properties in their therapeutic applications.     

重组腺相关病毒介导的BMP13和SOX9共转染促进骨髓间充质干细胞向类髓核细胞分化

目的观察骨形态发生蛋白13(bone morphogenetic protein 13,BMP13)和性别决定区Y框蛋白9(sex determining region Y box protein 9,SOX9)基因共转染对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向类髓核细胞分化的影响。方法通过腺相关病毒(adeno-associated virus,AAV)将目的基因导入BMSCs中;细胞分为4组:对照组、AAV-SOX9组、AAV-BMP13组和共转染组(AAV-SOX9+AAV-BMP13);应用实时荧光定量PCR技术检测mRNA表达水平;Western blot法检测蛋白表达水平;糖胺聚糖检测试剂盒检测糖胺聚糖的合成水平;MTT法检测细胞增殖。结果共转染组BMP13和SOX9的表达水平明显高于AAV-SOX9组、AAV-BMP13组和对照组(P0.05);共转染组的糖胺聚糖合成水平、角蛋白19(keratin 19,KRT19)及蛋白聚糖(Aggrecan)和Ⅱ型胶原蛋白(typeⅡcollagen,Col2a1)的表达水平明显高于AAV-SOX9组、AAV-BMP13组和对照组(P0.05);另外,AAV-SOX9和AAV-BMP13及共转染组的细胞增殖活性明显高于对照组(P0.05),但AAV-SOX9组和AAV-BMP13组与共转染组的增殖活性相比差异无显著性(P0.05)。结论 BMP13和SOX9双基因转染的BMSCs向类髓核细胞分化的能力明显高于单基因转染组,为椎间盘突出疾病的细胞治疗提供理论依据和参考。     

慢性再生障碍性贫血患者间充质干细胞对T细胞的抑制作用

目的比较来源于正常志愿者和慢性再生障碍性贫血(CAA)患者骨髓间充质干细胞(MSCs)免疫抑制作用的区别。方法培养5例正常志愿者和10例CAA患者的骨髓MSCs,比较两组MSCs的形态、细胞表型、细胞因子的表达,通过PHA刺激的T细胞增殖试验、混合淋巴细胞培养和T细胞周期检测比较两组MSCs对T细胞的抑制作用。结果两组MSCs形态、表型基本相同。CAA来源的MSCs对PHA和同种异体抗原诱导的T细胞的抑制作用均低于正常志愿者来源的MSCs,加入正常志愿者的MSCs后有更多的T细胞阻滞在G0/G1期,但CAA来源的MSCs作用较弱。两组MSCs表达的肝细胞生长因子(HGF)和转化生长因子(TGF-β2)无明显区别,但CAA患者的MSCs表达的TGF-β1、3较正常志愿者MSCs表达的明显减少。结论虽然CAA的MSCs在形态、增殖和细胞表型上基本正常,但其对T细胞的抑制作用减弱,在经过免疫抑制剂治疗后仍然存在,其异常是否与CAA的发病机制有关需要进一步研究。     

Chondrogenesis of human mesenchymal stem cells in fibrin constructs evaluated in vitro and in nude mouse and rabbit defects models

In this study, hMSCs encapsulated in a fibrin hydrogel containing heparinized NPs loaded with TGF-β3 (100?ng/ml), or TGF-β3 (100?ng/ml) alone, were subjected to growth factor release and denaturation tests at one, two and four weeks in in vitro culture systems. Additionally, stem cell differentiation was assessed via RT-PCR, real-time quantitative PCR (qPCR), histology, and immunohistochemical assays. In the in vivo studies with nude mouse, when transplanted into nude mice, hMSCs embedded in fibrin hydrogels survived and proliferated more readily in those samples containing TGF-β3-loaded NPs, or TGF-β3 alone, compared to those containing only NPs or the fibrin hydrogel alone. Additionally, RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses revealed that chondrocyte-specific extracellular matrix (ECM) genes and their proteins were expressed at high levels by hMSCs embedded in hydrogels containing TGF-β3-loaded NPs. Finally, the results observed in the rabbit animal model treated with hMSCs embedded in a fibrin hydrogel containing TGF-β3-loaded NPs were also evaluated by the RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses. The in vitro and in vivo results indicated that transplanted hMSCs together with TGF-β3 may constitute a clinically efficient method for the regeneration of hyaline articular cartilage.     

Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is characterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR)?<5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differentially expressed genes revealed the upregulation of genes categorized as 'neurological system process', 'cell adhesion', 'apoptosis', 'cell surface receptor linked signal transduction', 'intrinsic to membrane' and 'extracellular region'. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NB.     

RGD多肽修饰的改性PLGA仿生支架材料对骨髓间充质干细胞粘附、增殖及分化影响的研究

目的：探索RGD多肽修饰的改性PLGA支架材料上骨髓基质细胞的增殖、粘附及分化情况。方法用异型双功能交联剂Sulfo-LC-SPDP将GRGDSPC多肽共价结合到改性PLGA支架材料上，以未接多肽的改性PLGA材料做对照，取第三代MSC接种到材料上，培养1d、2d、3d、4d后比较材料上的细胞密度来反映细胞的增殖程度；取第三代MSC接种到材料上，培养4h、12h后沉淀法定量检测粘附的细胞数，培养24h后摄光镜图像比较粘附细胞的数量和形态，并用FITC连接的鬼笔环肽对细胞骨架染色，在荧光显微镜下观察细胞骨架的组织情况；取第三代MSC接种到材料上，用成骨性培养基培养7d、14d、21d，检测细胞中ALP活性来了解MSC分化情况。结果：培养1d、2d、3d、4d后细胞的增殖程度无显著性差异；培养4h、12h后实验组细胞粘附率均显著高于对照组，且24h后细胞的粘附质量、细胞骨架的组织情况也较对照组为好；培养14d后实验组细胞表达显著高的ALP活性。结论：RGD多肽修饰对细胞增殖无明显促进作用，但能提高改性PLGA支架材料对骨髓基质细胞的粘附性，对MSC向成骨细胞分化有显著促进作用。     

Effect of human thrombopoietin-modified bone marrow mesenchymal stem cells mediated by recombinant adeno-associated virus on megakaryocytopoiesis

Recently, the research of recombinant thrombopoietin (TPO) and its subsequent use in treating thrombocytopenia following radiation therapy and chemotherapy have become more important in clinics. Our study was to determine the feasibility of recombinant adeno-associated virus (rAAV)-mediated TPO gene transfer into bone marrow-derived mesenchymal stem cells (MSCs) and to evaluate the conditioned medium (CM) obtained from TPO-transduced human (h) hMSCs for promoting the process of megakaryocytopoiesis. We constructed recombinant adeno-associated viruses expressing TPO successfully, and TPO mRNA and protein were both strongly expressed in TPO-transduced hMSCs. There was no decrease in green fluorescent protein (GFP) fluorescence expression of the transduced cells with continuous passaged culturing in vitro. The CM of TPO-transduced hMSCs has been shown to enhance the number of CD41(+) cells and megakaryocytic progenitors (colony-forming unit-megakaryocyte) significantly as compared to the nontransduced control. In this study, a novel safe and efficient method of promoting the megakaryocytopoiesis was established following the TPO-transduced hMSCs. These results provide a basis for the future studies on hematopoietic regulation by hMSCs transfected with TPO.     

Migration of human mesenchymal stem cells under low shear stress mediated by mitogen-activated protein kinase signaling

Human mesenchymal stem cells (hMSCs) are attractive candidates for cell-based tissue repair approaches and have been used as vectors for delivering therapeutic genes to sites of injury. It is believed that hMSCs are able to detect and respond to shear stress due to blood and interstitial fluid flow through mechanotransduction pathways after transplantation. However, information regarding hMSC migration under shear stress and its mechanism is still limited. In this study, we examined the effect of shear stress on hMSC migration and the role of mitogen-activated protein kinases (MAPKs) in their migration. Shear stress between 0.2 and 10 Pa, which was produced by the flow medium, was exerted on fluorescently labeled hMSCs. Cell migration was evaluated using the scratch wound assay, and images were captured using a microscope equipped with a digital 3CCD camera. The results showed that hMSCs subjected to a shear stress of 0.2 Pa caused notably faster wound closure than statically cultured hMSCs, while migration in the 0.5- and 1-Pa shear stress group did not differ significantly from that in the control group. Shear stress >2 Pa markedly inhibited hMSC migration. hMSCs subjected to a shear stress of 0.2 Pa displayed an increase in extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 MAPK activation for up to 60?min, while a shear stress of 2 Pa abrogated the activation. JNK and p38 MAPK inhibitors completely abolished the effect of shear stress on hMSC migration, while significant differences were observed between the ERK1/2 inhibitor-treated static control and shear stress groups. Taken together, these results demonstrate that low shear stress effectively induces hMSC migration and that JNK and p38 MAPK play more prominent roles in shear stress-induced migration than ERK1/2.     

Continuing differentiation of human mesenchymal stem cells and induced chondrogenic and osteogenic lineages in electrospun PLGA nanofiber scaffold

Nanofibers have recently gained substantial interest for potential applications in tissue engineering. The objective of this study was to determine whether electrospun nanofibers accommodate the viability, growth, and differentiation of human mesenchymal stem cells (hMSCs) as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives. Poly(d,l-lactide-co-glycolide) (PLGA) beads with a PLA:PGA ratio of 85:15 were electrospun into non-woven fibers with an average diameter of 760+/-210 nm. The average Young's modulus of electrospun PLGA nanofibers was 42+/-26 kPa, per nanoindentation with atomic force microscopy (AFM). Human MSCs were seeded 1-4 weeks at a density of 2 x 10(6)cells/mL in PLGA nanofiber sheets. After 2 week culture on PLGA nanofiber scaffold, hMSCs remained as precursors upon immunoblotting with hKL12 antibody. SEM taken up to 7 days after cell seeding revealed that hMSCs, hMSC-Ob and hMSC-Ch apparently attached to PLGA nanofibers. The overwhelming majority of hMSCs was viable and proliferating in PLGA nanofiber scaffolds up to the tested 14 days, as assayed live/dead tests, DNA assay and BrdU. In a separate experiment, hMSCs seeded in PLGA nanofiber scaffolds were differentiated into chodrogenic and osteogenic cells. Histological assays revealed that hMSCs continuously differentiated into chondrogenic cells and osteogenic cells after 2 week incubation in PLGA nanofibers. Taken together, these data represent an original investigation of continuous differentiation of hMSCs into chondrogenic and osteogenic cells in PLGA nanofiber scaffold. Consistent with previous work, these findings also suggest that nanofibers may serve as accommodative milieu for not only hMSCs, but also as a 3D carrier vehicle for lineage specific cells.