Long noncoding RNA CCDC144NL-AS1 knockdown induces na?ve-like state conversion of human pluripotent stem cells

摘要

Human na?ve pluripotency state cells can be derived from direct isolation of inner cell mass or primed-to-na?ve resetting of human embryonic stem cells (hESCs) through different combinations of transcription factors, small molecular inhibitors, and growth factors. Long noncoding RNAs (lncRNAs) have been identified to be crucial in diverse biological processes, including pluripotency regulatory circuit of mouse pluripotent stem cells (PSCs), but few are involved in human PSCs’ regulation of pluripotency and na?ve pluripotency derivation. This study initially planned to discover more lncRNAs possibly playing significant roles in the regulation of human PSCs’ pluripotency, but accidently identified a lncRNA whose knockdown in human PSCs induced na?ve-like pluripotency conversion. Candidate lncRNAs tightly correlated with human pluripotency were screened from 55 RNA-seq data containing human ESC, human induced pluripotent stem cell (iPSC), and somatic tissue samples. Then loss-of-function experiments in human PSCs were performed to investigate the function of these candidate lncRNAs. The na?ve-like pluripotency conversion caused by CCDC144NL-AS1 knockdown (KD) was characterized by quantitative real-time PCR, immunofluorescence staining, western blotting, differentiation of hESCs in vitro and in vivo, RNA-seq, and chromatin immunoprecipitation. Finally, the signaling pathways in CCDC144NL-AS1-KD human PSCs were examined through western blotting and analysis of RNA-seq data. The results indicated that knockdown of CCDC144NL-AS1 induces na?ve-like state conversion of human PSCs in the absence of additional transcription factors or small molecular inhibitors. CCDC144NL-AS1-KD human PSCs reveal na?ve-like pluripotency features, such as elevated expression of na?ve pluripotency-associated genes, increased developmental capacity, analogous transcriptional profiles to human na?ve PSCs, and global reduction of repressive chromatin modification marks. Furthermore, CCDC144NL-AS1-KD human PSCs display inhibition of MAPK (ERK), accumulation of active β-catenin, and upregulation of some LIF/STAT3 target genes, and all of these are concordant with previously reported traits of human na?ve PSCs. Our study unveils an unexpected role of a lncRNA, CCDC144NL-AS1, in the na?ve-like state conversion of human PSCs, providing a new perspective to further understand the regulation process of human early pluripotency states conversion. It is suggested that CCDC144NL-AS1 can be potentially valuable for future research on deriving higher quality na?ve state human PSCs and promoting their therapeutic applications.