Optimization of adipogenic differentiation conditions for canine adipose-derived stem cells

BackgroundCanine adipose-derived stem cells (cADSCs) exhibit various differentiation properties and are isolated from the canine subcutaneous fat. Although cADSCs are valuable as tools for research on adipogenic differentiation, studies focusing on adipogenic differentiation methods and the underlying mechanisms are still lacking.ObjectivesIn this study, we aimed to establish an optimal method for adipogenic differentiation conditions of cADSCs and evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) and estrogen receptor (ER) signaling in the adipogenic differentiation.MethodsTo induce adipogenic differentiation of cADSCs, 3 different adipogenic medium conditions, MDI, DRI, and MDRI, using 3-isobutyl-1-methylxanthine (M), dexamethasone (D), insulin (I), and rosiglitazone (R) were tested.ResultsMDRI, addition of PPARγ agonist rosiglitazone to MDI, was the most significantly facilitated cADSC into adipocyte. GW9662, an antagonist of PPARγ, significantly reduced adipogenic differentiation induced by rosiglitazone. Adipogenic differentiation was also stimulated when 17β-estradiol was added to MDI and DRI, and this stimulation was inhibited by the ER antagonist ICI182,780.ConclusionsTaken together, our results suggest that PPARγ and ER signaling are related to the adipogenic differentiation of cADSCs. This study could provide basic information for future research on obesity or anti-obesity mechanisms in dogs.     

神经浸出液对兔脂肪干细胞向雪旺细胞分化的影响

胶原酶消化法分离获得家兔脂肪源性干细胞(adipose-derived stem cells,ADSCs),传代培养并进行生物学鉴定。取家兔坐骨神经,并经过无菌剪碎、浸出和过滤制备家兔周围神经浸出液。将家兔神经浸出液作为诱导剂,作用于第3代ADSCs,7d后通过倒置显微镜观察形态变化、RT-PCR和免疫荧光检测雪旺细胞(Schwann-like cells,SCs)特异性标记物S-100和GFAP mRNA和蛋白的表达情况。通过神经浸出液诱导前后的ADSCs与神经元前体细胞PC12共孵育12d后,利用形态观察和Western blot检测PC12细胞的轴突生长情况。形态观察显示,经坐骨神经浸出液处理过的家兔ADSCs形态由长梭形逐渐变为双极形,类似SCs的形态;RT-PCR和免疫荧光结果均显示诱导后ADSCs表达SCs标志物S-100和GFAP蛋白。神经浸出液诱导ADSCs后能够促进PC12细胞长出较长轴突,且阳性表达神经元标志物β3-tubulin和MAP-2蛋白。结果表明,神经浸出液能有效诱导家兔ADSCs向SCs分化,且分化后的ADSCs具有真正SCs的功能。     

山羊毛囊干细胞的分离及体外培养

试验采用2．4U／mL Dispase酶消化和机械切割法分离毛囊隆突部,经胰酶（0．5mg／mL胰酶＋0．2mg／mL EDTA）消化从山羊耳部皮肤分离得到的毛囊干细胞,然后进行形态学观察、细胞生长曲线测定、克隆形成率及免疫组化染色检测,并对毛囊干细胞的基础培养基进行了筛选。结果表明：DMEM／F12是一种适合毛囊干细胞体外增殖培养的培养基;在以DMEM／F12为基础培养液的培养体系中细胞可传代至19代。     

Sequential sub-passage decreases the differentiation potential of canine adipose-derived mesenchymal stem cells

Adipose-derived mesenchymal stem cells (AD-MSCs) are abundant in adipose tissue from animals of all ages, are easily isolated, can differentiate into multi-lineage cells, and have a clinical application. This promising potential may only be achieved if the cells are expanding in a large number while maintaining their stemness in sequential passages. In this study, canine AD-MSCs (cAD-MSCs) were individually isolated from five dogs and subjected to proliferative culture with seven sub-passages. The cells at each sub-passage were characterized for properties associated with multipotent MSCs such as proliferation kinetics, expression of MSCs-specific surface markers, expression of molecules associated with self-renewal and differentiation capabilities into mesodermal lineage cells. Proliferation of the cells plateaued at passage 5 by cumulative population doubling level, while cell doubling time gradually increased with passage. MSCs surface markers (CD44, CD90, and CD105) and molecules (Oct 3/4, Sox-2, Nanog and HMGA2) associated with self-renewal were all expressed in the cells between passages 1 to 6 by RT-PCR. In addition, the cells at passage 1, 3 or 6 underwent adipogenic and chondrogenic differentiation under specific induction conditions. However, the level of adipogenic and chondrogenic differentiation was negatively correlated with the number of sub-passage. The present study suggests that sequential sub-passages affect multipotent properties of cAD-MSCs, which should be considered in their therapeutic application in regenerative medicine.     

Isolation,characterization, and in vitro differentiation of ovine amniotic stem cells

Challenge tests with Artemia four different development stages (nauplii, metanauplii, pseudoadults and adults) to white spot syndrome virus was carried out by immersion challenge and virus-phytoplankton adhesion route in order to asses the possibility of Artemia acting as a vector of WSSV to penaeid shrimp Litopenaeus vannamei postlarvae. The WSSV succeeded in infecting four stages Artemia, and nested-PCR detection for WSSV revealed positive results to virus-phytoplankton adhesion route. No mass mortalities were observed in penaeid shrimp postlarvae fed with WSSV-positive Artemia which exposed to WSSV by virus-phytoplankton adhesion route, whereas WSSV DNA detected in penaeid shrimp postlarvae by nested-PCR. By contrary, no WSSV-positive was detected in any animal fed with WSSV-negative Artemia. These results indicated that Artemia could serve as a vector in WSSV transmission.     

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。     

Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Background

There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.

Results

Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.

Conclusions

Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.

Electronic supplementary material

The online version of this article (doi:10.1186/2049-1891-6-1) contains supplementary material, which is available to authorized users.     

Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Background: There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells(MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.Results: Goat MSCs isolated from bone marrow(BM-MSCs) and adipose tissue(ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency(CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection.BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture,exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.Conclusions: Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.     

Ghrelin对鸡脂肪间充质干细胞增殖及分化为脂肪细胞的影响

为探讨Ghrelin对鸡脂肪间充质干细胞(AMSCs)增殖及分化为脂肪细胞的影响。本研究采用CCK-8法检测不同浓度的Ghrelin对AMSCs增殖的影响,再通过实时荧光定量PCR检测Ghrelin对AMSCs中c-myc和胸苷激酶1(TK1)基因mRNA表达水平的影响。然后,采用化学法对AMSCs进行成脂分化诱导,在此过程中添加不同浓度的Ghrelin,观察AMSCs的形态学变化,油红染色测定甘油三酯的累积情况,并通过实时荧光定量PCR检测脂肪细胞分化转录因子过氧化物酶体增殖剂活化受体γ(PPARγ)和CAAT/增强子结合蛋白α(C/EBPα)基因mRNA表达水平的变化。结果显示,10-7~10-11 mol/L浓度的Ghrelin均能显著或极显著促进AMSCs的增殖,其中10-9 mol/L浓度的Ghrelin的促增殖作用最强;Ghrelin也能显著或极显著升高c-myc和TK1基因mRNA的表达量。同时,Ghrelin促进AMSCs分化为脂肪细胞过程中甘油三酯的累积和脂滴的形成,显著或极显著升高PPARγ和C/EBPα基因mRNA的表达水平。结果说明,Ghrelin能够促进AMSCs增殖及分化为脂肪细胞。其分子调节机制可能是,Ghrelin通过增加c-myc的含量,进而引起TK1的活化,从而导致细胞周期的激活,促进AMSCs增殖;Ghrelin可能通过促进PPARγ和C/EBPα的表达,从而促进AMSCs分化为脂肪细胞。     

奶山羊睾丸组织冷冻保存及复苏后精原干细胞的分离培养

以关中奶山羊为材料,比较了2种玻璃化冷冻法和1种慢速冷冻法对睾丸组织保存的效率,复苏后发现3种冻存方法均有较高的成活率(超过50%),其中玻璃化Ⅱ组达65%以上,培养及检测后均有精原干细胞存活,在体外培养可以形成胚胎干细胞样克隆,其表达精原干细胞和多能性干细胞的相关特异性标记:碱性磷酸酶(AP)、CD49f、CD133、C-Myc、Oct4和Klf4阳性,证明睾丸组织冷冻可以提供一种有效的奶山羊精原干细胞的保存方法。试验中所使用的低温保存方法简便易行,效果良好,表明这些方法可为癌症治疗等造成的无精症及少精症等不育症患者的医治以及一些珍稀濒危物种和优良畜禽的生殖细胞保存提供一种简捷有效的途径。     

Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells

It has been reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. Since DFAT cells can be prepared from a small quantity of adipose tissue, they could facilitate cell-based therapies in small companion animals such as cats. The present study examined whether multipotent DFAT cells can be generated from feline adipose tissue, and the properties of DFAT cells were compared with those of adipose-derived stem cells (ASCs). DFAT cells and ASCs were prepared from the floating mature adipocyte fraction and the stromal vascular fraction, respectively, of collagenase-digested feline omental adipose tissue. Both cell types were evaluated for growth kinetics, colony-forming unit fibroblast (CFU-F) frequency, immunophenotypic properties, and multilineage differentiation potential.DFAT cells and ASCs could be generated from approximately 1 g of adipose tissue and were grown and subcultured on laminin-coated dishes. The frequency of CFU-Fs in DFAT cells (35.8%) was significantly higher than that in ASCs (20.8%) at passage 1 (P1). DFAT cells and ASCs displayed similar immunophenotypes (CD44⁺, CD90⁺, CD105⁺, CD14^?, CD34^? and CD45^?). Alpha-smooth muscle actin-positive cells were readily detected in ASCs (15.2 ± 7.2%) but were rare in DFAT cells (2.2 ± 3.2%) at P1. Both cell types exhibited adipogenic, osteogenic, chondrogenic, and smooth muscle cell differentiation potential in vitro. In conclusion, feline DFAT cells exhibited similar properties to ASCs but displayed higher CFU-F frequency and greater homogeneity. DFAT cells, like ASCs, may be an attractive source for cell-based therapies in cats.     

妊娠中期猪胎儿神经干细胞分离培养及分化

从妊娠中期猪胎儿(胎龄60 d)脑组织分离培养神经干细胞并诱导其贴壁分化,采用RT-PCR技术检测干细胞及其分化细胞的表面标志.结果显示,神经干细胞中DCX、Hes1、Oct4、CD-90、Nanog、Sox2和Nestin表达阳性;体外诱导的神经干细胞可以分化为星形胶质细胞(表达GFAP)、少突胶质细胞(表达GalC)和神经元细胞(表达NF、NSE和MAP2).结果表明,从妊娠中期猪胎儿脑组织可以分离神经干细胞,神经干细胞具有自我更新和分化潜能.     

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.     

Myogenic and adipogenic properties of goat skeletal muscle stem cells

The aims of the present study were to establish a culture system for goat skeletal muscle stem cells and to examine their myogenic and adipogenic properties in vitro. Cells were isolated from the skeletal muscle of the Shiba goat and cultured in vitro. Most of the cells were positive for myogenic markers, such as Pax7, MyoD, and desmin, and immunocytochemistry revealed they differentiated to form myotubes expressing myosin heavy chain, indicating they were highly myogenic. Myogenic differentiation was strongly suppressed by the addition of basic fibroblast growth factor, while proliferation was unaffected. When the cells were cultured in adipogenic differentiation medium, some of the cells differentiated into mature adipocytes that stained with Oil Red-O. These cells were immunocytochemically positive for adipogenic markers, including peroxisome proliferator-activated receptor-gamma (PPAR gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP alpha). These results clearly demonstrate the presence of both myogenic and adipogenic stem cells in goat skeletal muscle.     

Mammary stem cells: expansion and animal productivity

Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally.     

羊驼毛囊干细胞分离、培养与鉴定

采用0.2%中性蛋白酶Ⅱ和0.25%胰酶∶0.02%EDTA(1∶1)两步酶消化法从羊驼背部皮肤分离得到羊驼毛囊干细胞。用无血清角质细胞培养基(K-SFM)培养进行形态学观察、细胞生长曲线克隆形成率及免疫组化染色检测。结果显示,细胞生长曲线表明,不同代次毛囊干细胞接种前3d生长缓慢,4⁶d进入倍增期,有无限增殖的趋势,所分离的羊驼毛囊干细胞具备毛囊干细胞特征(体积小、立体感强),呈现未分化特征;CK19、CK15、β1-integrin和CD34免疫组化染色阳性;第3、5、7、9代克隆形成率分别为(32.7±2.27)%、(47.0±3.46)%、(46.3±3.18)%和(43.3±3.76)%。结果表明,用两步酶消化法成功分离获得羊驼毛囊干细胞,无血清角质细胞培养基(K-SFM)可使羊驼毛囊干细胞体外维持未分化状态并传代至12代。     

山羊皱胃平滑肌细胞的体外分离与培养

近年来,随着奶产业的迅速发展,奶牛疾病的发病率逐年升高.皱胃变位是一种多发于围产期的代谢性疾病,严重影响奶牛的生产性能,给奶产业造成了严重的经济损失.皱胃动力不足和气体积聚是引起皱胃变位的两个先决条件[1].平滑肌是皱胃动力的基本单位.因此,分离培养皱胃平滑肌细胞,可为研究反刍动物皱胃疾病的发病机理提供试验材料.本试验在参照血管平滑肌细胞培养方法的基础上,成功培养出皱胃平滑肌细胞.报告如下.     

羊驼毛囊干细胞分离、培养与鉴定

采用0．2％中性蛋白酶Ⅱ和0．25％胰酶：0．02％EDTA（1：1）“两步酶”消化法从羊驼背部皮肤分离得到羊驼毛囊干细胞。用无血清角质细胞培养基（K-SFM）培养进行形态学观察、细胞生长曲线克隆形成率及免疫组化染色检测。结果显示,细胞生长曲线表明,不同代次毛囊干细胞接种前3d生长缓慢,4～6d进入倍增期,有无限增殖的趋势,所分离的羊驼毛囊干细胞具备毛囊干细胞特征（体积小、立体感强）,呈现未分化特征;CK19、CK15、81-integrin和CD34免疫组化染色阳性;第3、5、7、9代克隆形成率分别为（32．7±2．27）％、（47．0±3．46）％、（46．3±3．18）％和（43．3±3．76）％。结果表明,用“两步酶”消化法成功分离获得羊驼毛囊干细胞,无血清角质细胞培养基（K—SFM）可使羊驼毛囊干细胞体外维持未分化状态并传代至12代。