胚胎干细胞饲养层培养的改良法

198 1年Martin[1] 等首次分离培养了小鼠的胚胎干细胞(embyonicstemcell)后 ,胚胎干细胞已成为当今生物医学最热门和最为前沿的研究课题之一 ,而饲养层细胞对于胚胎干细胞的增殖和维持未分化状态具有重要的作用。目前 ,多熟研究采用小鼠成纤维细胞作为饲养层 ,也有用人胚胎成纤维细胞做饲养层[2 ] 。用传统的方法制备饲养层 ,耗时较长 ,成纤维维细胞的质与量不稳定 ,为此 ,对原方法进行了改良 ,力求在较短时间内 ,培养出优质高产的小鼠成纤维细胞。1　材料和方法1 1　培养液和消化液　用DMEM的培养液 (Gibco公司 ) ,补加NaHCO32g/L及H…     

小鼠胚胎成纤维细胞的原代培养及饲养层制备

目的建立小鼠胚胎成纤维细胞(MEF)饲养层,用于胚胎干细胞的培养。方法取孕13～15d的昆明种小鼠,分离原代成纤维细胞,在37℃时,用0.25%胰蛋白酶(含0.04%EDTA)消化组织块5min,重复消化多次,24h首次换液,培养3d后传代。采用10μg/mL丝裂霉素C处理胚胎成纤维细胞3h,制备出的饲养层细胞能有效地抑制胚胎干细胞的分裂,且不影响其活力。结果胎鼠分离原代成纤维细胞经10μg/mL丝裂霉素C处理后,细胞仍保持分泌多种生长因子的能力,在10d内既不增殖,也不死亡,能够很好的维持胚胎干细胞克隆的生长。结论该方法制备的饲养细胞层适用于胚胎干细胞的培养。     

人胚胎成纤维细胞饲养层的制备

背景：极小胚胎样干细胞是近年来发现的一种具有类似胚胎干细胞生物学特性的非造血干细胞,但对其体外培养扩增的方法报道极少。有研究推测,人胚胎成纤维细胞能为人骨髓极小胚胎样干细胞体外培养扩增提供良好的微环境。 目的：从人胚胎躯干中分离、培养人胚胎成纤维细胞,制备人胚胎成纤维细胞饲养层用于人骨髓极小胚胎样干细胞的培养。 方法：利用胰酶消化法从孕5-9周龄人胚胎躯干中分离培养人胚胎成纤维细胞。制作饲养层,使用不同浓度丝裂霉素C处理后,用于培养分选后的人骨髓极小胚胎样干细胞,以细胞形态、生长曲线作为胚胎成纤维细胞和饲养层的评价指标。 结果与结论：从人胚胎中成功分离培养出人胚胎成纤维细胞,该细胞可传代24代以上,且经过传代及冻存复苏后生物学特性无改变。丝裂酶素C低于12 mg/L时,人胚胎成纤维细胞增殖不能完全抑制;高于14 mg/L,人胚胎成纤维细胞可能死亡。12 mg/L丝裂霉素C作用3 h后能较好地抑制人胚胎成纤维细胞的增殖,并且保持其活力约2周,可以在很长一段时间内用作人骨髓极小胚胎样干细胞的饲养层。     

高效制备昆明鼠胚胎成纤维细胞饲养层

背景：小鼠胚胎成纤维细胞作为饲养层是胚胎干细胞培养最常用的方法,能有效抑制胚胎干细胞分化并促进其增殖,但其制备过程繁琐,工作量大,准备周期长。 目的：探索建立小鼠胚胎成纤维细胞饲养层简单、高效的培养体系。 方法：取13.5 d胎龄胎鼠用改良组织块法及简化酶消化法分离培养原代成纤维细胞,倒置显微镜下观察不同方法培养的原代和传代鼠胚胎成纤维细胞的生长形态、结构及细胞数量变化。收集鼠胚胎成纤维细胞进行冻存,复苏后细胞经不同浓度作用时间的丝裂霉素C处理,制备饲养层。 结果与结论：两种简化方法培养的原代鼠胚胎成纤维细胞生长状态良好,得到高效优质足量细胞,操作过程简单,省去了多次消化、离心、细胞计数等繁琐操作,均适宜于鼠胚胎成纤维细胞的原代培养。简化复苏法复苏后的细胞,按其生长汇合情况,以丝裂霉素C 10 mg/L作用1.5~2.0 h或1 mg/L培养过夜,省时并可获得细胞生长状态最佳的饲养层。     

人胚胎成纤维细胞对人胚胎干细胞生长的作用

目的：比较人和小鼠胚胎成纤维细胞对人胚胎干细胞生长的作用,为胚胎干细胞定向诱导各系统细胞应用于临床,消除异种蛋白污染打下基础。方法：分别采用人胚胎成纤维细胞和小鼠胚胎成纤维细胞为饲养层细胞,支持人受精卵的培养,观察其增殖和分化情况。结果：人和小鼠胚胎成纤维细胞分别加入白血病抑制因子（hLIF）均能很好支持人胚胎干细胞生长增殖,并保持未分化状态。结论：完全可以使用人胚胎成纤维细胞支持人胚胎干细胞增殖,消除异种蛋白污染的可能性,为胚胎干细胞定向诱导分化发育应用于临床打下坚实基础。     

人胚胎干细胞饲养层培养体系的研究进展

人胚胎干细胞(hES细胞)来源于着床前人囊胚内细胞团(ICM),由于具有体外无限增殖和分化成3个胚层来源的各种细胞的潜能,使其成为当今生命科学的研究热点.建立一个理想的hES细胞培养体系是利用它的前提.目前,最常用的hES细胞的体外培养方式是将其培养在饲养层细胞上.迄今为止,已经有多种细胞用于hES细胞的体外培养.饲养...     

人胚胎成纤维细胞与小鼠胚胎成纤维细胞生物学特性比较

探讨人胚胎成纤维细胞用于人胚胎干细咆体外长期培养的可行性，以含10％胎牛血清的DMEM(低糖)溶液为培养基，对人胚胎成纤维细胞的生长形态、对胰酶的敏感性、生长曲线及细胞周期进行研究，并与小鼠胚胎成纤维细胞作比较。结果显示，人胚胎成纤维细胞和小鼠胚胎成纤维细胞在体外均为贴壁生长型细胞，与小鼠胚胎成纤维细胞相比，人胚胎成纤维细胞生长更旺盛，且细胞寿命更长；在室温条件下，对0．25％胰酶更敏感，消化时间不宜超过3min。提示人胚胎成纤维细胞不仅在生长状况上与小鼠胚胎成纤维细胞类似，而且作为人胚胎干细胞体外长期培养的饲养层，在使用期限上优于后者，值得进一步研究。     

人胚胎干细胞系HUES4的培养和染色体核型分析

目的建立人胚胎干细胞无动物源性饲养层培养方法,同时对长时间体外培养的人胚胎干细胞核型变化进行分析。方法人胚胎干细胞系HUES4细胞分别培养于小鼠胚胎成纤维细胞和人包皮成纤维细胞饲养层,并对其干细胞特性进行鉴定;在培养传代过程中,收获P27、P34、P41和P44细胞进行染色体核型分析,P27细胞还进行DNA短串联重复序列多态性分析。结果生长于人包皮成纤维细胞饲养层的HUES4细胞碱性磷酸酶染色以及SSEA-4、TRA-1-60和TRA-1-81抗原阳性,SSEA-1抗原阴性。所检测的4代细胞中均见46,XY/46,XY,t（9;15）（q22;q26）核型嵌合现象,且异常核型百分比随传代次数增加有上升的趋势。结论培养人胚胎干细胞的饲养层细胞可由无动物源性的饲养层细胞替代;长期体外培养有增加细胞染色体核型异常的风险。     

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SSEA-1和SSEA-4;钙-钴法检测碱性磷酸酶活性;RT-PCR检测转录因子Oct-4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SSEA—1、SSEA-4呈阳性,碱性磷酸酶活性呈强阳性,Oct-4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。     

人胚胎干细胞HuES17向造血干细胞的分化

背景：人类胚胎干细胞是来源于着床前囊胚的内细胞团,能在长期培养中无限增殖并保持未分化状态,且具有分化成人体组织各种细胞类型能力的细胞。 目的：进一步验证人胚胎干细胞HuES17细胞株向造血干细胞分化的能力。 方法：人胚胎干细胞HuES17采用与人包皮成纤维细胞二维共培养的方式培养,采用人胚胎干细胞与小鼠骨髓基质细胞(OP9) 二维共培养的方法诱导胚胎干细胞向造血干细胞分化。 结果与结论：人胚胎干细胞与小鼠骨髓基质细胞(OP9) 二维共培养诱导造血分化的第四五天即开始出现OP9细胞逐渐老化,很快死亡;可以观察到人胚胎干细胞分化,然而,随着OP9细胞死亡,分化的人胚胎干细胞亦死亡,不能诱导人胚胎干细胞向造血干细胞分化。提示人胚胎干细胞HuES17细胞株可能不能向造血干细胞分化,或向造血干细胞分化的能力较低。     

E-cadherin-expressing feeder cells promote neural lineage restriction of human embryonic stem cells

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article, we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule, E-cadherin, to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin, but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase, suggesting that E-cadherin promotes rapid neuronal differentiation. Further, these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin, neurofilament, and neural cell adhesion molecule. Moreover, blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation, we are able to utilize a specific cell-cell adhesion molecule, E-cadherin, to influence the nature and degree of neural specialization.     

A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture

Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.     

Human feeder cells support establishment and definitive endoderm differentiation of human embryonic stem cells

Mouse embryonic fibroblasts (MEFs) have been extensively used as feeder cells to support the in vitro propagation of human embryonic stem cells (hESCs). However, owing to the risk of cross-contamination with animal or other unknown pathogens, the use of MEFs does not meet requirements for the clinical application of hESCs. Moreover, the actual role played by the feeders in the differentiation of hESCs is still unclear. In this study, human embryonic fibroblasts (HEFs) were used as feeder cells to support the establishment and undifferentiated growth of hESCs, and the capability of HEFs to induce the differentiation of definitive endoderm (DE) was evaluated. Three new hES cell lines were derived. These cell lines exhibited and maintained the common features of traditional hESCs after prolonged culture in vitro. Furthermore, DE differentiation of the newly established hES cell lines was performed using 100 ng/ml activin A, and the effects were compared among HEFs, MEFs, and feeder-free systems. On day 5 of induction, DE (SOX17(+)) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0 +/- 8.9% and 78.7 +/- 3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7 +/- 5.6%). The SOX17(+) cells tended to differentiate into an endodermal lineage in vivo and could be further induced into glucagon and C-peptide double positive islet-like clusters in vitro. Our studies suggest that, in terms of therapeutic application, HEFs can be an effective substitute for MEFs for sustaining the derivation and DE differentiation of hESCs.     

No evidence for infection of human embryonic stem cells by feeder cell-derived murine leukemia viruses

Until recently, culture and expansion of nondifferentiated human embryonic stem cells (hESCs) depended on coculture with murine embryonic fibroblasts. Because mice are known to harbor a variety of pathogens, such culture conditions implicate the risk of xenozoonoses. Among these pathogens, endogenous retroviruses, including murine leukemia viruses (MuLVs), are of special importance. It is well known that some strains cause pathogenic (e.g., leukemic) effects and that xenotropic, polytropic, and amphotropic MuLVs are able to infect human cells.In view of potential clinical applications of hESC lines, it is therefore imperative to investigate potential infection of hESCs by mouse feeder cell-derived viruses. As a first step towards a comprehensive infection risk assessment, we have analyzed embryonic fibroblasts derived from different mouse strains for expression and release of xenotropic, polytropic, and amphotropic MuLVs. Moreover, several hESC lines have been investigated for expression of specific receptors for xenotropic/polytropic MuLVs, as well as for MuLV infection and expression.Evidence for expression of humantropic MuLVs was found in cultures of mouse embryonic fibroblasts (MEFs). Moreover, expression of specific receptors for xenotropic/ polytropic MuLV on human HEK293 and hESC lines and infection after coculture with an MuLV-producing mink cell line could be demonstrated. In contrast, no evidence of MuLV transmission from MEFs to human HEK293 cells or to the hESC lines I-3, I-6, I-8, and H-9 has been obtained.Our results suggest that recently established hESC lines are free of MuLV infections despite long-term close contact with MEFs.     

Leukemia inhibitory factor-expressing human embryonic lung fibroblasts as feeder cells for human embryonic germ cells

A robust culture system is critical for maintaining both proliferation and the developmental potential of human embryonic germ (hEG) cells. Here, we use human embryonic lung fibroblasts (hELF) overexpressing leukemia inhibitory factor (LIF) as feeder cells to support the self-renewal of hEG cells. We examine the morphology, gene expression, and developmental potential of hEG cells grown on a feeder layer of LIF-expressing hELF (hELF/lif) cells. hEG cells were positive for alkaline phosphatase (AP), stage-specific embryonic antigen (SSEA)-1, SSEA-4, tumor rejection antigen (TRA)-1-60, and TRA-1-81. In addition, hEG cells maintained on hELF/lif expressed higher levels of pluripotency genes such as Oct4 and Nanog. In addition, hEG cells maintained on hELF/lif cells gave rise to differentiated tissues when grown as embryoid bodies, consistent with the broad developmental potential of the starting population. Our results suggest that a hELF/lif feeder layer can support the proliferation of hEG cells, and that LIF signaling plays an essential role in this process. This human-derived culture system provides an attractive alternative to more commonly used mouse-derived feeder layers for use in clinical applications.     

Generation of T lineage cells from human embryonic stem cells in a feeder free system

Human embryonic stem cells (hESC) have the potential to revolutionize certain medical treatments, including T-cell-based therapies. However, optimal approaches to develop T cells from hESC are lacking. In this report, we show that T-cell progenitors can be derived from hESC cultured as embryoid bodies (EBs). These EB-derived T-cell progenitors give rise to phenotypically and functionally normal cells of the T lineage when transferred into human thymic tissue implanted in immunocompromised mice, suggesting that introduction of these progenitors into patients may also yield functional T cells. Moreover, hematopoietic progenitors demonstrating T-cell potential appeared to be CD45+/CD34+, resembling those found in normal bone marrow. In contrast to T cells developed from hESC cocultured on murine stromal cells, the EB-derived T cells also expressed normal levels of CD45. Importantly, the EB system eliminates the previous need for murine cocultures, a key impediment to developing a protocol for T-cell progenitor derivation suitable for clinical use. Furthermore, following lentiviral-mediated introduction of a vector expressing enhanced green fluorescent protein into hESC, stable transgene expression was maintained throughout differentiation, suggesting a potential for gene therapy approaches aimed at the augmentation of T-cell function or treatment of T-cell disorders.     

A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells

BACKGROUND: Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. METHODS: We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. RESULTS: Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. CONCLUSIONS: We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.     

无血清无饲养层人胚胎干细胞向胶质前体细胞的分化

目的 建立无血清无饲养层人胚胎干细胞(hESCs)胶质前体细胞分化体系.方法将生长在层黏连蛋白包被的培养板上的人胚胎干细胞,悬浮培养诱导拟胚体(EBs)形成,将25d的EBs转移至含有胰岛素、5μg/L碱性成纤维细胞生长因子(bFGF)、20μg/L表皮生长因子(EGF)和5μg/L三碘甲状腺素(T3)的胶质细胞分化培...