High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells

// Hui Li 1, 2, 3 , Shuai Gao 3 , Hua Huang 5 , Wenqiang Liu 4 , Huanwei Huang 3 , Xiaoyu Liu 4 , Yawei Gao 4 , Rongrong Le 4 , Xiaochen Kou 4 , Yanhong Zhao 4 , Zhaohui Kou 3 , Jia Li 3 , Hong Wang 4 , Yu Zhang 3 , Hailin Wang 2, 5 , Tao Cai 3 , Qingyuan Sun 1, 2 , Shaorong Gao 4 and Zhiming Han 1 1 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People’s Republic of China 2 University of Chinese Academy of Sciences, Chinese Academy of Science, Beijing, People’s Republic of China 3 National Institute of Biological Sciences, NIBS, Beijing, People’s Republic of China 4 Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, People’s Republic of China 5 State Key Laboratory of Environment Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Science, Beijing, People’s Republic of China Correspondence to: Zhiming Han, email: hanzm@ioz.ac.cn Shaorong Gao, email: gaoshaorong@tongji.edu.cn Keywords: nuclear transfer reprogramming, induced pluripotent reprogramming, Dlk1-Dio3 region, imprinted gene, RNA-seq Received: February 10, 2017     Accepted: March 24, 2017     Published: April 18, 2017 ABSTRACT Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.