Highlights
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Generation of human-monkey chimeric embryos ex vivo with hEPSCs
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hEPSCs differentiated into hypoblast and epiblast lineages
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scRNA-seq analyses revealed developmental trajectories of human and monkey cells
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The approach may allow for enhancing chimerism between evolutionarily distant species
Summary
Graphical abstract
Keywords
Introduction
Results
Generation of human-monkey chimeric blastocysts in vitro
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https://www.cell.com/cms/asset/8ab73bb1-1924-44f1-bdbc-dfdfff9a2ff1/mmc4.mp4Loading ...Video S1. Time-lapse video of chimeric embryo development, related to Figure 1
The movement and mitosis of hEPSCs within the host monkey embryo can be clearly observed.
Chimeric contribution of hEPSCs to peri- and post-implantation monkey embryos
Transcriptional landscape of human-monkey chimeric embryos
Transcriptome dynamics of hEPSCs during human-monkey chimera development
Chimeric human EPI-like cells display a distinct developmental trajectory
Discussion
- Zheng C.
- Hu Y.
- Sakurai M.
- Pinzon-Arteaga C.A.
- Li J.
- Wei Y.
- Okamura D.
- Ravaux B.
- Barlow H.R.
- Yu L.
- et al.
- Aksoy I.
- Rognard C.
- Moulin A.
- Marcy G.
- Masfaraud E.
- Wianny F.
- Cortay V.
- Bellemin-Ménard A.
- Doerflinger N.
- Dirheimer M.
- et al.
- Zheng C.
- Hu Y.
- Sakurai M.
- Pinzon-Arteaga C.A.
- Li J.
- Wei Y.
- Okamura D.
- Ravaux B.
- Barlow H.R.
- Yu L.
- et al.
Limitations of study
STAR★Methods
KEY RESOURCES TABLE
REAGENT or RESOURCE | SOURCE | IDENTIFIER |
---|---|---|
Antibodies | ||
Oct-3/4 (C-10) | Santa Cruz Biotechnologies | Cat#sc-5279; RRID: AB_628051 |
NANOG | R&D systems | Cat#AF1997; RRID: AB_355097 |
GATA-6 (D61E4) XP Rabbit mAb | Cell Signaling Technology | Cat#5851; RRID: AB_10705521 |
GATA4 | Abcam | Cat#Ab124265; RRID: AB_11000793 |
SOX17 | R&D systems | Cat#AF1924; RRID: AB_355060 |
PDGFRa | Abcam | Cat#Ab203491 |
Brachyury (H-210)/T | Santa Cruz Biotechnologies | Cat#sc-20109; RRID: AB_2255702 |
OTX2 | Santa Cruz Biotechnologies | Cat#sc-514195 |
AP-2γ (6E4/4)/TFAP2C | Santa Cruz Biotechnologies | Cat#sc-12762 |
Anti-Collagen VI antibody/COL6A1 | Abcam | Cat#ab6588; RRID: AB_305585 |
Anti-Cytokeratin 7 | Abcam | Cat#ab68459; RRID: AB_1139824 |
OCT3-4 | R&D systems | Cat#AF1759; RRID: AB_354975 |
SOX2 | Abcam | Cat#ab137385; RRID: AB_2814892 |
Donkey anti goat IgG Alexa Fluor@647 | Abcam | Cat#ab150135; RRID: AB_2687955 |
Donkey anti goat IgG Alexa Fluor@488 | Abcam | Cat#ab150129; RRID: AB_2687506 |
Goat Anti-Rabbit IgG H&L (Alexa Fluor 647) | Abcam | Cat#ab150079 |
Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 488 | ThermoFisher | Cat#35552; RRID: AB_844398 |
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | ThermoFisher | Cat#A21235; RRID: AB_2535804 |
Goat anti-Mouse IgG / IgM (H+L) 488 | ThermoFisher | Cat#A10680; RRID: AB_2534062 |
Biological samples | ||
Macaca fascicularis embryos | State Key Laboratory of Primate Biomedical Research | N/A |
Chemicals, peptides, and recombinant proteins | ||
Recombinant Human LIF | Peprotech | Cat#AF-300-05 |
CHIR 99021 | Tocris | Cat#4423; CAS: 252917-06-9 |
Y-27632 dihydrochloride | Tocris | Cat#1254; CAS: 129830-38-2 |
(S)-(+)-Dimethindene maleate | Tocris | Cat#1425; CAS: 136152-65-3 |
Minocycline, Hydrochloride | Selleck | Cat#S4226; CAS: 13614-98-7 |
IWR-1-endo | Peprotech | Cat#1128234; CAS: 1127442-82-3 |
KnockOut Serum Replacement | ThermoFisher | Cat#A3181502 |
Neurobasal Medium | ThermoFisher | Cat#21103-049 |
DMEM/F-12 | ThermoFisher | Cat#11320-033 |
N-2 Supplement | ThermoFisher | Cat#17502-048 |
B-27 Supplement, minus vitamin A | ThermoFisher | Cat#12587-010 |
GlutaMAX | ThermoFisher | Cat#35050-061 |
Non-Essential Amino Acids Solution | ThermoFisher | Cat#11140-050 |
2-Mercaptoethanol | ThermoFisher | Cat#21985-023 |
Penicilin-Streptomycin | ThermoFisher | Cat#15140-122 |
Mitomycin C from Streptomyces caespitosus | Sigma-Aldrich | Cat#M4287; CAS: 50-07-7 |
Trypsin-EDTA (0.05%), phenol red | ThermoFisher | Cat#25300-062 |
DMEM-Dulbecco’s Modified Eagle Medium, High Glucose | ThermoFisher | Cat#11965092 |
TrypLE Express Enzyme (1X), no phenol red | ThermoFisher | Cat#12604021 |
heat-inactivated FBS | Corning | Cat#35-076-CV |
L-glutamine, 100 × | ThermoFisher | Cat#25030 |
Insulin-Transferrin-Selenium-Ethanolamine (ITS -X) (100X) | ThermoFisher | Cat#51500-056 |
β-estradiol | Sigma-Aldrich | Cat#E8875; CAS: 50-28-2 |
N-acetyl- L-cysteine | Sigma-Aldrich | Cat#A7250; CAS: 616-91-1 |
Progesterone | Sigma-Aldrich | Cat#P0130; CAS: 57-83-0 |
KnockOut Serum Replacement | ThermoFisher | Cat#10828010 |
4',6-diamidino-2-phenylindole (DAPI) | Sigma-Aldrich | Cat# D9542CAS: 28718-90-3 |
Recombinant human follitropin alpha | Merck Serono | GONAL-F |
Recombinant human chorionic gonadotropin alpha | Merck Serono | OVIDREL |
CMRL-1066 | ThermoFisher | Cat#11530037 |
PBS | Biological Industries | Cat#02-020-1A |
4% Paraformaldehyde (PFA) | Biosharp | Cat#BL539A |
Sucrose | meilunbio | Cat# BGC005CAS:57-50-1 |
optimal cutting temperature compound (Tissue-Tek OCT) | SAKURA | Cat#4583 |
FBS | Biological Industries | Cat# 04-001-1A |
BSA | Sigma-Aldrich | Cat#B2064CAS: 9048-46-8 |
PBS | Meilunbio | Cat#MA0008 |
Trypsin-EDTA (0.25%), phenol red | Thermo Fisher | Cat#25200-072 |
Triton X-100 | Sigma-Aldrich | Cat#T9284; CAS: 9002-93-1 |
FBS | Biological Industries | Cat# 04-002-1A |
Critical commercial assays | ||
Superscript II reverse transcriptase | Invitrogen | Cat#18064-014 |
KAPA HiFi HotStart ReadyMix | KAPA Biosystems | Cat#KK2601 |
Betaine | Sigma-Aldrich | Cat#61962; CAS: 107-43-7 |
Triton X-100 | Sigma-Aldrich | Cat#T9284; CAS: 9002-93-1 |
Magnesium chloride | Sigma-Aldrich | Cat#M8266; CAS: 7786-30-3 |
EB solution | QIAGEN | Cat#19086 |
dNTP mix | TAKARA | Cat#4030Q |
Recombinant RNase inhibitor | TAKARA | Cat#2313A |
RNase-OFF | TAKARA | Cat#9037 |
Agencourt Ampure XP beads | Beckman Coulter | Cat#A63881 |
HiSeq X Ten Reagent Kit v2.5 | Illumina | Cat#FC-501-2521 |
QuBit dsDNA HS Assay Kit (used with QuBit 3.0) | Invitrogen | Cat#Q32851 |
TruePrep DNA Library Prep Kit V2 for Illumina | Vazyme | Cat#TD501-503 |
High sensitivity DNA reagents (used with Agilent Bioanalyzer 2100 system) | Agilent Technologies | Cat#5067-4626 |
Deposited data | ||
Single cell RNAseq data generated in this study | This paper | GEO: GSE155381 |
Experimental models: cell lines | ||
iPS1-EPS | Yang et al., 2017 | N/A |
Experimental models: organisms/strains | ||
Macaca fascicularis | State Key Laboratory of Primate Biomedical Research | N/A |
Oligonucleotides | ||
Oligo-dT30VN/ISPCR oligo/TSO | TSINGKE oligo store (
Picelli et al., 2014
) |
N/A |
Software and algorithms | ||
HISAT2 version 2.2.1 |
Kim et al., 2019
|
https://daehwankimlab.github.io/hisat2/ |
StringTie version 2.1.2 |
Pertea et al., 2015
|
https://ccb.jhu.edu/software/stringtie/index.shtml |
SPRING_dev |
Weinreb et al., 2018
|
https://github.com/AllonMKlein/SPRING_dev |
R package Seurat version 3.1.1 |
Butler et al., 2018
|
https://cran.r-project.org/web/packages/Seurat/index.html |
R package monocle version 2.12.0 |
Trapnell et al., 2014
|
https://github.com/cole-trapnell-lab/monocle-release |
R package SCENIC version 1.1.1-10 |
Aibar et al., 2017
|
https://github.com/aertslab/SCENIC |
R package velocyto.R version 0.6 |
La Manno et al., 2018
|
https://github.com/velocyto-team/velocyto.R |
R package clusterProfiler version 3.16.0 |
Yu et al., 2012
|
https://www.bioconductor.org/packages//2.10/bioc/html/clusterProfiler.html |
CellPhoneDB version 2.0.1 |
Vento-Tormo et al., 2018
|
https://github.com/Teichlab/cellphonedb |
R package slingshot version 1.1-2 |
Street et al., 2018
|
http://www.bioconductor.org/packages/release/bioc/html/slingshot.html |
Resource availability
Lead contact
Materials availability
Data and code availability
Experimental model and subject details
Animals
Culture of human extended pluripotent stem (hEPS) cells
Animal, stem cell, and ethical approvals
Method details
Oocyte collection and in vitro fertilization
Microinjection of human EPS cells into monkey blastocysts and in vitro embryo culture
Human-monkey chimeric embryos culture in vitro
-
IVC1: DMEM/F12 (11320-033, Thermo Fisher Scientific) supplemented with 20% (vol/vol) heat-inactivated FBS (35-076-CV, Corning), 2mM L-glutamine (25030; Thermo Fisher Scientific), Penicillin (25units/ml)/streptomycin (25μg/ml) (15140-122; ThermoFisher Scientific), 1X ITS-X (51500-056; ThermoFisher Scientific), 8nM β-estradiol, 200ng/ml progesterone and 25μM N-acetyl- L-cysteine.
-
IVC2: 20% (vol/vol) heat-inactivated FBS of IVC1 was replaced with 30% (vol/vol) KnockOut Serum Replacement (KoSR, 10828010, Thermo Fisher Scientific).
Single cell collection and single cell RNA-seq (scRNA-seq)
Immunofluorescence (IF) analysis
Quantification and statistical analysis
Statistical analysis
Cell origin identification, scRNA-seq data pre-processing and quality control
Cell clustering, lineage identification
Identification of differentially expressed genes
Pseudotime alignment and pseudotime-dependent differentially expressed genes analysis
GO and KEGG pathway analysis
Single-cell transcription factor regulation networks construction and clustering
RNA velocity analysis
Identification of cross-talks between human and monkey cells within chimeric embryos
Acknowledgments
Author contributions
Declaration of interests
Inclusion and diversity
Supplemental information
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Document S1. Tables S1 and S2, related to Figures 2 and 3.
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Table S3. The statistically significant ligand-receptor interactions between EPI-EPI, EPI-HYP, and EPI-EXMC in the chimeric and control non-chimeric monkey cells, and the interactions of ligands and receptors between chimeric human and host monkey cells, related to Figure 4
EPI, epiblast; HYP, hypoblast; TE, trophectoderm; EXMC, extra-embryonic mesenchyme cell.
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Table S4. Differentially expressed genes from different comparisons, related to Figure 5
The 315 genes (genes that are putatively involved in making chimeric human EPI-like cells similar to host monkey EPI cells) are subsets from the DEGs between chimeric human and control non-chimeric human cells.
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- Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo
CellJune 24, 2021Open Archive