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

Yingying Wang,Haijun Lin,Zengrong Xiao,Xuehu Gao,Yalei Li,Yueqiang Song,Baosen Guo,Yuping Luo,Zhihua Shao,Jinjun Yu,Xuekun Li,Xi Zhang,Siguang Li

doi:10.1186/s13287-019-1323-9

Abstract

BackgroundHuman naïve pluripotency state cells can be derived from direct isolation of inner cell mass or primed-to-naïve 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 naïve 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 naïve-like pluripotency conversion.MethodsCandidate 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 naïve-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.ResultsThe results indicated that knockdown of CCDC144NL-AS1 induces naïve-like state conversion of human PSCs in the absence of additional transcription factors or small molecular inhibitors. CCDC144NL-AS1-KD human PSCs reveal naïve-like pluripotency features, such as elevated expression of naïve pluripotency-associated genes, increased developmental capacity, analogous transcriptional profiles to human naïve 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 naïve PSCs.ConclusionsOur study unveils an unexpected role of a lncRNA, CCDC144NL-AS1, in the naïve-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 naïve state human PSCs and promoting their therapeutic applications.

Highlights

Human naïve pluripotency state cells can be derived from direct isolation of inner cell mass or primed-to-naïve resetting of human embryonic stem cells through different combinations of transcription factors, small molecular inhibitors, and growth factors
Continuous exogenous expression of different combinations of pluripotency-associated factors, such as octamer-binding protein 4 (OCT4, known as POU5F1) and Kruppel-like factor 4 (KLF4), or KLF2 and KLF4, or NANOG and KLF2, allowed the derivation and expansion of human cells encompassing ground pluripotency attributes of mouse embryonic stem cells (ESCs) in “2i/Leukemia inhibitory factor (LIF)” conditions supplemented with Mitogen-activated protein kinase kinase (MEK) inhibitor (PD0325901), glycogen synthase kinase-3 (GSK3) inhibitor (CHIR99021), and human LIF [13, 14]
Candidate Long noncoding RNAs (lncRNAs) tightly correlated with human pluripotency were screened through bioinformatic and experimental approaches We initially attempted to characterize lncRNAs that support pluripotency maintenance of human pluripotent stem cells (PSCs)

Summary

Introduction

Human naïve pluripotency state cells can be derived from direct isolation of inner cell mass or primed-to-naïve resetting of human embryonic stem cells (hESCs) through different combinations of transcription factors, small molecular inhibitors, and growth factors. Continuous exogenous expression of different combinations of pluripotency-associated factors, such as octamer-binding protein 4 (OCT4, known as POU5F1) and Kruppel-like factor 4 (KLF4), or KLF2 and KLF4, or NANOG and KLF2, allowed the derivation and expansion of human cells encompassing ground pluripotency attributes of mouse ESCs in “2i/LIF” conditions supplemented with MEK inhibitor (PD0325901), GSK3 inhibitor (CHIR99021), and human LIF [13, 14]. Culture conditions containing 2i/LIF in company with inhibitors of Jun N-terminal kinase (JNK), P38, aPKC, Rho-associated protein kinase (ROCK), and growth factors FGF2 and TGFβ were described for inducing and maintaining human naïve PSCs [17]. Human naïve PSCs can be derived through directly culturing isolated cells of human inner cell mass in “t2iLGöY” medium which contained inhibitors for GSK3, MEK, PKC, and ROCK; growth factor human LIF; and ascorbic acid [19]. All artificial human naïve PSCs raise the feasibility and practical avenues to acquire an earlier pluripotency state than conventional primed human PSCs in vitro

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