用囊胚内细胞团建立小鼠胚胎干细胞系的尝试

目的建立小鼠129品系胚胎干细胞(ES)系。方法分离囊胚内细胞团接种至小鼠胚胎成纤维细胞,培养、传代以获得细胞系,通过形态观察、碱性磷酸酶检查、体内外分化实验予以鉴定。结果共获取12个囊胚,培养后有9个内细胞团增殖,消化传代,分离出2株克隆;形态观察具有明显的ES细胞形态,染色体核型检查为XY,碱性磷酸酶检查细胞呈阳性,体外分化实验表明可自发分化为外、中和内胚层细胞,体内分化实验获得典型的畸胎瘤,并具备小鼠特异性表面标志物Oct-4、SSEA-1。结论应用本方法建立了两株129品系胚胎干细胞系,并证实其符合小鼠胚胎干细胞的特征。     

小鼠孤雌胚胎干细胞系的建立

目的利用化学激活的技术,建立小鼠孤雌胚胎干细胞系。方法用钙离子载体A23187和6-二甲基氨基嘌呤（6-DMAP）处理小鼠卵母细胞,待囊胚形成后,分离内细胞团,细胞传代、扩增。待传至37代时,核型检测查染色体,用微卫星技术进行纯合性鉴定,用免疫荧光技术检测胚胎干细胞表面标记Oct-4、SSEA-1及SSEA-4的表达情况,并用畸胎瘤试验检测其多向分化能力。结果该细胞株为孤雌来源,表现为正常二倍体核型,表达ALK,表面标志物Oct-4、SSEA-1阳性,SSEA-4阴性。体内注射后,在局部形成含三个胚层组织的畸胎瘤。结论通过钙离子载体A23187和6-DMAP化学激活小鼠卵母细胞,可以得到具有胚胎干细胞特性的细胞株。     

人精原干细胞特异性标志的初步筛选

目的:寻求可能用于人精原干细胞(SSC)分离和纯化的特异性表面标志。方法:通过免疫组化方法,应用造血干细胞(HSC)表面标志c-kit、Thy-1,人胚胎干细胞(ES)表面标志阶段特异性胚胎抗原SSEA-3、SSEA-4、碱性磷酸酶(ALP),原始生殖细胞(PGC)标志SSEA-1以及小鼠SSC表面标志α6和β1整合素对成人及胎儿睾丸SSC的特异性表达进行筛选和鉴定。结果:在成人睾丸组织中,α6整合素在生精小管生殖细胞表面存在较广泛而显著阳性表达,而β1整合素主要在生精小管基底部细胞存在显著阳性染色,Thy-1在成人睾丸生精小管基底部细胞可见散在阳性染色,少量间质细胞中亦可见阳性染色。上述3种抗原标志在成人生殖细胞表面的表达具有一定的特异性。在胎儿睾丸生精小管中,可见SSEA-1在生殖细胞表面存在显著阳性表达,具有明显特异性。结论:α6、β1整合素和Thy-1可作为阳性标志用于人SSC的分选。SSEA-1可作为识别胎儿SSC的特异性标志。     

胚胎干细胞的增殖分化及应用研究

胚胎干细胞(embryonicstemcell,ESC)是从早期胚胎中发现的可在体外培养并无限增殖的一种高度未分化细胞,具有正常核型,表达干细胞典型标记物及高端粒酶活性。Oct3/4基因及许多细胞因子对ESC自我更新、多向分化潜能的维持具有重要作用,一定条件下ESC在体内、体外都可诱导分化为几乎机体所有类型细胞,在人类组织器官移植和基因治疗、人类胚胎发育机制研究、新药研制与开发等领域有着广阔的应用前景。本文就胚胎干细胞生物学特性、细胞标记物、增殖分化的可能机制及应用研究等进行综述。     

大鼠骨髓间充质干细胞作为滋养层培养小鼠胚胎干细胞

目的:探讨大鼠骨髓间充质干细胞(mesenchymal stemcells,MSCs)作为滋养层体外培养小鼠胚胎干细胞(embryonic stemcells ESCs)的可能。方法:收集BALB/C小鼠3·5d胎龄的囊胚,接种于大鼠骨髓间充质干细胞的滋养层上,培养5~6d后挑取胚胎干细胞集落,胰酶消化传代。观察细胞集落生长情况,通过碱性磷酸酶染色、细胞核型分析对细胞生物学特性进行检测。结果:ES细胞呈集落性生长,碱性磷酸酶组织化学染色阳性,具有正常核型及多向分化潜能。结论:大鼠MSCs可以作为滋养层体外培养小鼠胚胎干细胞,并能保持未分化状态。     

胚胎干细胞的增殖分化及应用研究

胚胎干细胞（embryonic stem cell，ESC）是从早期胚胎中发现的可在体外培养并无限增殖的一种高度未分化细胞，具有正常核型，表达干细胞典型标记物及高端粒酶活性。Oct3／4基因及许多细胞因子对ESC自我更新、多向分化潜能的维持具有重要作用，一定条件下ESC在体内、体外都可诱导分化为几乎机体所有类型细胞，在人类组织器官移植和基因治疗、人类胚胎发育机制研究、新药研制与开发等领域有着广阔的应用前景。本文就胚胎干细胞生物学特性、细胞标记物、增殖分化的可能机制及应用研究等进行综述。     

诱导人孤雌胚胎干细胞向类间充质干细胞分化的实验研究

目的探索人孤雌胚胎干细胞在体外向类间充质干细胞诱导分化的方法 ,并鉴定所得细胞的生物学特性。方法人孤雌胚胎干细胞在无血清条件下悬浮培养,形成拟胚体,10d后在含血清条件下使拟胚体贴壁生长,7d后胰酶消化,所得细胞在含血清的培养液中传代、扩增。观察传代、扩增后细胞的形态学变化;用免疫荧光染色和流式细胞技术进行细胞表型分析;取第9代细胞进行成脂、成骨和成软骨诱导,9～28d后行特殊染色及RT-PCR分析。结果人孤雌胚胎干细胞在诱导分化后,形态与骨髓间充质干细胞相似,多次扩增传代后仍保持细胞形态和扩增能力。免疫荧光染色发现,细胞表达中胚层标志波形蛋白(Vimentin)。流式细胞分析显示,细胞表达CD29、CD105、CD166、CD44等间充质干细胞表面标志。特殊染色及RT-PCR分析显示:成骨诱导后,细胞碱性磷酸酶和茜素红染色阳性,碱性磷酸酶和Cbfa-1表达增加;成软骨诱导后,细胞Ⅱ型胶原染色阳性,Ⅱ型胶原和软骨寡聚基质蛋白(COMP)表达增强;成脂诱导后,细胞油红染色阴性,脂蛋白酶和Leptin无表达。结论人孤雌胚胎干细胞可以诱导、分化为间充质干细胞,并具有成骨、成软骨分化潜能。     

人胚胎成纤维细胞饲养层支持人精原干细胞的生长

目的:探讨人精原干细胞分离、纯化及以人胚胎成纤维细胞为饲养层培养的方法和条件。方法:利用两步酶法和Percoll不连续密度离心法分离、纯化人精原干细胞,在人胚胎成纤维细胞饲养层上培养;用免疫组织化学方法检测精原干细胞表面标志SSEA-1和OCT4;检测精原干细胞克隆碱性磷酸酶活性;逆转录聚合酶链反应(RT-PCR)检测精原干细胞相关基因的表达。结果:精原干细胞在人胚胎成纤维细胞饲养层上可以存活并增殖形成集落。集落未分化标志检测显示SSEA-l、OCT4呈阳性,碱性磷酸酶活性呈强阳性,并表达精原干细胞相关基因。结论:人胚胎成纤维细胞饲养层可以支持人精原干细胞的生长。     

长期体外培养对人脂肪源性间充质干细胞生物学特性的影响

目的通过观察长期体外培养对人脂肪源性间充质干细胞(ADSCs)生物学特性的影响,鉴定ADSCs作为组织工程种子细胞的优越性。方法通过流式细胞技术观察长期体外培养对人ADSCs表面抗原表达和凋亡的影响。通过碱性磷酸酶染色、Von Kossa染色及RT-PCR检测长期体外培养对人ADSCs成骨分化潜能的影响。结果原代ADSCs表面高表达间充质干细胞表面标记物,而不表达血源性细胞表面标记物,标记物不随传代次数变化。早期ADSCs凋亡率为1%～2%,随传代次数增多凋亡率逐渐增加．但幅度不大。碱性磷酸酶染色、Von Kossa染色和RT-PCR检测显示ADSCs传至第8代时仍能保持成骨分化潜能。结论ADSCs生物学特性稳定,是较为理想的组织工程及再生医学研究的种子细胞。     

利用冷冻胚胎建立人胚胎干细胞系

目的利用辅助生殖技术中废弃的冷冻胚胎建立人胚胎干细胞系。方法将体外受精一胚胎移植周期中废弃的冷冻胚胎解冻,序贯培养至囊胚,分离出内细胞团,接种至混合饲养层,进行传代培养,对可连续传代的细胞系进行鉴定。结果共收集废弃冷冻胚胎44枚,形成囊胚15枚,原代接种内细胞团10枚,其中一枚已稳定传至25代,目前生长状态良好,经鉴定证实为胚胎干细胞。结论利用辅助生殖技术中废弃的冷冻胚胎,可成功建立人胚胎干细胞系。     

Human embryonal carcinoma cells and their differentiation in culture

Based on the study of two clonal cell lines, 2102Ep and TERA-2, isolated from human germ cell tumours, we have identified several properties that are commonly expressed by human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas. These properties include the expression of surface antigens SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and the presence of cytokeratin in the cytoplasm. However, some human EC cells lack expression of SSEA-3 and -4, although glycolipids containing these epitopes can be extracted from such variant cells. Analysis of the glycolipid composition of TERA-2 cells suggests that the switching of oligosaccharide core structure synthesis from globo-series to lacto- and ganglio-series is a key event during the differentiation of these cells. Variant, SSEA-3 and -4-negative EC cells may have already initiated these changes while retaining other features of the EC phenotype. Other studies have indicated that human EC cells variably express class I MHC antigens. We have shown that interferon induces expression of these surface molecules in EC cells without inhibiting their growth or inducing their differentiation or resistance to viral infection.     

Embryonic stem cell derived and adult hematopoietic stem cell transplantation for tolerance induction in a renal allograft recipient:--a case report

We generated an human embryonic stem cell (hESC) line to augment chimerism-associated tolerance. A 40-year-old African with chronic glomerulonephritis-chronic renal failure with 100% G6PD enzyme deficiency presented for renal transplantation with a 27-year-old, 6/6 HLA-matched sister as a willing donor. METHOD: We generated an hESC line from the donor's oocytes using long ovarian stimulation protocol simultaneously with tolerance induction protocol. A nuclear transfer (NT)-hESC line was derived by transferring a donor cumulus cell into an enucleated oocyte, subjected to electrical fusion, and cultured for 5 days. ESCs hatched from the blastocyst on day 6 were cocultured with her unmodified bone marrow for 2 days and suspended in Ringer's lactate. Five milliliters of suspension were collected for cell counting, viability, pluripotency, flow cytometry, and karyotyping. The remaining suspension was infused into the periphery of the recipient. Transplantation was performed 1 week later following a negative lymphocytotoxicity cross-match test using no immunosuppression. Peripheral blood chimerism (PBC) was studied using fluorescent in situ hybridization technique. Allograft biopsy was performed on day 7. RESULTS: NT-hESC CD34+ count was 7.6%, viability 100%, karyotyping normal, pluripotency markers: SSEA-1, SSEA-4, OCT-3/4, TRA-1/60:positive; 12% PBC was noted at 1 week after transplantation. Serum creatinine was 1.2 mg%, graft biopsy was unremarkable, and G6PD enzyme deficiency was corrected to 0% at 100 days posttransplant. Liver function tests and hematology profile were unremarkable for graft-versus-host disease. CONCLUSION: This is the first report of tolerance induction using NT-hESC-induced hematopoietic chimerism with synergistic use of adult bone marrow. It was safe and effective.     

Stable maintenance of monkey embryonic stem cells in the absence of bFGF

Monkey embryonic stem (ES) cells are useful tools in preclinical studies of gene therapy and tissue engineering as well as in primate developmental biology. However, their maintenance is not easy, requiring addition of bFGF to the medium. Herein, we have described a stable, cost-effective method that does not require bFGF. We used a high-density (1 to 1.5x10(5) cells/cm2) of mouse embryonic fibroblasts (MEF) as feeder cells to successfully maintain undifferentiated monkey ES cells for 2 years (approximately 150 passages). Furthermore, these ES cells were competent for electroporation of enhanced green fluorescent protein (EGFP) and subsequent drug selection procedures. We were able to establish EGFP-expressing cell lines using this culture condition. These cell lines expressed undifferentiated markers, such as alkaline phosphatase, SSEA-4, TRA-60, and TRA-81. In addition, strong EGFP expression was observed after differentiation into cardiomyocytes, neurons, or adipocytes, suggesting that these cell lines are a useful tool to study cell transplantation. This method simplifies the culture of monkey ES cells.     

Generation of induced pluripotent stem cells from human kidney mesangial cells

Glomerular injury and podocyte loss leads to secondary tubulointerstitial damage and the development of fibrosis. The possibility of genetically reprogramming adult cells, termed induced pluripotent stem cells (iPS), may pave the way for patient-specific stem-cell-based therapies. Here, we reprogrammed normal human mesangial cells to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4 and c-Myc). The kidney iPS (kiPS) cells resembled human embryonic stem-cell-like colonies in morphology and gene expression: They were alkaline phosphatase-positive; expressed OCT3/4, TRA-1 to 60 and TRA-1 to 81 proteins; and showed downregulation of mesangial cell markers. Quantitative (qPCR) showed that kiPS cells expressed genes analogous to embryonic stem cells and exhibited silencing of the retroviral transgenes by the fourth passage of differentiation. Furthermore, kiPS cells formed embryoid bodies and expressed markers of all three germ layers. The injection of undifferentiated kiPS colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. These results suggest that reprogrammed kidney induced pluripotent stem cells may aid the study of genetic kidney diseases and lead to the development of novel therapies.     

孤雌生殖的胚胎干细胞研究进展

人类胚胎干细胞可以在体外诱导分化成为人体的各种细胞 ,用于医学临床和科学研究。由于胚胎干细胞来源于胚胎 ,在某些国家和地区受到宗教、道德和伦理的制约。孤雌生殖的胚胎干细胞具有与胚胎干细胞相同的全能性和增殖性 ,可以在体外诱导分化成为体内的各种细胞。本文就孤雌生殖的胚胎干细胞的体外建系、定向分化的研究进展作一综述     

Importance of a new tumor market TRA-1-60 in the follow-up of patients with clinical stage I nonseminomatous testicular germ cell tumors

Background: TRA-1-60 is a new tumor marker for embryonal carcinoma-positive nonseminomatous testicular germ cell tumors (NSTGCT). Upper normal reference value (RV) and serum half-life (t_1/2) were determined. The value was determined in the follow-up of 154 patients with stage I NSTGCT. Methods: TRA-1-60 was measured in normal controls (n=100) to determine RV and in patients without recurrence for t_1/2. In all patients, TRA-1-60 was determined at the time of orchidectomy. In 42 patients with recurrence, values were also evaluated 1 month before and at the time of computed tomography-confirmed recurrence. Predictive values and survival probability were examined and compared with values for -fetoprotein (AFP) and human chorionic gonadotropin (hCG). Results: RV was 230 U/ml and t_1/2 9.5 days. Elevated TRA-1-60 at the time of orchidectomy was not associated with recurrence. One month before recurrence, 21 of 42 patients had elevated TRA-1-60 levels (50%); 10 were negative for both AFP and hCG. At the time of recurrence, 24 patients had elevated TRA-1-60 levels (57.1%); 9 were negative for AFP/hCG. Patients with TRA-1-60 levels of >500 U/ml had a poorer recurrence-free survival probability (p=0.015). Conclusions: TRA-1-60 is useful in the follow-up of stage I NSTGCT. The combination of AFP, hCG, and TRA-1-60 may improve the early detection of recurrence.Presented at the 49th Annual Cancer Symposium of The Society of Surgical Oncology, Atlanta, Georgia, March 21–24, 1996.