Single-cell analyses reveal distinct expression patterns and roles of long non-coding RNAs during hESC differentiation into pancreatic progenitors

Luo，Hai Tao1,3,4,5; He，Qian1,2,3,4,5; Yang，Wei1,3,4; He，Fei1,3,4; Dong，Jun5; Hu，Chao Feng5; Yang，Xiao Fei1,3,4; Li，Ning1,3,4; Li，Fu Rong1,3,4,5

2023-12-01

10.1186/s13287-023-03259-x

Stem Cell Research and Therapy   Impact Factor & Quartile

1757-6512

Background: Deep understanding the differentiation process of human embryonic stem cells (hESCs) is essential for developing cell-based therapeutic strategy. Substantial efforts have been made to investigate protein-coding genes, yet it remains lacking comprehensive characterization of long non-coding RNAs (lncRNAs) during this process. Methods: hESCs were passaged every 5–6 days and had maintained stable karyotype even until the 50th generation. Pancreatic progenitor specification of in vitro differentiation from hESCs was performed and modified. The nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI). Droplet-based platform (10X Genomics) was applied to generate the single-cell RNA sequencing (scRNA-seq) data. The quality of the filtered read pairs was evaluated by using FastQC. Batch effects were removed using the size factor method. Dimension reduction and unsupervised clustering analyses were performed using Seurat R package. The Monocle 2 and MetaCell algorithms were used to order single cells on a pseudotime course and partition the scRNA-seq data into metacells, respectively. Co-expression network was constructed using WGCNA. Module- and hub-based methods were adopted to predict the functions of lncRNAs. Results: A total of 77,382 cells during the differentiation process of hESCs toward pancreatic progenitors were sequenced. According to the single-cell map, the cells from different time points were authenticated to constitute a relatively homogeneous population, in which a total of 7382 lncRNAs could be detected. Through further analyzing the time course data, conserved and specific expression features of lncRNAs during hESC differentiation were revealed. Based upon pseudotime analysis, 52 pseudotime-associated lncRNAs that grouped into three distinct expression patterns were identified. We also implemented MetaCell algorithm and network-based methods to explore the functional mechanisms of these lncRNAs. Totally, 464 lncRNAs, including 49 pseudotime-associated lncRNAs were functionally annotated by either module-based or hub-based methods. Most importantly, we demonstrated that the lncRNA HOTAIRM1, which co-localized and co-expressed with several HOX genes, may play crucial role in the generation of pancreatic progenitors through regulation of exocytosis and retinoic acid receptor signaling pathway. Conclusions: Our single-cell analyses provide valuable data resources for biological researchers and novel insights into hESC differentiation processes, which will guide future endeavors to further elucidate the roles of lncRNAs.

Functional annotation hESC differentiation LncRNA Metacell Pancreatic progenitors scRNA-seq

SCI

English

First

WOS:000948984800001

2-s2.0-85150187346

Scopus

1.Translational Medicine Collaborative Innovation Center,Shenzhen People’s Hospital (The Second Clinical Medical College,Jinan University; The First Affiliated Hospital,Southern University of Science and Technology),Shenzhen,Guangdong,518020,China
2.School of Food and Drug,Shenzhen Polytechnic,Shenzhen,518055,China
3.Guangdong Engineering Technology Research Center of Stem Cell and Cell Therapy,Shenzhen Key Laboratory of Stem Cell Research and Clinical Transformation,Shenzhen Immune Cell Therapy Public Service Platform,Shenzhen,518020,China
4.Health Medicine Institute,Southern University of Science and Technology,Shenzhen,518055,China
5.Integrated Chinese and Western Medicine Postdoctoral Research Station,Jinan University,Guangzhou,510632,China

Luo，Hai Tao,He，Qian,Yang，Wei,et al. Single-cell analyses reveal distinct expression patterns and roles of long non-coding RNAs during hESC differentiation into pancreatic progenitors[J]. Stem Cell Research and Therapy,2023,14(1).

Luo，Hai Tao.,He，Qian.,Yang，Wei.,He，Fei.,Dong，Jun.,...&Li，Fu Rong.(2023).Single-cell analyses reveal distinct expression patterns and roles of long non-coding RNAs during hESC differentiation into pancreatic progenitors.Stem Cell Research and Therapy,14(1).

Luo，Hai Tao,et al."Single-cell analyses reveal distinct expression patterns and roles of long non-coding RNAs during hESC differentiation into pancreatic progenitors".Stem Cell Research and Therapy 14.1(2023).