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Single-Cell RNA-seq Analysis of a Human Embryonic Stem Cell to Endothelial Cell System Based on Transcription Factor Overexpression

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Abstract

Background

Human embryonic stem cell (hESC)-derived endothelial cells (ECs) possess therapeutic potential in many diseases. Cytokine supplementation induction and transcription factor overexpression have become two mainstream methods of hESC-EC induction. Single-cell RNA-seq technology has been widely used to analyse dynamic processes during hESC-EC induction and components of induced endothelial cells. However, studies that used single-cell RNA-seq are mainly based on cytokine supplementation methods. In this study, we used a high-efficiency human embryonic stem cell-endothelial cell line (hESC-EC) called the “FLI1-PKC system” as a research model and employed single-cell RNA sequencing (scRNA-seq) to investigate the transcriptional landscape and cellular dynamics.

Methods

The high-efficiency hESC-EC induction (FLI1-PKC) system was established in our previous study. We applied single-cell RNA sequencing (scRNA-seq) of the differentiated cells at different time points and investigated the gene expression profiles.

Results

The FLI1-PKC induction system can directionally differentiate hESCs into mature endothelial cells with all the requisite functions. Unlike other hES-EC induction protocols, the FLI1-PKC method follows a different induction route; nonetheless, the transcriptome of induced endothelial cells (iECs) remains the same. The elevated number of activated transcription factors may explain why the FLI1-PKC system is more effective than other hES-EC protocols.

Conclusion

Our study has presented a single-cell transcriptional overview of a high-efficiency hESC-EC induction system, which can be used as a model and reference for further improvement in other hESC induction systems.

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Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

hESC :

Human embryonic cells

ECs :

Endothelial cells

iEC :

Induced endothelial cells

TF :

Transcriptional factor

UMAP :

Uniform Manifold Approximation and Projection

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Acknowledgements

We thank Genergy Bio-Technology (Shanghai) Co. for providing sequencing service and helping with data analyses.

Funding

This research was supported by grants from the National Natural Science Foundation of China (Grant number: 81873478) and Hunan Provincial Grant for Innovative Province Construction (Grant number: 2019SK4012). The funding body played no role in the design of the study and collection, analysis, and interpretation of data.

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Authors and Affiliations

  1. Institute of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, School of Basic Medical Science, Central South University, Xiangya Road 88#, Changsha, 410008, Hunan, People’s Republic of China

    XiangWang Xu, YuZe Pi, Ge Lin & Liang Hu

  2. Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-XIANGYA, Hunan, 410008, Changsha, China

    JunRu Chen, Ge Lin & Liang Hu

  3. Xiangya Hospital of Central South University, Changsha, Human, China

    Hao Zhao

  4. Hunan International Scientific and Technological Cooperation Base of Development and Carcinogenesis, National Engineering and Research Center of Human Stem Cell, Changsha, 410008, Hunan, China

    Ge Lin & Liang Hu

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  1. XiangWang Xu
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Contributions

LH designed the study and revised the manuscript. XWX performed most of the experiments. HZ build the FLI1-PKC hES-EC induction system and provide technical support for the experiments. GL revised the manuscript. JRC analyzed the single-cell sequencing data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Liang Hu.

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Ethics Approval and Consent to Participate

(1) Title of the approved project: Stem cell bank of the institution of National Engineering and Research Center of Human Stem Cell; (2) Name of the institutional approval committee or unit: ethical committee of Reproductive and Genetic Hospital of CITIC-Xiangya; (3) Approval number: LL-SC-2022–031; (4) Date of approval: 8 October 2022.

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Xu, X., Chen, J., Zhao, H. et al. Single-Cell RNA-seq Analysis of a Human Embryonic Stem Cell to Endothelial Cell System Based on Transcription Factor Overexpression. Stem Cell Rev and Rep 19, 2497–2509 (2023). https://doi.org/10.1007/s12015-023-10598-y

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