Li Steven Shoei-Lung, Liu Yung-Hsien, Tseng Chao-Neng, Chung Tung-Liang, Lee Tzi-Yi, Singh Sher
Department of Biotechnology, Kaohsiung Medical University, Kaohsiung City 807, Taiwan.
Stem Cells Dev. 2006 Aug;15(4):532-55. doi: 10.1089/scd.2006.15.532.
Many human embryonic stem cell (hESC) lines have been reported, but only a few of them have been fully characterized. In this report, five new hESC lines were derived from 32 discarded blastocysts in Taiwan, and these lines were continuously cultured on mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer in the hESC medium for more than 44 passages and underwent freezing/thawing processes. All five hESC lines expressed characteristic undifferentiated hESC markers, such as SSEA-4, TRA-1-81, alkaline phosphatase, TERT, and the transcription factors POU5F1 (OCT4) and NANOG. 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). hESC lines T1, T2, T3, and T5 were able to produce teratomas in severe combined immunodeficient (SCID) mice, and line T4 could only form embryoid bodies (EBs) in vitro. Global gene expression profiles of these five newly derived hESC lines were analyzed using the 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 teratomas produced by the 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 transforming growth factor-beta (TGF-beta)/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. 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 EBs. These cell lines will be useful for human embryonic stem cell biology and drug development.
已有许多人类胚胎干细胞（hESC）系的报道，但其中只有少数得到了充分表征。在本报告中，从台湾的32个废弃囊胚中获得了5个新的hESC系，这些细胞系在hESC培养基中，于有丝分裂失活的小鼠胚胎成纤维细胞（MEF）饲养层上连续培养超过44代，并经历了冻融过程。所有5个hESC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）以及转录因子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（EB）。使用Affymetrix人类基因组U133 plus 2.0基因芯片分析了这5个新获得的hESC系的全基因组表达谱。结果显示，在所有5个hESC系中检测到4145个转录本，其中19%功能未知。将这5个hESC系中共同表达的4145个基因与hESC系T1产生的畸胎瘤和胎盘表达的基因进行比较，发现有40个基因仅在所有5个hESC系中表达。这40个基因包括先前报道的干性基因，如负责自我更新和多能分化的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（TGF-β）/激活素分支成分抑制素BC、ACVR2A、ACVR1（ALK2）、TGFBR1（ALK5）和SMAD2在这5个hESC系的未分化状态下高度表达，分化时表达下降。简而言之，这5个hESC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EB的能力。这些细胞系将有助于人类胚胎干细胞生物学研究和药物开发。
