Osteoblast differentiation of amniotic fluid-derived stem cells irradiated with visible light

The effect of visible light irradiation on the expression of pluripotent genes (Oct-4, Sox2, and Nanog) in amniotic fluid-derived stem cells (AFSCs) and on the osteogenic differentiation ability of AFSCs was investigated using light-emitting diodes (LEDs) at 0-2 mW/cm(2) in various wavelengths: [blue (470 nm), green (525 nm), yellow (600 nm), and red (630 nm)]. Pluripotent gene expression in AFSCs was up-regulated by visible light irradiation from a LED for more than 6 h. Green light irradiation of AFSCs up-regulated the expression of pluripotent genes more significantly than irradiation with other light. The osteogenic differentiation of AFSCs was facilitated by green and blue light irradiation. Facilitated differentiation into osteogenic cells by visible light irradiation was not mediated by reactive oxygen species (ROS); alkaline phosphatase activity (a marker of early osteogenic differentiation) and gene expression of osteopontin (a marker of late osteogenic differentiation) did not change significantly between AFSCs in differentiation medium with or without a ROS scavenger (vitamin C). The mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway, as well as other unknown signaling pathways, may be responsible for the activation of signaling pathways that facilitate the differentiation of AFSCs into osteogenic cells on light irradiation.     

胚胎干细胞向成骨、软骨细胞定向分化研究进展

骨、软骨组织工程的热点之一是寻找合适的种子细胞.胚胎干细胞因具有全能性和无限增殖的能力有望成为组织工程中的种子细胞新来源.主要介绍了胚胎干细胞定向诱导分化为成骨、软骨细胞研究所取得的进展,并展望了胚胎干细胞作为组织工程种子细胞的前景和所面临的困难.     

Osteoblastic differentiation potential of human amniotic fluid-derived mesenchymal stem cells in different culture conditions

Osteoporosis is a bone degenerative disease characterized by a decrease in bone strength and an alteration in the osseous micro-architecture causing an increase in the risk of fractures. These diseases usually happen in post-menopausal women and elderly men. The most common treatment involves anti-resorptive agent drugs. However, the inhibition of bone resorption alone is not adequate for recovery in patients at the severe stage of osteoporosis who already have a fracture. Therefore, the combination of utilizing osteoblast micro mimetic scaffold in cultivation with the stimulation of osteoblastic differentiations to regain bone formation is a treatment strategy of considerable interest. The aims of this current study are to investigate the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid and to compare the monolayer culture and scaffold culture conditions. The results showed the morphology of cells in human amniotic fluid as f-type, which is a typical cell shape of mesenchymal stem cells. In addition, the proliferation rate of cells in human amniotic fluid reached the highest peak after 14 days of culturing. After which time, the growth rate slowly decreased. Moreover, the positive expression of specific mesenchymal cell surface markers including CD44, CD73, CD90, and also HLA-ABC (MHC class I) were recorded. On the other hand, the negative expressions of the endothelial stem cells markers (CD31), the hematopoietic stem cells markers (CD34, 45), the amniotic stem cells markers (CD117), and also the HLA-DR (MHC class II) were also recorded. The expressions of osteoblastogenic related genes including OCN, COL1A1, and ALP were higher in the osteogenic-induced group when compared to the control group. Interestingly, the osteoblastogenic related gene expressions that occurred under scaffold culture conditions were superior to the monolayer culture conditions. Additionally, higher ALP activity and greater calcium deposition were recorded in the extracellular matrix in the osteogenic-induced group than in the culture in the scaffold group. In summary, the mesenchymal stem cells derived from human amniotic fluid can be induced to be differentiated into osteoblastic-like cells and can promote osteoblastic differentiation using the applied scaffold.     

人羊水来源间充质干细胞向成肌细胞诱导的体外实验研究

目的 探讨成肌细胞条件培养液体外诱导人羊水来源间充质干细胞向成肌细胞分化的可行性.方法 B超引导下穿刺抽得孕中期羊水,体外培养、分离得到羊水来源间充质干细胞.鼠成肌细胞体外培养后收集上清液,检测上清液中半乳糖凝集素-1（Galectin-1）含量,并制备成肌细胞条件培养液.实验组于成肌细胞条件培养液中培养,对照组于成肌细胞诱导培养液中培养.观察2组细胞形态学变化,免疫荧光染色、RT-PCR检测成肌细胞特异性标志物Pax7、MyoD、肌结蛋白（Desmin）、肌钙蛋白Ⅰ（Tn Ⅰ）及mRNA表达情况.结果 倒置相差显微镜下可见实验组细胞诱导第18天出现折光性强、体积较小的细胞,呈多角形,并带有突起,且逐渐成长条形;可见少量多核细胞.对照组细胞呈扁平多角形,胞体较大.诱导24 d免疫荧光染色及RT-PCR提示实验组细胞不同程度表达Pax7、MyoD、Desmin、TnⅠ及mRNA;对照组呈阴性.成肌细胞培养上清液中半乳糖凝集素-1含量较低.结论 成肌细胞条件培养液能诱导人羊水来源间充质干细胞向成肌细胞样细胞分化.     

Mouse adipose-derived stem cells undergo multilineage differentiation in vitro but primarily osteogenic and chondrogenic differentiation in vivo

Human, rat, and mouse studies have demonstrated the existence of a population of adipose-derived adult stem (ADAS) cells that can undergo multilineage differentiation in vitro. However, it remains unclear whether these cells maintain their multilineage potential in vivo. The aim of this study was to examine the in vitro and in vivo characteristics and behavior of a potential population of murine ADAS (muADAS) cells isolated from the visceral fat of the abdominal cavity of C57BL/10J mice. We used flow cytometry to examine the cells' expression of CD29, CD31, CD45, CD34, CD44, CD144, CD146, Flk1, and Sca-1. The isolated cell population was CD45 negative, which precludes contamination by hematopoietic cells, but was partially positive for Sca-1 and CD34: 2 stem-cell markers. After induction in conditioned medium, the muADAS cells gained the ability to undergo adipogenic, osteogenic, chondrogenic, myogenic, and hematopoietic differentiation in vitro. The muADAS cells readily differentiated to form bone and cartilage in vivo for up to 24 weeks, but their ability to regenerate muscle or reconstitute bone marrow was found to be limited.     

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.     

Comparing hydroxyapatite with osteogenic medium for the osteogenic differentiation of mesenchymal stem cells on PHBV nanofibrous scaffolds

Having advantageous biocompatibility and osteoconductive properties known to enhance the osteogenic differentiation of mesenchymal stem cells (MSCs), hydroxyapatite (HA) is a commonly used material for bone tissue engineering. What remains unclear, however, is whether HA holds a similar potential for stimulating the osteogenic differentiation of MSCs to that of a more frequently used osteogenic-inducing medium (OIM). To that end, we used PHBV electrospun nanofibrous scaffolds to directly compare the osteogenic capacities of HA with OIM over MSCs. Through the observation of cellular morphology, the staining of osteogenic markers, and the quantitative measuring of osteogenic-related genes, as well as microRNA analyses, we not only found that HA was as capable as OIM for differentiating MSCs down an osteogenic lineage; albeit, at a significantly slower rate, but also that numerous microRNAs are involved in the osteogenic differentiation of MSCs through multiple pathways involving the inhibition of cellular proliferation and stemness, chondrogenesis and adipogenesis, and the active promotion of osteogenesis. Taken together, we have shown for the first time that PHBV electrospun nanofibrous scaffolds combined with HA have a similar osteogenic-inducing potential as OIM and may therefore be used as a viable replacement for OIM for alternative in vivo-mimicking bone tissue engineering applications.     

流体静压力及雌激素共同作用对骨髓间充质干细胞成骨及成软骨分化的影响

目的观察流体静压力联合雌激素对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖、细胞骨架及成骨、成软骨向分化的影响,检测这两种刺激共同作用是否具有生物叠加效应。方法采用全骨髓贴壁分离法分离培养BMSCs,流式细胞仪进行BMSCs表面标志物分子鉴定。细胞分为对照组(C组)、1 nmol/L17β-雌二醇作用组(E组)、1 nmol/L雌激素受体拮抗剂TAM作用组(T组)、90 kPa压力加载1 h组(P组)、17β-雌二醇预处理12 h后再加压1 h组(P+E组)以及TAM预处理12 h后再加压1 h组(P+T组)。流式细胞仪检测各组细胞的细胞周期变化;FITC-鬼笔环肽染色后激光共聚焦显微镜下观察F-actin细胞骨架表达与重组情况;各组细胞经成骨诱导7 d和14 d后茜素红染色观察钙结节形成量、Real-time PCR检测成骨细胞标志基因Col I、ON、OPN及BSP的mRNA表达;成软骨诱导14 d和28 d后甲苯胺蓝染色检测蛋白多糖表达、Real-time PCR检测成软骨细胞标志基因Sox9、Aggrecan及ColⅡ的mRNA表达。采用SPSS 16.0对数据进行单因素方差分析,后续Dunnett t检验进行各时间点上实验组与空白对照组数据之间及各实验组之间的组间比较。结果流体静压力(90 kPa,1 h)及1 nmol/L 17β-雌二醇作用下细胞增殖能力以及细胞骨架的活性均增强,但并不具有生物整合效应。成骨诱导14 d后,钙结节形成。Real-time PCR结果显示,17β-雌二醇作用组中成骨标志基因Col I、ON、OPN及BSP的表达水平显著增加。两种刺激在成骨诱导分化7 d时均可提高标志基因的表达,具有生物协同效应,但成骨诱导14 d时两者则表现出相互拮抗的作用。成软骨诱导28 d后,甲苯胺蓝染色阳性。成软骨诱导过程中,单纯压力刺激组成软骨标志基因Sox9、Aggrecan及ColⅡ的表达显著升高,雌激素预调则削弱压力对间充质干细胞的促成软骨分化作用。结论流体静压力及雌激素仅在骨髓间充质干细胞成骨分化早期表现生物整合效应,而在增殖、细胞骨架活性方面则无此效应。在成骨分化晚期及成软骨分化中两种刺激因素表现出拮抗作用,雌激素可促成骨分化,而压力则表现为促成软骨分化的作用。     

人羊水间充质干细胞分离培养方法的优化

背景：目前关于人羊水间充质干细胞的体外分离培养方法不一,获得较多数量的间充质干细胞仍有困难。 目的：分离80份羊水标本,比较不同的体外培养方法,优化筛选到一种合适的培养纯化条件,为人羊水间充质干细胞的基础研究和临床广泛应用奠定基础。 方法：无菌条件下采集足月分娩和终止妊娠引产的羊水,分离羊水细胞,比较孕期、不同培养基、接种密度、首次换液时间对人羊水间充质干细胞原代培养过程的影响,通过细胞化学染色方法检测细胞代谢情况,观察人羊水间充质干细胞的生物学特性。 结果与结论：相同培养条件下,采用AmnioMAXⅡcomplete羊水专用细胞培养基,以5×10⁴/cm² 的密度接种,首次换液时间为6 d时,人羊水间充质干细胞原代培养成功率较高。孕期为14~20周的人羊水间充质干细胞培养成功率高于晚期妊娠(21~36周)及足月分娩(37~42周)的羊水。人羊水间充质干细胞强表达过碘酸-雪夫、酸性磷酸酶、酸性α-醋酸萘酚酯酶染色,弱表达中性粒细胞碱性磷酸酶,不表达过氧化酶、苏丹黑染色。提示,优化筛选到一种合适的人羊水间充质干细胞培养纯化条件。     

In vitro and in vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage

Recent findings have shown that amniotic fluid (AF) could be a putative new source of multipotent stem cells (SC). We investigated whether these human SC could efficiently differentiate into myogenic lineage in vitro and integrate in vivo skeletal muscle in severe combined immunodeficiency (SCID) mice. C/kit immunomagnetic-sorted AF (AF c/kit+) SC were characterized by immunocytochemistry and Southern blotting for myogenic markers (desmin, MyoD). In vitro, AF c/kit+ SC phenotypic conversion into myogenic cells was assayed by myogenic-specific induction media. AF c/kit+ SC without ex vivo manipulation were transplanted into the tibialis anterior (TA) of (SCID) mice. Acquisition of a myogenic-like phenotype (desmin, MyoD) in AF c/kit+ SC was observed after culture in myogenic-specific induction media. In vivo, transplanted AF c/kit+ SC showed an engraftment in the skeletal muscle of SCID mice, but with unexpected tubular glandular tissue-like differentiation. Importantly, no immuno-rejection, inflammatory response or tumorigenicity of these cells was found. Within these experimental conditions, AF c/kit+ SC were able to differentiate into myogenic cells in vitro, but not in vivo after their transplantation into the skeletal muscle of SCID mice. Because AF c/kit+ SC survived and differentiated into tubular gland-like cells after their transplantation in the TA, an ex vivo engagement in myogenic pathway prior their transplantation could favor their differentiation into myogenic cells in vivo.     

Expansion and chondrogenic differentiation of human mesenchymal stem cells

The potential of human mesenchymal stem cells (hMSC) to differentiate into various types of mesenchymal tissue, such as chondrocytes, makes them a potential cell source in cartilage tissue engineering. Because of the requirement of high cell amounts for the generation of cartilage implants or for the extensive experimental studies to investigate the culture parameters, the initial cells have to be expanded, which leads to high population doubling numbers. It is known that hMSC can differentiate into chondrocytes at least up to the 15th population doubling. To monitor the differentiation status, the protein MIA (melanoma inhibitory activity), which is only synthesized by malignant melanomas and chondrocytes, can be used. In this study the chondrogenic differentiation potential of hMSC beyond the 15th population doubling was investigated using MIA as a chondrocyte marker. A chondrogenic potential of hMSC at higher population doubling numbers may be of interest due to the requirement of less frequent isolations of cells. Therefore hMSC were cultured in a monolayer until the 37th population doubling. Cells of different passages were cultured as pellets for two weeks in transforming growth factor (TGF)-beta3 containing differentiation medium. The MIA contents in medium on the last three cultivation days were measured for each case using an MIA-ELISA-kit. A significant difference between MIA content in medium of the pellet and nonstimulated monolayer reference cultures was detectable until the 32nd population doubling. In addition, the hMSC were seeded at lower densities to investigate whether the cells may be expanded faster and with less amount of work due to higher population doubling numbers per passage. The reduced inoculation density led to an increased growth rate.     

诱导胚胎干细胞向软骨细胞分化研究进展

胚胎干细胞因其具有体外无限增殖能力和体内外分化发育的全能性,有望为细胞治疗或组织工程化组织构建提供可靠的种子细胞来源.如何诱导胚胎干细胞向目的细胞分化是目前面临的一大难题.首先回顾软骨细胞的体内发生、发育过程,继而对目前已知的能在体外培养环境中影响胚胎干细胞向软骨细胞分化的各类因素进行分析综述,并探讨进一步的研究方向.     

The influence of three-dimensional nanofibrous scaffolds on the osteogenic differentiation of embryonic stem cells

Embryonic stem cells represent a potentially unlimited cell source for tissue engineering applications. However, in order to be used for such applications, embryonic stem cells' differentiation must be controlled to only the desired lineages. In this study, we examine the effects of nanofibrous architecture and biochemical cues on the osteogenic differentiation of embryonic stem cells compared to the more traditional architecture without the nanofibrous features in two dimensions (thin matrix or flat films) and three dimensions (scaffolds) in vitro. After three weeks of culture the nanofibrous thin matrices were capable of supporting mRNA expression of osteogenic differentiation markers in embryonic stem cells without osteogenic supplements, while solid films required osteogenic supplements and growth factors to achieve mRNA expression of osteogenic differentiation markers. Nanofibrous scaffolds substantially enhanced mRNA expression of osteogenic differentiation markers compared to solid-walled scaffolds, nanofibrous thin matrices or solid films. After 4 weeks of culture, nanofibrous scaffolds were found to contain 3 times more calcium and stronger osteocalcin stain throughout the scaffolds than the solid-walled scaffolds. Overall, the nanofibrous architecture enhanced the osteogenic differentiation and mineralization of embryonic stem cells compared to the solid-walled architecture in both two and three-dimensional cultures.     

羊水间充质干细胞体外培养方案的优化及生物学特性***

背景：目前干细胞工程种子来源多样化,羊水中存在胎儿脱落细胞,可分离培养出间充质来源干细胞。 目的：探索不同成分培养基对人羊水间充质干细胞(human amniotic fluid-derived mesenchymal stem cells,h-AFMSCs)体外培养的影响,并分析最适培养基体外培养的 h-AFMSCs 的生物学特性,建立优化的h-AFMSCs培养方案。 方法：孕16~24周的孕妇产前检查中获得羊水进行原代及传代培养并采用6种不同的培养基成分对h-AFMSC进行培养。 结果与结论：从孕中期羊水中可分离出h-AFMSCs,体外使用低糖DMEM培养基(L-DMEM)+体积分数10%胎牛血清与合成培养基MESEN PRO体积比1∶1配制的培养基对其扩增促进作用最强。h-AFMSCs高表达CD29、CD73、CD90、CD105、CD166,低表达CD14、CD34、CD45及HLA-DR;分离培养的h-AFMSCs高表达的干细胞基因为OCT-4和Nanog;h-AFMSCs体外具有向成骨、成脂细胞分化的潜能且具有抑制淋巴细胞增殖的作用。提示孕中期羊水是低免疫原性的h-AFMSCs的良好细胞来源。 关键词：羊水;间充质干细胞;优化培养;生物学特性;培养基 doi:10.3969/j.issn.1673-8225.2012.10.016     

人脂肪来源间充质干细胞成骨及成软骨的潜能

背景：人脂肪来源间充质干细胞是种具有较强的体外增殖和多系分化能力的成体干细胞,可以从美容吸脂手术中获得,取材方便,原料来源丰富,在生物治疗应用方面蕴藏着巨大的价值。 目的：体外分离、培养人脂肪来源间充质干细胞,探讨其基本生物学特性及成骨成软骨的潜能。 方法：取美容吸脂获得的脂肪组织,采用Ⅱ型胶原酶消化法分离人脂肪来源间充质干细胞并进行体外培养;观察细胞形态、测定细胞周期、流式细胞仪鉴定细胞表面标志;取第3代细胞,分别加入成骨诱导培养基及成软骨诱导培养基行体外成骨及成软骨诱导。 结果与结论：体外培养人脂肪来源间充质干细胞呈纤维样形态,原代细胞24 h内贴壁,培养5~7 d 后开始形成细胞集落;经细胞周期检测显示G₀/G₁,S和G₂/M所占比例分别为(88±2)%,(12±2)%和0.03%。经流式细胞仪检测CD29和CD105呈阳性表达,CD34和CD45呈阴性表达。RT-PCR检测显示,人脂肪间充质干细胞经成骨诱导分化后细胞中骨桥蛋白mRNA呈阳性表达,经软骨诱导分化后细胞中Ⅱ型胶原mRNA呈阳性表达。结果证实,实验成功体外分离、培养人脂肪来源间充质干细胞,其具有向成骨细胞和软骨细胞分化的潜能。     

体外构建人羊膜间充质干细胞膜片及成骨分化潜能

文题释义：细胞膜片技术：该技术避免了蛋白酶的消化和外源性支架材料的应用,通过细胞外基质分泌形成膜片组织,然后将膜片用于修复组织缺损和改善器官功能。该技术保留了大量自体细胞分泌的细胞外基质,为细胞的增殖和分化提供与体内极度相似的微环境,目前该技术已经用于临床眼角膜和食管损伤的修复。人羊膜间充质干细胞：取自于废弃的胎盘,贴壁生长,具有低免疫原性和生长周期短等特点,不仅具有成体间充质干细胞的特性还具有部分胚胎间充质干细胞的特性。  摘要背景：人羊膜间充质干细胞属于成体干细胞,其来自于废弃的胎盘,来源广泛,可以无创获取,具有免疫原性低、生长周期短等特点,是组织工程种子细胞的重要来源,目前人羊膜间充质干细胞已经用于临床糖尿病的治疗。目的：探索一种简便的方法构建人羊膜间充质干细胞膜片,并研究其成骨分化潜能。方法：将第3代人羊膜间充质干细胞高密度接种于普通培养皿中,加入成膜片诱导培养基以构建人羊膜间充质干细胞膜片,通过组织学染色以及扫描电镜观察细胞膜片的特性。取第3代人羊膜间充质干细胞高密度接种于培养皿中,加入成膜片诱导培养基培养7 d,再换用成骨诱导培养基培养14 d以构建成骨诱导的人羊膜间充质干细胞膜片。通过茜素红染色、免疫组化染色、碱性磷酸酶活性、RT-PCR以及Western blot检测人羊膜间充质干细胞膜片的成骨分化潜能。结果与结论：①苏木精-伊红染色可见人羊膜间充质干细胞膜片由多层细胞累积而成,细胞分布均匀;②扫描电镜观察可见人羊膜间充质干细胞膜片呈复层结构,胞外有大量的胞外基质产生,细胞包埋于胞外基质中;③人羊膜间充质干细胞膜片成骨诱导14 d,茜素红染色后可见橘红色沉淀,免疫组化染色后细胞周围有大量Ⅰ型胶原产生;④与未诱导的人羊膜间充质干细胞膜片相比,成骨诱导14 d后人羊膜间充质干细胞膜片碱性磷酸酶活性显著升高(P < 0.01),Ⅰ型胶原、骨钙蛋白、Runt相关转录子2的mRNA和蛋白表达量显著升高(P < 0.05);⑤该实验应用一种简便、经济的方法在普通培养皿上成功构建了人羊膜间充质干细胞膜片,体外研究证实人羊膜间充质干细胞膜片具有良好的成骨分化潜能。ORCID: 0000-0003-2163-3897(邹刚)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     

Proteomic characterization of the conditioned media produced by the visceral endoderm-like cell lines HepG2 and END2: toward a defined medium for the osteogenic/chondrogenic differentiation of embryonic stem cells

Visceral endoderm (VE) is an extraembryonic cell layer formed before gastrulation that secretes critical factors involved in embryonic development with inductive effects on embryonic stem cell (ESC) differentiation. We utilized the conditioned media (CM) from the VE-like cell lines, HepG2 and END2, to enhance lineage-specific differentiation of murine ESCs (mESCs) toward the osteogenic lineage. Previously, we have demonstrated that use of the HepG2-CM resulted in the efficient osteogenic/chondrogenic differentiation of mESCs without embryoid body (EB) formation. In this study, we demonstrate, for the first time, the osteogenic-inducing activity of END2-CM, suggesting a potential shared protein profile between HepG2- and END2-CM. To identify the active factors in the CM, proteomic analysis using differential gel electrophoresis coupled with matrix-assisted laser desorption/ionization was performed on the two CM, resulting in six proteins being established to be present in both CM, including ones that may function on the epithelial-mesenchymal transition (EMT). Our results represent the first study on the VE-like cell line secretome and provide information on VE proteins identifying possible candidate proteins to be used for mesoderm-specific osteogenic differentiation.     

In vitro chondrogenic differentiation of human mesenchymal stem cells in collagen microspheres: influence of cell seeding density and collagen concentration

Given the inadequacies of existing repair strategies for cartilage injuries, tissue engineering approach using biomaterials and stem cells offers new hope for better treatments. Recently, we have fabricated injectable collagen-human mesenchymal stem cell (hMSC) microspheres using microencapsulation. Apart from providing a protective matrix for cell delivery, the collagen microspheres may also act as a bio-mimetic matrix facilitating the functional remodeling of hMSCs. In this study, whether the encapsulated hMSCs can be pre-differentiated into chondrogenic phenotype prior to implantation has been investigated. The effects of cell seeding density and collagen concentration on the chondrogenic differentiation potential of hMSCs have been studied. An in vivo implantation study has also been conducted. Fabrication of cartilage-like tissue micro-masses was demonstrated by positive immunohistochemical staining for cartilage-specific extracellular matrix components including type II collagen and aggrecan. The meshwork of collagen fibers was remodeled into a highly ordered microstructure, characterized by thick and parallel bundles, upon differentiation. Higher cell seeding density and higher collagen concentration favored the chondrogenic differentiation of hMSCs, yielding increased matrix production and mechanical strength of the micro-masses. These micro-masses were also demonstrated to integrate well with the host tissue in NOD/SCID mice.