Establishing a 3D Culture System for Early Organogenesis of Monkey Embryos Ex vivo and Single-cell Transcriptome Analysis of Cultured Embryos

Overview

Journal STAR Protoc

Publisher Cell Press

Specialties Biology
Biomedical Engineering
Science

Date 2024 Jan 15

PMID 38224493

Authors

Nianqin Sun

Honglian Shao

Youyue Zhang

Baiquan Ci

Hui Yao

Bing Bai

Tao Tan

Affiliations

Soon will be listed here.

Abstract

Creating in vitro culture platforms for monkey embryos is crucial for understanding the initial 4 weeks of early primate embryogenesis. Here, we present a protocol to culture cynomolgus monkey embryos in vitro for 25 days post-fertilization and to delineate the key developmental events of gastrulation and early organogenesis. We describe steps for culturing with a 3D system, immunofluorescence analysis, single-cell RNA sequencing, and bioinformatic analysis. For complete details on the use and execution of this protocol, please refer to Gong et al. (2023)..

References

La Manno G, Soldatov R, Zeisel A, Braun E, Hochgerner H, Petukhov V . RNA velocity of single cells. Nature. 2018; 560(7719):494-498. PMC: 6130801. DOI: 10.1038/s41586-018-0414-6. View

Bergen V, Lange M, Peidli S, Wolf F, Theis F . Generalizing RNA velocity to transient cell states through dynamical modeling. Nat Biotechnol. 2020; 38(12):1408-1414. DOI: 10.1038/s41587-020-0591-3. View

McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A . The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9):1297-303. PMC: 2928508. DOI: 10.1101/gr.107524.110. View

Wolf F, Angerer P, Theis F . SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018; 19(1):15. PMC: 5802054. DOI: 10.1186/s13059-017-1382-0. View

Danecek P, Bonfield J, Liddle J, Marshall J, Ohan V, Pollard M . Twelve years of SAMtools and BCFtools. Gigascience. 2021; 10(2). PMC: 7931819. DOI: 10.1093/gigascience/giab008. View

Niu Y, Sun N, Li C, Lei Y, Huang Z, Wu J . Dissecting primate early post-implantation development using long-term in vitro embryo culture. Science. 2019; 366(6467). DOI: 10.1126/science.aaw5754. View

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N . The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16):2078-9. PMC: 2723002. DOI: 10.1093/bioinformatics/btp352. View

Luecken M, Theis F . Current best practices in single-cell RNA-seq analysis: a tutorial. Mol Syst Biol. 2019; 15(6):e8746. PMC: 6582955. DOI: 10.15252/msb.20188746. View

Wang J, Chen Y, Zou Q . Inferring gene regulatory network from single-cell transcriptomes with graph autoencoder model. PLoS Genet. 2023; 19(9):e1010942. PMC: 10519590. DOI: 10.1371/journal.pgen.1010942. View

10.

Qiu X, Mao Q, Tang Y, Wang L, Chawla R, Pliner H . Reversed graph embedding resolves complex single-cell trajectories. Nat Methods. 2017; 14(10):979-982. PMC: 5764547. DOI: 10.1038/nmeth.4402. View

11.

Korsunsky I, Millard N, Fan J, Slowikowski K, Zhang F, Wei K . Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods. 2019; 16(12):1289-1296. PMC: 6884693. DOI: 10.1038/s41592-019-0619-0. View

12.

Hao Y, Hao S, Andersen-Nissen E, Mauck 3rd W, Zheng S, Butler A . Integrated analysis of multimodal single-cell data. Cell. 2021; 184(13):3573-3587.e29. PMC: 8238499. DOI: 10.1016/j.cell.2021.04.048. View

13.

McGinnis C, Murrow L, Gartner Z . DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors. Cell Syst. 2019; 8(4):329-337.e4. PMC: 6853612. DOI: 10.1016/j.cels.2019.03.003. View

14.

Dobin A, Davis C, Schlesinger F, Drenkow J, Zaleski C, Jha S . STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2012; 29(1):15-21. PMC: 3530905. DOI: 10.1093/bioinformatics/bts635. View

15.

Gong Y, Bai B, Sun N, Ci B, Shao H, Zhang T . Ex utero monkey embryogenesis from blastocyst to early organogenesis. Cell. 2023; 186(10):2092-2110.e23. DOI: 10.1016/j.cell.2023.04.020. View

16.

Van de Sande B, Flerin C, Davie K, De Waegeneer M, Hulselmans G, Aibar S . A scalable SCENIC workflow for single-cell gene regulatory network analysis. Nat Protoc. 2020; 15(7):2247-2276. DOI: 10.1038/s41596-020-0336-2. View