Li Steven Shoei-Lung
Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Methods Mol Biol. 2012;873:127-49. doi: 10.1007/978-1-61779-794-1_8.
Human embryonic stem cell (hESC) lines have been derived from the inner cell mass of blastocysts. Five hESC lines have been derived from 32 discarded blastocysts in Taiwan, and these lines have since been continuously cultured on mitotically inactivated mouse embryonic fibroblasts as feeder in the hESC medium for more than 44 passages and underwent freezing/thawing processes. All of five hESC lines expressed characteristic undifferentiated hESC markers such as SSEA-4, TRA-1-81, alkaline phosphatase, TERT, transcription factors POU5F1 (OCT4), and NANOG. The hESC lines T1 and T3 possess normal female karyotypes, whereas lines T4 and T5 are normal male, but line T2 is male trisomy 12 (47XY,+12). The hESC lines T1, T2, T3, and T5 were able to produce teratomas in SCID mice, and line T4 could only form embryoid bodies in vitro. Global gene expression profiles of single colonies of these five hESC lines were analyzed using Affymetrix human genome U133 plus 2.0 GeneChip. The results showed that 4,145 transcripts, including 19% of unknown functions, were detected in all five hESC lines. Comparison of the 4,145 genes commonly expressed in the five hESC lines with those genes expressed in teratoma produced by hESC line T1 and placenta revealed 40 genes exclusively expressed in all five hESC lines. These 40 genes include the previously reported stemness genes such as POU5F1 (OCT4), NANOG, TDGF1 (CRIPTO), SALL4, LECT1, and BUB1 responsible for self-renewal and pluripotent differentiation. The global gene expression analysis also indicated that the TGFβ/activin branch components inhibin BC, ACVR2A, ACVR1 (ALK2), TGFBR1 (ALK5), and SMAD2 were found to be highly expressed in undifferentiated states of these five hESC lines and decreased upon differentiation. The epigenetic states and expression of 32 known imprinted genes in these five hESC lines and/or differentiated derivatives were also investigated. In short, the hESC nature of these five hESC lines is supported by the undifferentiated state, extensive renewal capacity, and pluripotency, including the ability to form teratomas and/or embryoid bodies; and these cell lines will be useful for research on human embryonic stem cell biology and drug development/toxicity testing. The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.
人类胚胎干细胞（hESC）系已从囊胚的内细胞团中分离得到。台湾地区从32个废弃囊胚中获得了5个人类胚胎干细胞系，此后这些细胞系一直在丝裂霉素灭活的小鼠胚胎成纤维细胞作为饲养层的hESC培养基中连续培养超过44代，并经历了冻融过程。所有这5个人类胚胎干细胞系均表达特征性的未分化hESC标志物，如SSEA - 4、TRA - 1 - 81、碱性磷酸酶、端粒酶逆转录酶（TERT）、转录因子POU5F1（OCT4）和NANOG。hESC系T1和T3具有正常的女性核型，而T4和T5系为正常男性，但T2系为男性12三体（47XY，+12）。hESC系T1、T2、T3和T5能够在严重联合免疫缺陷（SCID）小鼠中产生畸胎瘤，而T4系仅能在体外形成胚状体。使用Affymetrix人类基因组U133 plus 2.0基因芯片分析了这5个人类胚胎干细胞系单个集落的全基因组表达谱。结果显示，在所有5个人类胚胎干细胞系中检测到4,145个转录本，其中19%功能未知。将这5个人类胚胎干细胞系中共同表达的4,145个基因与hESC系T1产生的畸胎瘤和胎盘中表达的基因进行比较，发现有40个基因仅在所有5个人类胚胎干细胞系中表达。这40个基因包括先前报道的负责自我更新和多能分化的干性基因，如POU5F1（OCT4）、NANOG、TDGF1（CRIPTO）、SALL4；LECT1和BUB1。全基因组表达分析还表明，转化生长因子β/激活素分支成分抑制素BC、激活素受体2A（ACVR2A）、激活素受体1（ALK2）、转化生长因子β受体1（ALK5）和SMAD2在这5个人类胚胎干细胞系的未分化状态下高表达，分化时表达下降。还研究了这5个人类胚胎干细胞系和/或分化衍生物中32个已知印记基因的表观遗传状态和表达。简而言之，这5个人类胚胎干细胞系的hESC特性得到了未分化状态、广泛的更新能力和多能性的支持，包括形成畸胎瘤和/或胚状体的能力；并且这些细胞系将有助于人类胚胎干细胞生物学研究以及药物开发/毒性测试。在长期培养后以及分化时，应深入研究hESC系中印记基因的表观遗传状态和表达，以便在其临床应用前了解hESC系中的表观遗传稳定性。
