Zhao Xing, Li Qi, Jiang Wei-Min, Liu Hong-Yan, Ma Ning, Zhou Zhi, Li Lin-Jiang, Huang Yuan-Hua, Ma Yan-Lin
First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China; Affiliated Hospital of Hainan Medical University, Haikou 570102, Hainan Province, China.
Affiliated Hospital of Hainan Medical University, Haikou 570102, Hainan Province, China.
Asian Pac J Trop Med. 2014 Aug;7(8):639-644. doi: 10.1016/S1995-7645(14)60107-3.
To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells (iPSCs), to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.
Four genes (Oct4, Sox2, Klf4, C-Myc) were introduced into human foreskin fibroblasts (HFFs) by retroviruses. The HFFs were induced to reprogramming. Different forms of colonies were picked up, analyzed, and compared with iPSCs from different aspects, including the morphology of clones, alkaline phosphatase (AP) staining, immuno-fluorescence, and Q-PCR.
In the reprogramming process, different colonies were emerged, some of them exhibited typical human embryonic stem cell morphology (eg., compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli). However, these colonies couldn't maintain these characters after passage. There was an intermediate state, named partially reprogramming. Through analysis and identification, AP staining results were weakly positive, compared with iPSC colonies. The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4. Q-PCR indicated that the expression of exogenous transcription factors was inappropriate, either at a high level or at a low level. Most of the endogenous pluripotency genes were expressed at a low level.
It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed, and successful reprogramming may depend on a specific stoichiometric balance of Oct4, Sox2, Klf4 and c-Myc.
比较不完全重编程克隆与诱导多能干细胞（iPSC）中多能性基因的表达水平，探讨多能性基因表达与不完全重编程之间的关系。
通过逆转录病毒将四个基因（Oct4、Sox2、Klf4、C-Myc）导入人包皮成纤维细胞（HFF）。诱导HFF进行重编程。挑选不同形态的克隆进行分析，并从克隆形态、碱性磷酸酶（AP）染色、免疫荧光和Q-PCR等不同方面与iPSC进行比较。
在重编程过程中出现了不同的克隆，其中一些表现出典型的人胚胎干细胞形态（如紧密的克隆、高核质比和明显的核仁）。然而，这些克隆传代后无法维持这些特征。存在一种中间状态，称为部分重编程。通过分析和鉴定，与iPSC克隆相比，AP染色结果呈弱阳性。免疫荧光染色显示这些克隆仅表达多能蛋白Oct4。Q-PCR表明外源转录因子的表达不合适，要么过高要么过低。大多数内源性多能性基因表达水平较低。
外源多能性基因低表达或高表达可能是不完全重编程的原因之一，成功的重编程可能依赖于Oct4、Sox2、Klf4和c-Myc的特定化学计量平衡。
