Lin28调节小鼠胚胎干细胞向原始生殖细胞分化

目的 探讨小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向原始生殖细胞(primordial germ cells,PGCs)分化过程中特异基因表达变化及可能机制.方法 mESCs分化形成拟胚体(embryoid bodies,EBs),不同浓度atRA(1μM,2 μ M,5 μ M)持续诱导EBs 16h、2d和5d,RT-PCR、和免疫荧光分别检测Lin28和Blimp1以及相应蛋白的表达变化.结果 atRA诱导16h的EBs其Lin28 mRNA表达量较高,随着诱导时间延长而逐渐降低,Blimp1则无明显变化.WB结果显示EBs的Lin28蛋白表达随诱导时间延长逐渐减弱,而Blimp1蛋白表达逐渐增强.免疫荧光显示Lin28和Blimp1阳性信号均定位于细胞胞浆,其变化特点与WB结果相一致.结论 EBs经atRA诱导后可影响Lin28的表达,Lin28和Blimp1的变化并不协调,其蛋白表达随着诱导会随之发生变化.atRA诱导可能使PGCs分化的Lin28低水平表达时期提前出现.     

不同基质蛋白对小鼠胚胎干细胞生殖细胞分化相关基因表达的影响

目的:探讨不同基质蛋白对小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)分化形成拟胚体(embry-oid bodies,EBs)后生殖细胞分化相关基因表达的影响及机制。方法:mESCs分化形成EBs 3 d后接种于不同基质蛋白包被的培养皿中,包括人工基底膜(matrigel,M组)、纤维连接蛋白(fibronectin,F组)、层粘蛋白(laminin,L组)、胶原(collagen,C组)和非生物活性底物琼脂糖(agarose,A组),同时设不加任何基质蛋白的空白对照组(B组)。RT-PCR检测EBs在不同基质蛋白上d1～d4期间生殖细胞分化相关基因的表达,以及mESCs内源性基质蛋白FN(fibronectin)、LN(laminin)以及整合素受体β1基因的表达。结果:L组和F组EBs易贴壁分化,RT-PCR结果也证实原始生殖细胞(PGCs)分化基因Blimp-1、Stella、Mvh和减数分裂启动基因Stra8在L组和F组表达总体趋势为逐渐增强;M组和C组的表达趋势不明显;A组基因表达水平整体偏低;空白对照B组基因表达水平整体偏高但均呈下降趋势。L组和空白对照组细胞表达内源性基质蛋白FN、LN以及整合素受体β1。结论:层粘蛋白/β1信号途径可能对mESCs向PGCs分化具有指导作用,外源性层粘蛋白可能通过其受体β1亚单位传递诱导信号,调节mESCs来源的EBs向PGCs分化,FN可能通过其他整合素受体亚单位发挥作用。无任何外源性基质蛋白时,自身分泌的内源性基质蛋白也促进细胞分化。     

小鼠iPS细胞具备诱导性原始生殖细胞分化潜能的研究

目的:探讨小鼠诱导性多能干细胞IP14D-1是否具备诱导性原始生殖细胞(induced primordial germcells,iPGCs)分化潜能,以及特异基因表达变化及可能机制。方法:未分化IP14D-1培养扩增,分化形成诱导性拟胚体(induced embryoid bodies,iEBs)。RT-PCR和免疫荧光分别检测4、7、9 d的iEBs中Lin28、Blimp1、Stra8和Mvh的表达变化和蛋白定位情况。结果:未分化IP14D-1同小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)相同,Lin28表达较弱,Blimp1表达相对较强,Mvh和Stra8也在这两种细胞及其相应iEBs和EBs中表达,但均无明显差别。IP14D-1分化形成的iEBs从4 d生长到7 d时,Lin28表达逐渐增强,到9 d时表现为下降,Blimp1表达则随着iEB生长时间延长而逐渐降低。结论:建立了IP14D-1和相应拟胚体(iEBs)的完善、稳定的培养及分化体系;未分化IP14D-1与mESCs在Lin28、Blimp1、Mvh和Stra8表达方面无明显差别;iEB和EBs的Mvh和Stra8表达也无明显差别。IP14D-1及iEBs具有iPGCs分化潜能,且可能是7 d的iEBs内iPGCs分化数量较多,之后进入iPGCs分化的Lin28低表达时期。     

诱导多能干细胞体外向生精细胞自发分化潜能的研究

目的:探讨诱导多能干细胞(iPS)体外培养自发分化过程中生精细胞相关基因的表达,评估iPS体外向生精细胞自发分化的潜能。方法:经类胚体(EB)形成,体外诱导iPS向生精细胞分化,实时定量PCR和PCR检测生精细胞相关基因的表达。结果:实时定量PCR和PCR结果显示iPS经EB形成诱导分化后生精细胞不同时期的相关基因均有不同程度表达。iPS体外培养自发分化后生精细胞相关基因出现4种时间表达特征:Oct4基因表达量呈波浪状上升;Dppa3和Stra8基因表达量随诱导时间延长而下降;Dazl基因表达量呈波浪状下降;减数分裂前期基因Tex14、Msy2,减数分裂期基因Scp1、Scp3以及单倍体基因Akap3随着诱导时间延长先表达增加,而后表达下降。结论:iPS在经EB自发分化过程中表达生精细胞不同时期的相关基因,并且表达雄性配子单倍体基因,具有向雄性配子的分化潜能。     

诱导小鼠骨髓间充质干细胞向雄性生殖细胞的定向分化

目的探讨小鼠骨髓间充质干细胞是否能够在体外被诱导发生向雄性生殖细胞方向的分化。方法从雄性小鼠骨髓中分离能够长期贴壁生长的细胞,并鉴定其是否为间充质干细胞。对分离的细胞进行生殖细胞特异性报告基因标记(stra-8-GFP)。采用视黄酸诱导标记的细胞发生向生殖细胞方向的分化。通过观察报告基因表达和生殖细胞相关基因mRNA表达情况确定是否发生了分化。结果从小鼠骨髓中分离到的贴壁生长的细胞表达间充质干细胞的表面标志CD90、CD44、CD105和Sca-1;细胞在体外可以被诱导分化为成骨、成软骨及成脂肪细胞。报告基因标记的间充质干细胞在被视黄酸诱导2d后开始表达绿色荧光蛋白和生殖细胞相关基因Mvh、Fragilis和Stella的mRNA。未经视黄酸诱导的细胞不表达绿色荧光蛋白和生殖细胞相关基因。结论小鼠骨髓间充质干细胞在体外可以被视黄酸诱导发生向雄性生殖细胞方向的分化。     

Stra8:生殖细胞有丝分裂转变为减数分裂前特异表达的基因

Stra8(stimulated by retinoic acid gene 8)是哺乳动物生殖细胞由有丝分裂转变为减数分裂前特异表达的基因。Stra8在小鼠胚胎卵巢从前向后先后表达,从E12.5到E16.5持续约4d,Stra8表达后1d随之诱发减数分裂使减数分裂前期进行;在出生后睾丸开始Stra8的表达并诱发减数分裂,在胚胎性腺生殖细胞减数分裂的发生时间决定生殖细胞是朝雄性还是雌性的方向发育。视黄酸(retinoic acid,RA)的合成部位及CYP26b1的表达(降解RA的酶)调节Stra8基因表达。胞质Stra8蛋白增加导致生殖细胞进入减数分裂前期,但Stra8蛋白的功能还是未知的。     

多因子联合诱导人胚胎干细胞定向分化为肝细胞样细胞

目的 建立有效的体外诱导人胚胎干细胞(hESCs)分化为肝细胞样细胞的培养体系.方法 将H9 hESC细胞株接种到基底膜提取物包被的培养板上,序贯加入含有下列诱导因子的分化培养基诱导分化:100μg/L细胞因子活化素A( activin A)诱导3d,20 μg/L骨形成蛋白4(BMP-4)和10 μg/L碱性成纤维细胞生长因子(bFGF)诱导4d,10μg/L肝细胞生长因子(HGF)诱导5d,最后以含10 μg/L制瘤素M(OSM)及1×10-7 mol/L地塞米松(Dex)的培养液继续诱导4d.于细胞诱导分化第16天,采用逆转录-聚合酶链反应(RT-PCR)及免疫荧光检测分化细胞肝脏特异性基因和蛋白表达水平;过碘酸-雪夫反应(PAS)试验和吲哚氰绿(ICG)摄取试验检测分化细胞是否具备肝细胞功能.结果 activin A、BMP-4、bFGF、HGF、OSM和Dex联合诱导的分化细胞具有类似肝细胞的形态结构,呈多角形或卵圆形;RT-PCR结果显示分化第16天的细胞表达甲胎蛋白(AFP)、细胞角蛋白7(CK7)、细胞角蛋白19(CK19)、肝细胞核因子-4α(HNF4-α)、1-抗胰蛋白酶(AAT)等肝脏特异性基因;免疫荧光化学检测结果显示分化第16天的细胞表达AFP、ALB、CK19、CYP7A1等肝脏特异性蛋白;PAS试验及ICG试验显示分化细胞具备糖原合成和ICG摄取释放等肝细胞功能.结论 体外联合多种诱导因子可诱导hESCs定向分化为肝细胞样细胞.     

小鼠雌性生殖干细胞体内分化鉴定方法

目的建立一种小鼠卵巢雌性生殖干细胞(FGSCs)体内分化鉴定方法。方法从6周龄C57BL/6小鼠卵巢利用Fragilis抗体基于免疫磁珠分选(MACS)分离FGSCs细胞,并置于STO饲养层细胞上扩增培养,采用RT-PCR法检测生殖干性基因包括Fragilis、MVH、Prdm1和Tert,细胞免疫荧光法检测Fragilis和MVH蛋白表达,利用绿色荧光蛋白(GFP)慢病毒载体荧光标记FGSCs后将其植入受体鼠卵巢,在体内环境下促其分化发育为卵母细胞,至少12d后取卵巢置于载玻片上并压上盖玻片,直接置于荧光显微镜下观察。结果 RT-PCR结果显示FGSCs生殖干性基因包括Fragilis、MVH、Prdm1和Tert为阳性表达,而卵母细胞特异表达基因中Nobox为阴性,GDF9和ZP3为弱阳性;细胞免疫荧光结果显示FGSCs的MVH和Fragilis蛋白表达阳性,而且呈明显的膜表达。GFP-FGSCs植入6周龄雌性C57BL/6的卵巢中,至少12d以后将卵巢取出,压片后置于荧光显微镜下观测,结果显示有荧光阳性的卵母细胞。结论该鉴定方法简单直观,可作为FGSCs体内分化鉴定的可选方法。     

人脐血间充质干细胞在体外向肝样细胞的诱导分化

目的 探讨脐血间充质干细胞(MSCs)在体外能否分化成肝细胞.方法 分离人脐血MSCs,培养传代,流式细胞仪进行细胞表面标志检测.取培养至第三代的细胞,接种于六孔板内,分两个阶段进行细胞分化的诱导,第一阶段采用含地塞米松(终浓度为0.5 μmol/L,下同)、肝细胞生长因子(HGF,10 ng/ml)、表皮生长因子(10 ng/ml)及1×ITS(胰岛素-转铁蛋白-硒)的F12培养基诱导2周,第二阶段用含地塞米松(0.5 μmol/L)、HGF(10 ng/ml)、抑瘤素M(10 ng/ml)及1×ITS的F12培养基继续诱导2周.诱导期间于倒置显微镜下观察细胞的形态变化;采用逆转录聚合酶链反应检测分化细胞的甲胎蛋白(AFP)、白蛋白、人细胞角蛋白18(CK-18)及酪氨酸氨基转移酶(TAT)基因的表达,以免疫荧光染色法检测分化细胞胞浆中AFP、白蛋白、CK-18的表达;用电子显微镜观察分化细胞的超微结构.结果 培养的脐血MSCs不表达CD14、CD34及CD45,也不表达CD49f、CD54及HLA-DR;部分表达CD106;强表达CD13、CD29及CD44.未分化的脐血MSCs不表达AFP、TAT及白蛋白基因,弱表达CK-18基因;诱导分化1周后可检测到AFP基因的表达,诱导分化4周后,不仅表达AFP、CK-18和白蛋白基因,还表达TAT基因.免疫荧光染色显示,未分化的MSCs胞浆中无AFP、白蛋白及CK-18的表达;分化细胞则可以检测到上述物质的表达.诱导至第4周的分化细胞,可见核仁大且明显,细胞核周围有板层状的内质网,胞浆中可见脂滴及簇状糖原,线粒体丰富,细胞表面有微绒毛.结论 脐血MSCs在合适的诱导条件下可以分化为肝样细胞.     

苯乙酸和二甲基甲酰胺对胶质瘤同源盒基因表达影响的比较

目的　观察苯乙酸 (PA)和二甲基甲酰胺 (DMF)诱导分化胶质瘤细胞C6 过程中同源盒(Hox)基因表达的变化。方法　用逆转录 聚合酶链反应 (RT PCR)及图像分析法 ,检测PA、DMF对胶质瘤细胞C6 诱导分化过程中Hox基因组P1、P2、P3及特异Hox基因的表达。Hox基因(组 )表达水平用基因 (组 ) / β 肌动蛋白 (β actin)灰度比值表示。 结果　Hox基因组P2在胶质瘤细胞C6 中表达明显低于P1、P3组 [0 .682 5± 0 .0 987<0 .881 7± 0 .0 73 1 ,0 .860 8± 0 .0 881 ,(P<0 .0 0 1 ) ]。应用PA后 ,P2组HoxB2基因表达明显上调 [0 .776 3± 0 .1 2 4 1 >0 .483 9± 0 .1 34 3 ,(P <0 .0 0 1 ) ] ;应用DMF后 ,HoxB2基因表达无显著变化 ,差异无显著性 (P >0 .0 5)。HoxB4基因在应用PA和DMF前后均未见表达。结论　PA对HoxB2基因mRNA水平的表达有明显的上调作用 ,DMF对Hox基因表达无显著影响     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Germ cell differentiation of bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BM‐MSCs) were first cultured under induction of retinoic acid (RA), Sertoli cells conditioned medium and RA + con (conditioned medium) as treatment groups. The presence of Sertoli cells was confirmed by immunocytochemistry of follicle‐stimulating hormone receptor in Sertoli cells and flow cytometry by anti‐Gata4 antibody. Cell viability and morphology of nucleus and cytoplasm of BM‐MSCs were evaluated by MTT test and DAPI staining respectively. The expression of Oct4, Plzf, Scp3, Caspases 8, 9 and 3 genes was evaluated by RT‐PCR. For increasing the accuracy of experiment, the expression of Vasa and SCP3 genes was investigated quantitatively by real‐time PCR after 0, 5, 10, 15 days of culture. The results showed that the number of apoptotic cells increased in RA group. The expression of apoptosis genes (Caspases 3, 8 and 9) was also observed in this group all days of culture. Measurement of Vasa and Scp3 genes by RT‐PCR confirmed the positive effects of retinoic acid on increasing of genes expression. So, in this study, a group with maximum expression of differentiation genes and minimum expression of apoptotic genes was RA + conditioned medium group. DNA fragmentation was not observed in all groups.     

Differentiation of mesenchymal stem cells to germ‐like cells under induction of Sertoli cell‐conditioned medium and retinoic acid

The aim of this research was to find a way to differentiate germ cells from umbilical cord Wharton's jelly mesenchymal stem cells (MSCs) to support in vitro spermatogenesis. A small piece of Wharton's jelly was cultured in high‐glucose Dulbecco's modified Eagle's medium in present of 10% foetal calf serum. After the fourth passage, the cells were isolated and cultured in Sertoli cell‐conditioned medium under induction of two different doses of retinoic acid (10^?5, 10^?6 m ). The differentiation of MSC to germ‐like cells was evaluated by expression of Oct4, Nanog, Plzf, Stra8 and Prm1 genes during different days of culture through qPCR. The results showed that there were downregulation of Oct4 and Nanog and upregulation of pre‐meiotic germ cell marker (stra8) and haploid cell marker (Prm1) when MSCs are differentiated over time. The expression of Bax gene (an apoptotic marker) was significantly observed in high dosage of retinoic acid (RA). As a result, RA has positive effects on proliferation and differentiation of MSCs, but its effects are related to dosage. The success of this method can introduce umbilical cord MSC as a source of germ cells for treatment of infertility in future.     

Retinoic acid and 17β-estradiol improve male germ cell differentiation from mouse-induced pluripotent stem cells

This research aimed to explore the impacts of retinoic acid (RA)/17β-estradiol (E) induction and embryoid body formation to enhance differentiation of mouse-induced pluripotent stem cells (miPSCs) into male germ cells in vitro. Flow cytometry and qPCR were conducted to describe miPSCs differentiation process. Various temporal expression profiles of germ cell-related genes were traced. Stra8 gene expression increased in the RA group on the 4th day compared to other groups. The RA group experienced a more significant increase than E group. The expression of Sycp3 increased in RA + E group on 4th day compared with other groups. Expression of AKAP3 enhanced in the RA + E group than other groups on day 4. Moreover, miPSCs showed that this gene expression in the RA + E group was increased in comparison to RA and E groups on day 7. AKAP3 gene expression on day 7 of miPSCs decreased in RA and E groups. Flow cytometry data indicated that 3%–8% of the cells in sub-G1 stage were haploid after RA and E induction compared to other groups on day 4. This study showed that miPSCs possess the power for differentiating into male germ cells in vitro via formation of embryoid body by RA with/or E induction.     

Differentiation potential of adipose tissue-derived mesenchymal stem cells into germ cells with and without growth factors

This study aimed to culture the adipose tissue-derived mesenchymal stem cells (AT-MSCs) with and without leukaemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF) and retinoic acid (RA), and investigate their impact on the differentiation of these cells into germ cells. MSCs were separated from adipose tissue of mice, and the nature of these cells is confirmed by flow cytometry. The cells were cultured in different conditions, including MSCs grown in the presence of the growth factors, MSCs without the growth factors, MSCs cultured with combined growth factors and RA, and MSCs cultured with RA. After 2 weeks, the gene expression of c-Kit, Gcnf, Mvh and Scp3 and the protein expression of c-Kit and Gcnf were assessed by real-time PCR and Western blot, respectively. Scp3 was overexpressed in the groups supplemented with RA (p < .01). The expression of c-Kit and Mvh in the growth factor-supplemented groups was increased (p < .01). Western blot analysis confirmed the real-time PCR results. The use of the growth factors for the long-term culture of stem cells can be beneficial. However, to promote germ cell differentiation, the growth factors might be used by other meiosis inducer factors, such as RA.     

Retinoic acid inhibits HIV-1-induced podocyte proliferation through the cAMP pathway

HIV-associated nephropathy is characterized by renal podocyte proliferation and dedifferentiation. This study found that all-trans retinoic acid (atRA) reverses the effects of HIV-1 infection in podocytes. Treatment with atRA reduced cell proliferation rate by causing G1 arrest and restored the expression of the differentiation markers (synaptopodin, nephrin, podocin, and WT-1) in HIV-1-infected podocytes. It is interesting that both atRA and 9-cis RA increased intracellular cAMP levels in podocytes. Podocytes expressed most isoforms of retinoic acid receptors (RAR) and retinoid X receptors (RXR) with the exception of RXRgamma. RARalpha antagonists blocked atRA-induced cAMP production and its antiproliferative and prodifferentiation effects on podocytes, suggesting that RARalpha is required. For determination of the effect of increased intracellular cAMP on HIV-infected podocytes, cells were stimulated with either forskolin or 8-bromo-cAMP. Both compounds inhibited cell proliferation significantly and restored synaptopodin expression in HIV-infected podocytes. The effects of atRA were abolished by Rp-cAMP, an inhibitor of the cAMP/protein kinase A pathway and were enhanced by rolipram, an inhibitor of phosphodiesterase 4, suggesting that the antiproliferative and prodifferentiation effects of atRA on HIV-infected podocytes are cAMP dependent. Furthermore, both atRA and forskolin suppressed HIV-induced mitogen-activated protein kinase 1 and 2 and Stat3 phosphorylation. In vivo, atRA reduced proteinuria, cell proliferation, and glomerulosclerosis in HIV-1-transgenic mice. These findings suggest that atRA reverses the abnormal phenotype in HIV-1-infected podocytes by stimulating RARalpha-mediated intracellular cAMP production. These results demonstrate the mechanism by which atRA reverses the proliferation of podocytes that is induced by HIV-1.     

Evidence for hepatocyte differentiation from embryonic stem cells in vitro

We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.