人胚胎干细胞的培养条件与鉴别的方法学研究

目的：研究人胚肺成纤维细胞(human Embryonic Lung Fibroblast，HELF)饲养层对人胚胎干细胞(Human Embryonic Stem Cells，hES细胞)生长的作用，摸索适宜的hES细胞培养与识别方法。方法：以流产胎儿肺组织制备HELF饲养层，取生殖嵴或背肠系膜及其周围组织，获得由人原始生殖细胞(human Primordial Germ Cells，hPGCs)转化成的hES细胞。比较其在不同条件下的增殖行为，并通过生物学特性鉴别该细胞系。结果：HELF饲养层在LIF存在的条件下有利于支持hES细胞的体外生长；hES细胞碱性磷酸酶组织化学染色及端粒酶ELISA检测阳性；具有正常的核型；在体外发展为类胚体。结论：HELF饲养层和LIF的协同作用能支持hES细胞的生长；建立通过生物学特性鉴别hES细胞的方法。     

人胚胎干细胞系NS1的建立

目的:探讨利用长期冷冻的废弃胚胎建立人胚胎干(hES)细胞系.方法:将临床体外受精(IVF)及精子卵浆内注射(ICSI)周期中剩余冷冻5年以上的废弃胚胎复苏后继续培养至扩张囊胚,链霉蛋白酶去除透明带后采用全囊胚培养法获取原代类胚胎干细胞,再用机械法传代分离纯化hES细胞,命名为NS1.取不同代次的培养细胞,进行碱性磷酸酶染色、转录因子OCT-4、阶段特异性胚胎抗原SSEA-4、肿瘤排斥抗原TRA-1-60、TAR-1-81、核型及体内分化实验对NS1进行全能性鉴定.结果:12枚废弃胚胎在体外继续培养后,有4枚发育为囊胚,最终得到1个干细胞系,经鉴定,这些细胞都具有hES细胞的生物学特性.结论:长期冷冻的废弃胚胎可用来建立hES细胞系.     

饲养层体系的建立与胚胎干细胞的培养

胚胎于细胞(ES细胞)是南早期胚胎内细胞团或原始生殖细胞经体外分离、抑制分化培养获得的多潜能细胞。ES细胞的研究已在多种动物实验中获得了突破性进展。ES细胞在体外培养不仅要保持其增殖能力,而且要维持其未分化的状态。由于饲养层细胞能分泌白血病抑制因子(IAF)等抑制ES细胞分化的凶子,目前,ES细胞的培养主要是以饲养层来维持其干细胞特性。本实验探讨了胎鼠成纤维细胞(MEF)的分离培养、传代及其作为饲养层细胞的处理条件,建立快速稳定的MEF培养体系,并在此基础上探索了小鼠ES细胞的适宜培养、传代条件。     

人胚胎干细胞在血清和无血清培养体系中的特性比较

目的 探讨血清培养体系和无血清培养体系对人胚胎干细胞（hES cells）生长特性的影响。 方法 将人胚胎干细胞株BG02接种在丝裂霉素C处理灭活的小鼠胚胎成纤维细胞饲养层上,分别在含血清hES完全培养基或无血清培养基中连续培养25~30代。在不同培养体系下比较人胚胎干细胞形态、集落贴壁率;运用BrdU掺入法检测人胚胎干细胞增殖,用细胞计数法计算细胞倍增时间;采用免疫荧光染色检测人胚胎干细胞特异性分子标志的表达;用流式细胞仪检测人胚胎干细胞Oct-4,Nanog阳性的比例;RT-PCR检测成纤维细胞生长因子(FGFs)家族基因的表达。 结果 在两种培养体系的BG02细胞都具有人胚胎干细胞的形态特征。在含血清培养体系下,BG02细胞集落贴壁率和表达OCT-4,Nanog的阳性细胞明显高于无血清培养体系( P<0.05)。无血清培养体系中,BG02细胞生长速度明显高于含血清培养体系,细胞倍增时间分别为(33.8±4.3) h、(45.9±5.7) h,( P<0.05)。无血清培养体系的BG02细胞高表达 FGF2 、 FGFR2 、 FGFR4 。 结论 两种不同培养体系中人胚胎干细胞的体外培养特性存在一定的差异,可能与BG02细胞 FGFs 家族基因激活有关。     

人类胚胎干细胞在疾病治疗方面的研究进展

人类胚胎干细胞(hES细胞)对于疾病的治疗,尤其在器官移植和组织修复方面存在着广泛的应用前景。近来研究表明,虽然目前进行hES细胞的临床治疗为时尚早,但已获得令人瞩目的效果。本文就hES细胞在疾病治疗方面的进展以及存在的问题作一综述。     

胸腺上皮细胞体外培养及其功能研究进展

体外培养胸腺上皮细胞,分析其与胸腺细胞的直接相互作用是研究早期T 细胞发育的有效手段之一。胸腺上皮细胞体外建株培养的方法主要有饲养细胞法,选择性培养基法和逆转录病毒转染法等。体外培养的胸腺上皮细胞具有上皮细胞的特征,可分泌IL-1,IL-6,IL-7等多种细胞因子;与胸腺细胞直接结合,促进胸腺细胞增殖分化。同时胸腺细胞对胸腺上皮细胞具有反馈调节作用     

睾丸支持细胞促进体外培养神经元生长的研究

目的　探讨睾丸支持细胞 (SC)对体外培养神经元生长发育的促进作用。　方法　将SCs制备成饲养层和SCs条件培养液 (SCM)后与神经元共培养 ,观察培养细胞的存活数和突起数 ,并应用图像分析仪观察细胞的面积。　结果　SCs饲养层及SCM培养神经元的存活数、突起数和胞体面积明显高于对照组 (P <0 0 1)。　结论SCs对体外培养的神经元具有显著的促生长作用。     

Ad-LIF-OSM双基因转染饲养细胞对脐血造血细胞体外增殖和分化的影响

目的 构建表达人白血病抑制因子(LIF)和抑瘤素M(OSM)双基因的WI-38人胚肺成纤维细胞,并以此细胞作为饲养层细胞观察其对CD34+.造血干/祖细胞体外增殖和分化的影响.方法 以双启动子转移载体pAdTrack-CMV-LIF-polyA+promoterΔ为基础,将OSM基因片段酶切后插入,构建出转移质粒pAdTrack-CMV-LIF-polyA+promoterΔ -OSM.将构建正确的转移质粒与腺病毒骨架质粒共转化,获得重组腺病毒载体pAdEasy-1-pAdTrack-CMV-LIF-polyA+promoter△-OSM,通过包装,收获重组腺病毒(AdLIF-OSM).将重组腺病毒感染饲养层细胞,经RT-PCR、ELISA法检测外源基因在细胞中的表达;体外与CD34+造血干/祖细胞共培养后,通过Transwell法和细胞计数检测,比较各实验组干/祖细胞体外扩增与分化情况.结果 测序结果显示重组载体中的LIF和OSM基因序列正确;转基因饲养层细胞中能检测到外源LIF和OSM基因的转录和表达;外源LIF和OSM基因在造血干/祖细胞体外培养中能够发挥作用.结论 成功构建携带人LIF和OSM的双基因重组腺病毒载体(Ad-LIF-OSM),Ad-LIF-OSM在造血干/祖细胞体外培养的过程中能够有效地扩增CD34+造血干/祖细胞,并延缓其分化.     

子宫内膜基质细胞作为饲养层培养人胚原始生殖细胞

目的探讨子宫内膜基质细胞作为饲养细胞培养人胚原始生殖细胞的可能。方法从5—9周人胚胎生殖嵴、中肾嵴、肠系膜中消化分离原始生殖细胞(PGCs),种植于经丝裂霉素c处理的人子宫内膜基质细胞饲养层上培养。结果PGCs可以在体外培养3个多月,传14代。这样培养的PGCs表达胚胎干细胞的多种标志,如碱性磷酸酶染色、SSEA和TRA抗原等。另外细胞保持正常的染色体核型。结论人子宫内膜基质细胞可以作为饲养细胞在体外培养人胚原始生殖细胞,保持未分化状态。     

Ad-hLIF转基因饲养层细胞对CD34+造血干/祖细胞增殖和分化的影响

目的 用腺病毒载体介导的人白血病抑制因子基因(Ad-hLIF)感染WI-38人胚肺成纤维细胞制备饲养层细胞,体外观察转基因细胞对CD34+造血干/柑细胞增殖和分化的影响,体内研究对辐射损伤SCID小鼠造血功能恢复的效果.方法 用RT-PCR和ELISA法鉴定Ad-hLIF转基因饲养层细胞目的 基因的表达后,将经免疫磁珠法分离和流式细胞术检测后的CD34+细胞在饲养层和/或细胞因子培养体系中扩增28 d,检测不同时间点的单个核细胞(MNC)数量及CD34+细胞阳性率;扩增后的MNC经CFDA SE荧光标记后移植入辐射损伤SCID小鼠体内,RT-PCR和细胞荧光标记法检测小鼠内含Alu基因人源细胞的门巢情况.结果 感染50MOI(multiplicity of infection)Ad-hLIF的饲养层细胞均有绿色荧光,RT-PCR和ELISA法结果 显示hLIF目的 基因能在WI-38饲养层细胞中表达,免疫磁珠法分离的CD34+造血干/祖细胞经流式细胞术检测其纯度可达95.60%±2.58%,MNC在Ad-hLIF转基因饲养层培养体系持续扩增,最高可达356.95±0.87倍,其中CD34+细胞仪在0～14 d能维持较高水平,最高可扩增52.11±1.13倍,以后逐渐降低.将其移植辐射损伤SCID小鼠后,可明显提高小鼠存活率,4周内小鼠骨髓中不仅可观察到CFDA SE荧光标记的细胞,而且经RT-PCR法搭定后.还可检测到表达Alu人源基因的人脐血造血归巢细胞.结论 成功建立的Ad-hLIF转基因饲养层细胞不仅体外可以有效地扩增CD34+造血干/祖细胞,并延缓其分化.扩增的CD34+细胞对辐射损伤SCID小鼠具有造血功能恢复的功能.     

Transgenic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection

Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells.     

Propagation of undifferentiated human embryonic stem cells with nano-liposomal ceramide

Human embryonic stem (hES) cells, located on the periphery of the colonies, express the neuroectodermal markers nestin and Tuj1, suggesting a prematurely differentiated subgroup of cells. Here, we report that ceramide, a bioactive sphingolipid, selectively eliminates hES cells differentially expressing nestin and Tuj1. In contrast, undifferentiated cells are resistant to the apoptotic effects of ceramide. Ceramide-resistant hES cells express higher levels of the messenger RNA for ceramide-metabolizing enzymes that convert ceramide into pro-mitogenic metabolites. Based on these findings, we conducted long-term studies to determine whether liposomal ceramide can be used to maintain undifferentiated hES cells free of feeder cells. We continuously cultured hES cells on matrigel for 4 months with liposomal ceramide in a feeder cell-free system. Human ES cells treated with liposomal ceramide maintained their pluripotent state as determined by in vivo and in vitro differentiation studies and contained no chromosomal abnormalities. In conclusion, our findings suggest that exposure to ceramide provides a viable strategy to prevent premature hES cell differentiation and to maintain pluripotent stem cell populations in the absence of feeder cells.     

Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwell-patterned substrates

Human embryonic stem (hES) cells are generally cultured as cell clusters on top of a feeder layer formed by mitotically inactivated murine embryonic fibroblasts (MEFs) to maintain their undifferentiated state. This co-culture system, which is typically used to expand the population of undifferentiated hES cells, presents several challenges since it is difficult to control cell cluster size. Large cell clusters tend to differentiate at the borders, and clusters with different sizes may lead to heterogeneous differentiation patterns within embryoid bodies. In this work, we develop a new approach to culture hES cells with controlled cluster size and number through merging microfabrication, and biomaterials technologies. Polymeric microwells were fabricated and used to control the size and uniformity of hES cell clusters in co-culture with MEFs. The results show that it is possible to culture hES cells homogeneously while keeping their undifferentiated state as confirmed by the expression of stem cell markers octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP). In addition, these clusters can be recovered from the microwells to generate nearly homogeneous cell aggregates for differentiation experiments.     

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.     

Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders

BACKGROUND: Human embryonic stem (hES) cells are pluripotent cells usually derived from the inner cell mass (ICM) of blastocysts. Because of their ability to differentiate into all three embryonic germ layers, hES cells represent an important material for studying developmental biology and cell replacement therapy. hES cell lines derived from blastocysts diagnosed as carrying a genetic disorder after PGD represent in vitro disease models. METHODS: ICMs isolated by immunosurgery from human blastocysts donated for research after IVF cycles and after PGD were plated in serum-free medium (except VUB01) on mouse feeder layers. RESULTS: Five hES cell lines were isolated, two from IVF embryos and three from PGD embryos. All lines behave similarly in culture and present a normal karyotype. The lines express all the markers considered characteristic of undifferentiated hES cells and were proven to be pluripotent both in vitro and in vivo (ongoing for VUB05_HD). CONCLUSIONS: We report here on the derivation of two hES cell lines presumed to be genetically normal (VUB01 and VUB02) and three hES cell lines carrying mutations for myotonic dystrophy type 1 (VUB03_DM1), cystic fibrosis (VUB04_CF) and Huntington disease (VUB05_HD).     

Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts

It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles that of human embryonic stem (hES) cells. This was achieved by ectopic expression of four genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low, especially for postnatal human fibroblasts. We started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23-70-fold from both human adult and fetal fibroblasts. Discernible hES-like colonies also emerged 1-2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs with immortalized human feeder cells that we previously established for optimal hES cell growth. We further characterized individually picked hES-like colonies after expansion (up to 24 passages). The majority of them expressed various undifferentiated hES markers. Some but not all the hES-like clones can be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or bone morphogenetic protein 4 induction as classic hES cells. Using this improved approach, we also generated hES-like cells from homozygous fibroblasts containing the sickle cell anemia mutation Hemoglobin Sickle. Disclosure of potential conflicts of interest is found at the end of this article.     

Late-adhering human embryonic stem cell clumps during serial passage can yield morphologically 'normal-looking' colonies

During serial passage of human embryonic stem (hES) cells, freshly-seeded hES cell clumps that do not adhere to the newly-plated murine embryonic fibroblast (MEF) feeder layer after culturing overnight, are routinely discarded with the changing of fresh culture media. Preliminary observations revealed that non-adhered hES cell clumps that were still viable possessed a distinct, rounded morphology with 'sharp,' clearly-defined edges. Typically >90% of 'viable' hES cell clumps with such morphology adhered after a further 24 hr of culture on newly-plated MEF. The remaining non-adhered cell clumps that had ill-defined jagged edges could represent either non-viable hES cells or senescent MEF carried over from the previous sub-culture. Virtually none of these adhered to the newly-plated MEF feeder layer. The overwhelming majority of 'viable,' late-adhering hES cell clumps (typically >90%) gave rise to morphologically 'normal-looking' hES colonies, which appeared to remain undifferentiated. Additionally, hES cell clumps that were attached together with the majority of the passaged colonies, but were somewhat delayed in the adhesion process, compared to neighbouring colonies, also gave rise to morphologically 'normal-looking' undifferentiated colonies that were virtually indistinguishable from neighbouring colonies. Hence, it may be useful to retain late-adhering hES cell clumps during serial passage of hES cells, rather than discarding them.     

Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects

Human embryonic stem (hES) cells are pluripotent stem cells capable of self-renewal and differentiation into virtually all cell types. Thus, they hold tremendous potential as cell sources for regenerative therapies. The concurrent development of accurate, sensitive, and noninvasive technologies capable of monitoring hES cells engraftment in vivo can greatly expedite basic research prior to future clinical translation. In this study, hES cells were stably transduced with a lentiviral vector carrying a novel double-fusion reporter gene that consists of firefly luciferase and enhanced green fluorescence protein. Reporter gene expression had no adverse effects on cell viability, proliferation, or differentiation to endothelial cells (human embryonic stem cell-derived endothelial cells [hESC-ECs]). To compare the two popular imaging modalities, hES cells and hESC-ECs were then colabeled with superparamagnetic iron oxide particles before transplantation into murine hind limbs. Longitudinal magnetic resonance (MR) imaging showed persistent MR signals in both cell populations that lasted up to 4 weeks. By contrast, bioluminescence imaging indicated divergent signal patterns for hES cells and hESC-ECs. In particular, hESC-ECs showed significant bioluminescence signals at day 2, which decreased progressively over the following 4 weeks, whereas bioluminescence signals from undifferentiated hES cells increased dramatically during the same period. Post-mortem histology and immunohistochemistry confirmed teratoma formation after injection of undifferentiated hES cells but not hESC-ECs. From these data taken together, we concluded that reporter gene is a better marker for monitoring cell viability, whereas iron particle labeling is a better marker for high-resolution detection of cell location by MR. Furthermore, transplantation of predifferentiated rather than undifferentiated hES cells would be more suited for avoiding teratoma formation.     

Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers

BACKGROUND: Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells. METHODS: Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized. RESULTS: Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry. CONCLUSIONS: This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.