Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells

The difference between Noggin and basic fibroblast growth factor for the neural precursor differen- tiation from human embryonic stem cells has not been studied. In this study, 100 tJg/L Noggin or 20 IJg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen- tiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro- scope. Immunofluorescence staining revealed expression levels of Nestin, [3-111 Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in- creases the differentiation of neural precursors.

Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells

Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.

Development of neural precursor cells from mouse embryonic stem cells

Objective:To exploretheserum-freecultureconditionsfordifferentiatingmouseembryonicstemcells(ES cells)intoneuralprecursorcells(NPC)andcomparetheeffectsof humanembryonicfibroblasts(HEF)as thefeederlayer of ES withthatof mouseembryonicfibroblasts(MEF)in vitro.Methods:MouseES cellswereculturedin or notin feederlayer cellsmediumcontainingor notleukemiainhibitoryfactorto suppresstheirdifferentiation.Immunocytochemicalmethod was usedto identifyNPCby detectingnestinantigenandalkalinephosphatase.Results: TheES cellsculturedin HEF werepositiveto alkalinephosphatase.Serum-freemediumallowedthedifferentiationof ES cellsintoNPC.Conclusion:HEFcouldreplaceMEFandkeeptheundifferentiatedconditionof ES cellswithmorebenefits.NPCof highpuritycould be culturedfromEScellsby serum-freeculturemethod.

Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells

Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.

Cell therapy from bench to bedside:Hepatocytes from fibroblasts-the truth and myth of transdifferentiation

Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inheritedliver diseases and liver failure.It is a relatively less complicated surgical procedure,and has the advantage that it can be repeated several times if unsuccessful.Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation.Despite these advantages hepatocyte transplantation is less popular.Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes.Generation of "hepatocyte like cells"/i Heps from embryonic stem cells(ES) and induced pluripotent stem cells(iP SCs) by directed differentiation is an emerging solution to the latter issue.Direct conversation or trans-differentiation of fibroblasts to "hepatocyte like cells" is another way which is,being explored.However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or i PSC.There are several methods claiming to be "highly efficient" for generating "highly functional" "hepatocyte like cells".Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol.Directed differentiation protocols need to be designed,compared,analyzed and tweaked systematically and logically than empirically.There is a need for a wellcoordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.

Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application, we need to replace mouse embryonic fibroblasts with human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state. We success-fully use human fibroblasts derived from aborted fetus and adult prepuce as feeder layer to maintain human embryonic stem cells growth. During the passage and growth on this feeder layer, the human embryonic stem cells can keep their undifferentiated state.

Upregulation of FIk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.

Pcgf2促进鸡胚成纤维细胞重编程形成诱导多能干细胞

【目的】以提高诱导鸡多能干细胞的干性为目的,利用Pcgf2基因与传统诱导多能干细胞的重编程因子共同诱导鸡多能干细胞。【方法】以鸡早期胚胎为模板,对Oct4、Sox2、Nanog、Lin28、C-myc、Klf4这6种传统的重编程因子及Pcgf2进行基因扩增,并与慢病毒载体重组为慢病毒质粒,在包装慢病毒并测定相对应的病毒滴度后,分组感染鸡胚成纤维细胞,在诱导的过程中对干细胞标记物进行检测。【结果】利用传统的重编程因子组合与加入Pcgf2共同诱导后的细胞都较早地表达出磷酸酶活性。加入Pcgf2诱导多能干细胞可以使细胞更早地出现类干细胞形态,且内源性基因Oct4、Sox2、Nanog和Lin28的表达得到更稳定的提高。【结论】Pcgf2在加入诱导体系后可以有效提高细胞的重编程接近干细胞状态。该研究可为选择重编程因子诱导鸡细胞完全重编程及其机制研究提供理论参考。

3D培养诱导胚胎成纤维细胞为神经前体样细胞

目的探讨3D培养微环境和小分子组合(ATPV)对胚胎成纤维细胞诱导为神经前体样细胞的影响。方法在0.5%琼脂糖低粘附3D培养微环境和ATPV处理下,将小鼠胚胎成纤维细胞形成3D球体,分别通过RT-PCR、免疫荧光染色和碱性磷酸酶染色的方法检测神经前体细胞标记基因的表达水平。结果在3D环境下培养,随着成球培养时间的增长,小鼠胚胎成纤维细胞NPC特异性标记基因的表达明显上升,表现出神经前体样细胞的特征,并且3D ATPV培养微环境明显提高了小鼠胚胎成纤维细胞向神经前体样细胞的转化效率。结论小鼠胚胎成纤维细胞在3D和3D ATPV微环境下,聚集成球培养后逐渐获得了干细胞特征并诱导为神经前体样细胞,暗示着成球培养和小分子微环境可以给小鼠胚胎成纤维细胞创造一种良性的细胞聚集微环境,诱导神经前体样细胞的产生,这将为神经退行性病变的细胞替代疗法带来新的细胞来源。

Embryonic stem cell as nuclear donor could promote in vitro development of the heterogeneous reconstructed embryo

The nucleus of a somatic cell could be dedif-ferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellu-cida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic

小鼠胚胎成纤维细胞的分离与培养

目的 探讨影响小鼠胚胎成纤维细胞 (MEF)分离和培养的一些因素 ,以建立稳定的 MEF培养体系。方法 取 BAL B/ c小鼠胚胎分离成纤维细胞 ,利用体外培养体系 ,对 MEF的生长形态、生长曲线及细胞周期进行观察 ,并对不同胚胎日龄以及不同胰酶作用时间对 MEF分离及培养的影响进行研究。结果 13.5 d胎龄鼠胚的 MEF分离效果优于 10 .5 d、18.5 d胎龄鼠胚 ;MEF在体外为贴壁生长型细胞 ,第三代细胞增殖旺盛 ;在室温条件下 ,0 .2 5 %胰酶消化 MEF时间以 3～ 5 min为宜。结论 MEF在第 3代增殖旺盛 。

昆明鼠胚胎干细胞的分离培养与鉴定

目的:从昆明系小鼠的早期胚胎分离和培养胚胎干细胞(ES细胞).方法:收集小鼠3.5 d胚龄的囊胚,将其培养在小鼠胚胎成纤维细胞饲养层上,5-6d后取隆起生长的内细胞团块分离后再培养,观察集落的生长情况并通过碱性磷酸酶染色、原位杂交、细胞核型分析等对细胞集落进行鉴定.结果:ES细胞集落性生长,符合小鼠胚胎干细胞的一系列特性.结论:昆明系小鼠囊胚在胚胎成纤堆细胞饲养层上可以发育成ES细胞,并能进行传代培养.

小鼠胚胎成纤维细胞饲养层制备条件的研究

目的:建立小鼠胚胎成纤维细胞饲养层最佳分离培养及丝裂霉素C(MMC)处理的方法,用于培养胚胎干细胞。方法:取不同胎龄的胎鼠分离原代成纤维细胞,观察不同胎龄和培养、分离条件对细胞增殖的影响;观察不同浓度和不同作用时间下MMC对细胞增殖的抑制作用。用MTT法测定细胞增殖的数量。取妊娠3.5d的囊胚在经MMC处理的饲养层上培养,观察胚胎干细胞集落生成情况。结果:制备小鼠胚胎成纤维细胞饲养层的最佳胎龄是12.5～14.5d;室温条件下,0.25%胰蛋白酶消化时间最好在3～5min之内;原代细胞培养2～4d后即可传代,MMC能抑制胚胎成纤维细胞的增殖,最佳的作用浓度和时间是20μg/ml,2～4h,成纤维细胞饲养层可以维持10d。囊胚在饲养层上能发育成典型的“鸟巢”状干细胞集落。结论:从12.5～14.5d胎龄小鼠胚胎分离培养的胚胎成纤维细胞质量最佳,经20μg/mlMMC处理2～4h后,可以作为饲养层用于胚胎干细胞的分离克隆。

慢病毒介导的外源基因体外投递系统的建立

目的针对不同哺乳类细胞建立相应的慢病毒体外感染体系,以建立慢病毒介导的外源基因体外投递系统。方法按Invitrogen公司推荐的标准程序进行慢病毒(携带EGFP基因)包装(脂质体介导的瞬时转染)、超速离心浓缩和保存等,随后用病毒上清或浓缩后的病毒感染293FT细胞,24～48h后荧光显微镜下观察是否见绿色荧光以证实慢病毒是否成功生产;将携带EGFP基因的病毒上清或浓缩后的病毒分别加入内含293FT细胞、小鼠ES细胞、小鼠胚胎成纤维细胞(MEFs)或小鼠睾丸生殖细胞的培养板孔内,感染6～12h后,用相应培养基替换感染液,数天后荧光显微镜下观察是否见绿色荧光以证实慢病毒是否成功感染不同哺乳类细胞。结果按标准程序生产的携带EGFP基因慢病毒(病毒上清或浓缩后的病毒)成功高效率感染293FT细胞、MEFs或小鼠睾丸生殖细胞;用浓缩后的病毒(携带EGFP基因)感染小鼠ES细胞,亦可获得EGFP阳性的ES细胞克隆。结论熟练掌握了慢病毒包装、浓缩及鉴定等技术,同时针对不同哺乳类细胞建立了相应的慢病毒介导的外源基因体外传递系统,这些为相关后续研究打下了良好的基础。

小鼠胚胎干细胞体外分化为神经前体细胞的研究

目的 探索小鼠胚胎干细胞 (Embryonicstemcell ,EScell)体外分化为神经前体细胞 (Neuralprecursorcells ,NPC)的无血清培养条件 ,比较人胚胎成纤维细胞 (Humanembryonicfibroblasts ,HEF)与小鼠胚胎成纤维细胞 (Mouseembryonicfibroblasts ,MEF)对小鼠ES细胞生长的作用。方法 在MEF或HEF饲养层上培养ES细胞 ,培养液中含白血病抑制因子。采用无血清方法培养NPC ,免疫组化方法检测巢蛋白 (Nestin) ,用硝基四氮唑蓝 /5 溴 4 氯 3 吲哚基磷酸 (NBT/BCIP)显色检测碱性磷酸酶。结果 无血清培养可以获得 86 %的NPC。HEF与MEF一样能维持ES未分化状态。结论 无血清培养方法有利于ES细胞向NPC分化 ,HEF可用于小鼠ES细胞的培养 ,而且比MEF优越。

人胚胎成纤维细胞的分离、培养和鉴定及人胚胎生殖细胞体外生长所需细胞因子的表达

目的：从人胚胎生殖腺嵴和肠背系膜中分离、培养及鉴定人胚胎成纤维细胞（hEFs）,检测hEFs表达人胚胎生殖细胞（hEG）体外生长所需的重要细胞因子。方法：利用酶消化法从孕5-9周龄人胚胎的生殖腺嵴和肠背系膜中分离培养hEFs,检测其生物学特性（细胞形态、细胞生长特点、细胞周期）。采用逆转录多聚酶链反应（RT-PCR）检测hEFs表达成纤维细胞特异性标志（脯氨酰4-羟化酶β亚单位）和上皮细胞特异性标志（细胞角蛋白4）的情况,对该细胞进行鉴定。应用RT-PCR检测hEG生长所需的重要细胞因子的表达,即碱性成纤维细胞生长因子（bFGF）和白血病抑制因子（LIF）。结果：从人胚胎生殖腺嵴和肠背系膜中成功地分离培养出hEFs,该细胞可传25代以上,且经过传代及冻存复苏后生物学特性无改变;hEFs表达脯氨酰4-羟化酶β亚单位,不表达细胞角蛋白4,确定其为成纤维细胞;该成纤维细胞表达bFGF和LIF。结论：成功地分离和培养了人胚胎生殖腺嵴和肠背系膜来源的成纤维细胞,证明其表达对于hEG体外生长起重要作用的细胞因子。

人胚胎生殖细胞在人胚胎成纤维细胞饲养层上的生长

目的探讨以人胚胎成纤维细胞为饲养层分离、培养人胚胎生殖细胞的方法和条件。方法分离、培养3～4月胚胎成纤维细胞,取3～15代细胞经丝裂酶素处理后铺板备用;分离6～11周胚胎原始生殖细胞,将其置于人胚胎成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞;用免疫细胞化学方法检测胚胎生殖细胞表面标志SSEA1和SSEA4;钙钴法检测碱性磷酸酶活性;RT PCR检测转录因子Oct4的表达。结果人胚胎成纤维细胞可连续传代25代以上(6月),3～15代细胞可以用作饲养层细胞。分离的胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过8代。集落未分化标志检测显示SSEA1、SSEA4呈阳性,碱性磷酸酶活性呈强阳性,Oct4表达阳性。结论用人胚胎成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。

人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。

大鼠胚胎成纤维细胞与胰岛共培养的实验研究

目的探讨大鼠胚胎成纤维细胞(EFCs)与胰岛共培养对胰岛活性与功能的影响。方法制备SD大鼠胚胎饲养层;分离SD大鼠胰岛。实验分为单纯胰岛培养组、EFCs与胰岛共培养组和EFCs单纯培养组。胰岛活性以AO/PI、DTZ染色、线粒体活性检测,以胰岛素释放试验评价其功能。结果共培养组中,AO/PI双染显示胰岛活性高于95%,MTT显示共培养3、5、7、14d胰岛细胞吸光度明显高于单纯培养组(P<0.05或P<0.01),刺激指数SI也明显高于对照组。结论新鲜分离的胰岛与EFCs饲养层共培养可明显改善胰岛功能。

诱导胚胎干细胞分化为心肌细胞的实验研究

目的 :分别使用布法罗大鼠肝细胞 (buffaloratsliver,BRL)条件培养基或鼠胚成纤维细胞 (mouseembry onicfibroblast,MEF)饲养层体外培养 ,比较不同培养条件对胚胎干细胞 (embryonicstemcells,ESC)分化为心肌细胞的影响。方法 :用BRL条件培养基或MEF饲养层抑制ESC分化并保持其增殖 ,以维甲酸 (retinoicacid ,RA)、二甲基亚砜 (DMSO)、转化生长因子 β1(TGF β1)、激活素 A(activin A)为分化诱导剂 ,组合成 6种分化培养基 ,采用悬滴、悬浮、贴壁三步法诱导ESC分化为心肌细胞 ,比较各组分化比率。结果 :BRL及MEF饲养层两种条件均能促进ESC增殖 ,6种分化条件均能使ESC分化为心肌细胞 ;其中以BRL条件培养基培养 ,TGF β1(2ng·ml-1)、activin A(2 0ng·ml-1)及 2 0 %胎牛血清 (fetuscalfserium ,FCS)组成的分化培养基可以使 (92 .8± 2 .2 ) %的ESC分化为心肌细胞 ,分化比率显著高于其它各组 (P <0 .0 1)。结论 :以BRL条件培养基培养 ,TGF β1、activin A细胞因子组合是一种理想的ESC体外分化体系。