猪骨髓间充质干细胞分离培养和诱导分化脂肪细胞

采用全骨髓培养法分离猪骨髓间充质干细胞(Mesenchymal stem cells,MSCs)并传代培养;取第4代纯化的MSCs在成脂诱导培养基中诱导分化;分化的成脂细胞用形态学和油红O染色法进行鉴定;用实时荧光定量PCR(Real-time PCR)检测成脂分化标志基因PPARγ2和LPL mRNA的表达情况。结果显示,分离培养的猪MSCs细胞经连续传代形态上无明显改变;MSCs在成脂分化培养液中诱导分化2d开始有少量脂滴出现,油红O染色成阳性,诱导18d成脂转化率可达59.8%;在诱导分化第5、10、15天时,PPARγ2mRNA相对表达量分别是(5.065±0.159)、(6.268±0.340)、(9.277±0.261),LPL mRNA的相对表达量分别是(10.995±1.473)、(13.130±0.712)、(15.762±0.934)。结果表明,用本诱导条件诱导猪MSCs向脂肪细胞分化,经形态学和油红O染色鉴定,成脂细胞分化率可达60%,且随分化时间的延长,脂肪细胞标志基因表达增加。     

绵羊骨髓间充质干细胞分离培养与鉴定

旨在分离绵羊骨髓间充质干细胞(sheep bone marrow mesenchymal stem cells, SBMSC),建立绵羊骨髓间充质干细胞系,为相关研究提供种子细胞。取新生健康绵羊肋骨为试验材料,利用贴壁法分离获得SBMSC;取生长良好的P3代细胞通过免疫荧光,进行CD73、CD90、CD105、CD106表型鉴定;取生长良好的P3代细胞分别进行成软骨、成脂、成骨方向的诱导,对诱导结果分别进行阿利新蓝、油红O和茜素红染色,鉴定分化结果。SBMSC呈成纤维样贴壁生长;体外培养P3代细胞表面标记物CD73、CD90、CD105和CD106完全符合标准;P3代细胞经软骨、脂肪、骨方向诱导3周后,经阿利新蓝染色,细胞呈亮蓝色;经油红O染色,细胞质内出现橙红色脂滴;茜素红染色表明聚集的细胞团中央能形成钙化结节。说明SBMSC经体外诱导培养后可向软骨细胞、脂肪细胞和成骨细胞分化,并具有明显的成软骨、成脂和成骨表型,表明SBMSC具有多向分化潜能。通过贴壁培养法建立了SBMSC完整的体外培养体系,为绵羊育种等方面提供种子细胞。     

野猪骨髓间充质干细胞的分离培养与生物学特性

采用全骨髓培养法对野猪股骨中骨髓间充质干细胞（Bone mesenchymal stem cells,BMSC）进行分离、培养并传代,建立野猪骨髓间充质干细胞体外培养方法及对其生物学特性进行观察研究。结果显示,细胞形态呈梭形,漩涡状生长,生长曲线为典型S型,在诱导液作用下,可分化为成脂肪样细胞。结果表明,通过本方法能够分离到较纯的BMSC,为野猪资源保存和体细胞核移植提供技术支持。     

南阳牛骨髓间充质干细胞的分离培养及多向分化

本研究旨在建立南阳牛骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)体外分离培养方法,在此基础上研究其生物学特性和多向分化的能力。采用骨髓穿刺法取3月龄小牛的肋骨骨髓,分离培养BMSCs,传代培养并测定其生长曲线,RT-PCR检测Oct4、Nanog、Sox2基因的表达,然后取P3 BMSCs分别向神经和脂肪细胞进行诱导分化,并利用组织学染色技术和RT-PCR技术进行鉴定。结果表明,分离得到的BMSCs大小均匀,多呈梭形的成纤维细胞样生长;RT-PCR可检测到干细胞因子Oct4、Nanog、Sox2的表达;不同代次细胞生长曲线呈S型,一般在第3天时进入指数生长期,第7天后进入平台期;成神经诱导后,甲苯胺蓝染色可见明显的尼氏体结构,RT-PCR检测ENO2和GFAP基因表达呈阳性;成脂肪诱导后,油红O染色后可见大量的脂滴存在,RT-PCR检测Leptin和PPAR基因表达呈阳性。试验证明,成功分离得到了南阳牛BMSCs,且其具有多向诱导分化潜能。     

蒙古羊骨髓间充质干细胞的分离培养及多向诱导分化

本研究旨在建立蒙古羊骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)体外分离、纯化、增殖方法,诱导其向脂肪细胞、成骨细胞及软骨细胞分化。穿刺法抽取蒙古羊骨髓组织,采用密度梯度离心法结合贴壁培养法分离纯化BMSC,增殖,并测定其生长曲线及细胞倍增时间。对第3代蒙古羊BMSC进行成脂、成骨及成软骨诱导,观察诱导后的细胞形态。采用油红O、茜素红、HE等染色法在组织学水平对BMSC进行成脂、成骨和成软骨分化鉴定。结果显示,蒙古羊BMSC呈现均一梭形成纤维细胞样生长,生长曲线呈S型,生长增殖能力良好。在不同分化诱导之后,细胞呈现脂肪细胞、成骨细胞及软骨细胞的表型特征。表明获得的蒙古羊BMSC具有多向分化潜能,所分离细胞确为骨髓间充质干细胞。     

山羊骨髓间充质干细胞的分离培养

旨在对山羊的骨髓间充质干细胞(BMSCs)进行分离培养。将分离得到的BMSCs进行传代培养,采用RT-PCR法检测其干细胞转录因子oct4和sox2基因,以验证其干细胞特性;在此基础上绘制BMSCs的生长曲线,对其生物学特性进行直观了解。结果表明,分离到的山羊BMSCs原代细胞呈现出成纤维样,并能够表达oct4和sox2基因,证明了其干细胞特性;其生长曲线呈"S"型,在1~2 d时为潜伏期,第3天时进入指数生长期,第7天时进入平台期。结果提示,分离得到的细胞具有BMSCs的特性,能够用于后续的相关试验研究。     

马骨髓间充质干细胞向软骨细胞的诱导分化

试验旨在建立马骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）细胞系,并诱导其向软骨细胞分化。通过获取马BMSCs,进行细胞培养和纯化,对第3代（P3）细胞进行干细胞特性鉴定,并诱导其向软骨细胞分化,对分化后的细胞染色,并检测其软骨细胞特异性基因的表达。结果显示,获得的马BMSCs表达标记基因Sox2和Nanog,并表达间充质干细胞表面标记因子CD44、CD90和CD105,不表达造血细胞表面标志物CD34和CD45。P3代细胞经诱导培养后形态发生改变,阿尔新蓝染色为阳性,并表达软骨细胞特异基因Col,且其表达量随着诱导分化时间的增加而增高。综上表明,本试验建立了马BMSC细胞系,并成功诱导其分化为软骨细胞,为软骨损伤的干细胞治疗提供了试验依据。     

滩羊骨髓间充质干细胞分离与培养

利用全骨髓法与差速贴壁法分离和培养滩羊骨髓间充质干细胞（BMSCs）,观察细胞形态及成脂诱导分化实验进行鉴定。结果显示原代培养的细胞呈圆形或三角形,24h后大部分细胞均贴壁,7d左右可达80％-90％融合,体外成脂诱导分化获得脂肪样细胞,油红染色为阳性。因此,全骨髓法与差速贴壁法可有效分离培养滩羊的骨髓间充质干细胞,是一种比较理想的分离培养方法。     

崂山奶山羊永生化骨髓间充质干细胞的诱导分化研究

为研究崂山奶山羊永生化骨髓间充质干细胞(BMSCs)的诱导分化潜能,采用P3代BMSCs和P30代TERT-BMSCs进行体外成脂和成神经诱导分化。结果显示,当细胞传至P30代时,TERT-BMSCs生长旺盛;在成脂诱导后,P3代BMSCs和P30代TERT-BMSCs均能观察到细胞中透明脂滴的形成,油红O染料染色后可见脂滴被染成红色;在成神经诱导后,P3代BMSCs和P30代TERT-BMSCs均能形成树突样或三角形的形态,甲苯胺蓝染色后可观察到细胞中尼氏体的存在。综上提示,永生化的崂山奶山羊BMSCs在多次传代后仍具有较强的多向诱导分化能力,这为永生化MSCs的广泛应用提供了理论依据。     

不同代次大鼠骨髓间充质干细胞和脂肪间充质干细胞成骨分化比较

本研究旨在观察不同代次骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ADSCs)体外培养的生长特点和体外诱导成骨能力。通过密度梯度离心和贴壁培养法分离培养大鼠骨髓间充质干细胞和脂肪间充质干细胞,用含地塞米松、抗坏血酸、β-甘油磷酸钠的培养液定向诱导传代细胞向成骨细胞分化,并利用茜素红染色、碱性磷酸酶染色及PCR方法检测成骨细胞。结果表明骨髓及脂肪间充质干细胞呈成纤维细胞样生长,增殖能力强,生长迅速。第5、10、15、20代BMSCs及ADSCs经诱导培养后茜素红染色呈阳性并且出现"矿化"、碱性磷酸酶活性强,随着细胞代次的递增,诱导后细胞碱性磷酸酶活性呈递减趋势;诱导后的两类细胞传代后细胞仍能继续分化,并形成正常的"矿化"结节,且碱性磷酸酶染色均弱于初次诱导。结果提示,BMSCs及ADSCs易于分离培养及体外扩增,诱导条件下成骨能力强且成骨细胞传代培养仍具有成骨能力,适合作为再生医学骨组织工程的种子细胞。     

猪肌肉卫星细胞的分离培养及生物学特性鉴定

以猪胎儿为材料,采用胶原酶消化法或组织块法培养胎儿背部最长肌获得了肌肉卫星细胞,该细胞体外可以传到9代以上。培养的细胞多呈纺锤体型和梭型,具有明显的方向性,呈典型的长轴平行排列。流式细胞仪分析结果显示,该细胞呈CD29、CD166、CD45、CD44阳性,CD71、CD34阴性。RT-PCR检测发现其表达Desmin、C-Myc、Nanog、Pcna、Oct4、Klf4,弱表达Myog,不表达Sox2、MyoD。免疫组化染色发现其表达Desmin等肌肉细胞的特异性标记,同时表达Nanog、Pcna等多能性细胞标记。本试验建立了一种简便高效的猪肌肉卫星细胞体外分离和培养方法,得到的细胞具有肌肉卫星细胞的典型生物学特性,同时表达间质干细胞和多能性干细胞的部分标记。     

Comparative characteristic study from bone marrow-derived mesenchymal stem cells

Tissue engineering has been extensively investigated and proffered to be a potential platform for novel tissue regeneration. The utilization of mesenchymal stem cells (MSCs) from various sources has been widely explored and compared. In this regard, MSCs derived from bone marrow have been proposed and described as a promising cell resource due to their high yield of isolated cells with colony-forming potential, self-renewal capacity, MSC surface marker expression, and multi-lineage differentiation capacities in vitro. However, there is evidence for bone marrow MSCs (BM-MSCs) both in vitro and in vivo from different species presenting identical and distinct potential stemness characteristics. In this review, the fundamental knowledge of the growth kinetics and stemness properties of BM-MSCs in different animal species and humans are compared and summarized. Finally, to provide a full perspective, this review will procure results of current information studies focusing on the use of BM-MSCs in clinical practice.     

Isolation and characterization of equine amniotic membrane-derived mesenchymal stem cells

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco''s modified Eagle''s medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.     

小鼠脂肪间充质干细胞向胰岛素分泌细胞的诱导分化研究

为了通过特定转录因子将小鼠脂肪间充质干细胞(mADSCs)定向诱导分化为胰岛素分泌细胞(IPCs)。本研究分别构建Pdx1(胰十二指肠同源盒基因1)、MafA(V-maf肌肉腱膜纤维肉瘤癌基因同源物A)、NeuroD1(神经分化因子1)3种基因的慢病毒过表达载体,使用293T细胞对3种因子进行慢病毒包装,将3种慢病毒过表达载体以单因子侵染、双因子侵染、三因子联合侵染的方式对mADSCs进行定向分化诱导,于诱导分化第15天对不同方式诱导的IPCs进行检测鉴定,并对不同方式诱导组的IPCs进行高糖刺激,刺激后30~120min检测培养基中含糖量的变化。结果显示,构建的慢病毒过表达载体pHBLV-CMV-IRES-ZsGreen-Pdx1、pHBLV-CMV-PGK-RFPMafA、pHBLV-CMV-PGK-RFP-NeuroD1所含目的片段基因序列与小鼠全基因编码序列完全一致,三种基因慢病毒过表达载体构建成功;诱导分化第15天,三因子联合诱导组所形成的IPCs克隆双硫腙(DTZ)染色呈阳性,并可表达胰岛素生物合成及分泌相关基因;在高糖刺激条件下,三因子联合诱导组糖分解速度、分解量远优于单因子或双因子诱导组。结果表明,Pdx1、MafA、NeuroD1 3种因子联合作用,可以将小鼠脂肪间充质干细胞定向诱导分化为胰岛素分泌细胞,并可在高糖刺激下,有效发挥降糖作用。     

Comparing the osteogenic potential of canine mesenchymal stem cells derived from adipose tissues, bone marrow, umbilical cord blood, and Wharton's jelly for treating bone defects

Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton''s jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with β-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.     

Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III β tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.     

Isolation and biological characteristics of multipotent mesenchymal stromal cells derived from chick embryo intestine

ABSTRACT

1. Over the past decade, rapid advancement in isolation methods for identifying markers of the once elusive intestinal stem cell (ISC) populations has laid the foundation for unravelling their complex interrelationships during homeostasis. Study on ISC in avian intestinal tissue might play a pivotal foundation for further studies on the epithelial-to-mesenchymal transition (EMT) in gastrointestinal disease and cell-based therapy as well as intestinal tissue engineering.

2. The following experiment isolated a population of fibroblast-like, plastic adhering cells derived from chick embryo intestine, showing a strong self-renewing and proliferative ability, which was maintained in vitro up to passage 25. The findings included growth characteristics, detected expression of cell surface markers and characterised the capability of these cells to differentiate towards the osteogenic, adipogenic, and chondrogenic cell lineages.

3. RT-PCR analysis showed that these cells from chick embryos expressed mesenchymal stromal cell markers CD44, CD90 and VIMENTIN as well as ISC-specific genes LGR5, MI1, SMOC2, BMI1, and HOPX. Immunofluorescence and flow cytometry confirmed this biology characterisation further.

4. In conclusion, cells were isolated from the intestine of 18-day-old chicken embryos that exhibited the biological characteristics of mesenchymal stromal cells as well as markers of intestinal stem cells. Our findings may provide a novel insight for in vitro cell culture and characteristics of ISCs in avian species, which may also indicate a benefit for obtaining cell source for intestinal tissue engineering as well as cell-based investigation for gastrointestinal disease and treatment.     

Isolation,proliferation and characterization of endometrial canine stem cells

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.