单细胞克隆人胚胎干细胞系的建立及其生物学特性的鉴定

目的建立单细胞克隆人胚胎干细胞系,并对其生物学特性进行鉴定。方法采取分离自人囊胚内细胞团并传代20代的细胞,用胰酶消化将细胞分散成单细胞悬液,将单个细胞接种至96孔板,生长出的细胞集落用胶原酶消化传代,采用细胞化学法和免疫荧光法检测细胞表面标志物;采用常规G带法分析细胞染色体核型;将细胞接种至严重联合免疫缺陷小鼠后腿肌肉内,观察细胞多向分化潜能。结果接种的96个细胞生长出2个细胞集落,扩增建立了2个单细胞克隆人胚胎干细胞系,稳定增殖了12个月,传代48代。细胞呈集落状生长,细胞核大、核仁明显;具有稳定的46XX核型;细胞的碱性磷酸酶、阶段特异性胚胎抗原（SSEA）4、肿瘤识别抗原（TRA）1-60、TRA-1-81等特征性细胞表面标志物呈阳性;接种至严重联合免疫缺陷小鼠后腿肌肉内的细胞,形成了包含神经组织、软骨组织、鳞状上皮组织及柱状上皮组织等来源于3个胚层组织细胞的畸胎瘤。结论本研究成功建立了单细胞克隆人胚胎干细胞系,其生物学特性稳定。

Establishment and characteristics of clonal human embryonic stem cell lines

Objective To establish clonal human embryonic stem cell lines and investigate their biological characteristics. Methods Cells were derived from one inner cell mass of human blastocyst, multiplied for 20 passages, and then dissociated into single cell suspension by digestion with 0.5% trypsin. Single cell was picked up and plated into individual well of a 96-well plate containing feeder-layers directly under a dissection microscope. The outgrowth clones were passed by treatment with collagenase. Surface markers were detected by cytochemistry and histoimmunochemistry. Karyotypes were tested using standard G-banding techniques. The pluripotency was analyzed by inoculating cells into severe combined immunodeficient (SCID) mice. Results Two clonal human embryonic stem cell lines were established. Cells of these two lines possess the characteristics and differentiating potencies: normal 46 XX karyotypes; expressing a series of surface markers such as: alkaline phosphotase, stage-specific embryonic antigen (SSEA)-4, tumor recognition antigen (TRA)-1-60, TRA-1-81 etc; and forming teratomas comprising derivatives of three embryonic germ layers such as neural tissue, cartilage, squamous epithelium and columlar epithelium when injected into SCID mice. Conclusions The two single cell-cloned human embryonic stem (hES) cell lines were derived in our laboratory. The cells possess stable biological characteristics of undifferentiated hES cells.