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Defining cell populations and their interactions in the pig blastocyst

Defining cell populations and their interactions in the pig blastocyst. Journée Utilisateurs GeT, the 15th of december 2018 Hervé Acloque (herve.acloque@inra.fr). Failure to produce pig Pluripotent Stem Cells ( PSCs ) using conventional protocols. Culture from blastocysts

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Defining cell populations and their interactions in the pig blastocyst

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  1. Defining cell populations and their interactions in the pig blastocyst Journée Utilisateurs GeT, the 15th of december 2018 Hervé Acloque (herve.acloque@inra.fr)

  2. Failure to producepig Pluripotent Stem Cells (PSCs) usingconventionalprotocols Culture from blastocysts LIF or bFGF+Nodal Somatic Reprogramming Do not work in pigs

  3. A betterknowledge of pigembryonicPSCs and of their micro-environmentisrequired to maintainthemin vitro éclos > < 2mm D6-7 D9 D10 D12-13 D11-12 Fertilization to Blastocyst Blastocyst to Implantation Adapted from Brevini et al. 2007

  4. A betterknowledge of pigembryonicPSCs and of their micro-environmentisrequired Endometrium Interactions • Physical • Molecular • Hormonal Readouts: • Cell Adhesion • Differentiation • Proliferation • Growth • Apoptosis • … Ovoid Blastocyst (D8) EPIBLAST TE ExEnd ExMeso

  5. Experimental Design of the UNIPLURI project

  6. scRNA-seq of pigblastocystsfrom 2 different stages Three trips: D7_rep1 (30/08/2017), D7_rep2 (14/11/2017) and D10 (22/08/2018) D0 15h-20h Toulouse D1 Toulouse D0 9h-11h Lusignan D -1 Paris QC librairie ~15 embryos for D7 (rep1 and rep2) ~1 or 3 embryos for D10 Cell dissociation (Accutase) 10X Genomics 3’ mRNA seq V2

  7. scRNA-seq of pigblastocystsfrom 2 different stages: data analysis Quality of genome annotation is critical

  8. scRNA-seq of pigblastocystsfrom 2 different stages: D7

  9. scRNA-seq of pigblastocystsfrom 2 different stages: D10

  10. Assigning clusters: who’swho ?

  11. Assigning clusters: data fromhuman and mice Epiblast (EPI) Pluripotent OCT4, NANOG, SOX2,ID1, GDF3, NR5A2, DNMT3B, FBP1 Primitive Endoderm (PrE) GATA6, GATA4, SOX17, APOE, COL18A1, RSPO3, SERPINH1, HNF4A, ID2 Trophectoderm (TE) Stirparo et al. 2018 Wei et al. 2018 blastocyst embryo CDX2, GATA3, KRT8, GATA2, KRT18, TEAD3, DAB2, PTGES, ABCG2, ELF3

  12. Assigning clusters: Trophectoderm

  13. Assigning clusters: Extraembryonicendoderm

  14. Assigning clusters: Epiblast

  15. Assigning clusters: differencesbetween stages ?? ?? Trophectoderm: 58% Extra-Endoderm: 39% Epiblast : 3% Trophectoderm: 37% Extra-Endoderm: 59% Epiblast : 2%

  16. Cyclingcellswithin clusters TE_2 EPI ExEnd_1 ExEnd_2 TE_1 PrEnd EPI TE_2 TE_1

  17. Conclusions I Earlycelllineages are conserved in pigblastocysts: • Epiblastrepresents a small population (~3%) at D7 and D10 withspecific expression of pluripotency markers • Extraembryonicendoderm express known markers of Primitive endoderm: the proportion of ExtraEndincreasesbetween D7 to D10 • Trophectoderm: conserved expression of known markers fromhuman and mice, canbedividedintotwosub-populations

  18. Pigblastocysts are Prostaglandins’ and Estrogenfactories

  19. A betterknowledge of pigembryonicPSCs and of their micro-environmentisrequired Endometrium PGs E2 EPIBLAST TE ExEnd ExMeso

  20. Signallingpathways active in the blastocyst mouse ESCs human ESCs/mEpiSCs BMP4 (serum) ActivinA Nodal FGF4 (paracrine) FGF2 LIF GSK3 SMAD 1/5/7 ß-catenin STAT3 ERK1/2 (MAPK) ERK1/2 (MAPK) SMAD 2/3 DUSP9 TCF3 ID SELF RENEWAL / PLURIPOTENCY SELF RENEWAL / PLURIPOTENCY DIFFERENTIATION

  21. Signallingpathways: BMPs • BMPs (2/4/7) are expressed by the Extra-End in D7 and D10 blastocysts • SMAD1 and SMAD5 expression is detected in few cells • All blastocysts cell population may respond to BMP signaling through different ID genes: • ID1 in the epiblast • ID2 in the Extra-End. • ID3 and ID4 in the TE

  22. Signallingpathways: JAK/STAT3 • STAT3 is mostly detected in Extra-End and EPI • Signalling through IL6/LIF may occur only in Extra-End • No LIF and very few IL6 positive cells detected • Signalling through PDGFA and CNTF may occur also in EPI

  23. Signallingpathways: Nodal • SMAD2 is expressed in all tissues • Receptors : Extra-End and EPI • NODAL and GDF3 are mostly expressed by EPI (autocrine)

  24. Signallingpathways: FGF Only in Primitive endoderm

  25. Signallingpathways active in the blastocyst Mouse ESCs Pig PSCs PDGFa CNTF Para/autocrine GDF3 Nodal Autocrine BMP2 Paracrine GSK3 BMP4 (serum) FGF4 (paracrine) LIF GSK3 SMAD 2 SMAD 1 JAK1 SMAD 1/5/7 ß-catenin STAT3 ERK1/2 (MAPK) DUSP9 STAT3 TCF3 ID1 TCF3 ID DIFF. SELF RENEWAL / PLURIPOTENCY SELF RENEWAL / PLURIPOTENCY ???

  26. A betterknowledge of pigembryonicPSCs and of their micro-environmentisrequired Endometrium PGs E2 Nodal/GDF3/PDGFA BMP/CNTF EPIBLAST TE ExEnd ExMeso

  27. Conclusions II • LIF/IL6 seems not to beinvolved in the maintenance of pluripotency at these stages, CNTF and PGFA are better candidates to activate JAK/STAT3 pathway • Probable autocrine action of NODAL/GDF3 on epiblastcells • No clearevidence of FGF2/4 expressions in any tissue • BMP2/4 are expressed by the Extra-End and mayactivatespecific ID genes in each tissue • No clearevidencealso for Wnt (WNT6 in the TE, TCF3 a bit everywhere)

  28. Looking for markers of pigpluripotency: Differential expression analysis Top 12 DEGs from log2-fold change

  29. Looking for markers of pigpluripotency: Differential expression analysis Top DEGs with log2-fold change >1 + specificity (EPI-only) CD24 BEX4 UCHL1 PCSK1N SMIM1 CD24 PCSK1N TCEAL9 FSTL1 LZTS1 SOX15 ID1 ERAS KAT7 IGFBP2 UCHL1 TCEAL9 CITED1

  30. Conclusions III Single-cell transcriptomic study allow to perform differential expression analysis on subset of cells This is a powerful tool to find new markers for populations of interest It also helps to define proliferative or quiescent populations, to identify active signalling pathways. 3’Quant RNA-seq strategies used with DROP-seq based technologies (10X Chromium) do not provide full transcriptome and may affect the detection of weakly expressed markers (like SOX2 or NANOG) or poorly annotated genes (when the 3’UTR is not annotated)

  31. Perspectives Better characterization of extra-embryonic cell populations (mural trophectoderm, extraembryonic endoderm, extra-mesoderm, extra-ectoderm) Validation of the identified populations by In situ hybridization Determination of lineages’ origins with pseudotime analysis Potential Cell-cell interactions between populations identified by CellPhoneDB (Vento-Tormo et al. 2018) Defining a working hypothesis on a pig pluripotency network

  32. Aknowledgements All the team GenESI in Rouillé for their reactivity and their support to sample pig embryos. Frederic Martins, Claire Kuchly and Remy Serres from the genomic facility of Toulouse (GeT) The GenPhySE unit and Cytogen team for their support Sarah Djebali and Nathalie Vialaneix for their support regarding RNA-seq analysis Many thanks to GA Division for supporting and funding this project

  33. Looking for markers of pigpluripotency: Differential expression analysis Top 12 DEGs from log2-fold change

  34. Looking for markers of pigpluripotency: Differential expression analysis Genes identified by Bernardo et al. (2018) Bernardo et al. 2018 PSAP PSAP SCPEP1 GJB5 SPIC DUSP6 LIN28B TRIP6 TRIP6 JAKMIP2 DUSP6 LIN28B

  35. WNTs - D10

  36. WNT – D7

  37. Le projet UNIPLURI: mieux connaitre les cellules embryonnaires porcines pluripotentes Analyse transcriptome complet: 3 stades embryonnaires Sarah Djebali

  38. Le projet UNIPLURI: mieux connaitre les cellules embryonnaires porcines pluripotentes Analyse transcriptome complet: 3 stades embryonnaires D12 Wholeembryo Lignées PSCs D10 Wholeembryo D10 TE D7 Wholeembryo D12 TE Sarah Djebali

  39. Le projet UNIPLURI: mieux connaitre les cellules embryonnaires porcines pluripotentes Demultiplexage, alignement et réassignation (cellules + genes) avec CellRanger: Par Claire Kuchly à PlaGe

  40. Le projet UNIPLURI: mieux connaitre les cellules embryonnaires porcines pluripotentes D7bis summary D7 summary

  41. Le projet UNIPLURI: mieux connaitre les cellules embryonnaires porcines pluripotentes Assigner aux clusters une identité : les populations sont censées`être similaires entre réplicats biologiques d’un même stade Comment aggréger deux expérimentations en supprimant les biais expérimentaux ? Mirror effect using CellRanger --aggr Seurat package (CCA) Butler et al. 2018

  42. Le projet UNIPLURI: mieux connaitre les cellules embryonnaires porcines pluripotentes Définir les clusters: K-nearest neighbor (KNN) graph based on euclidean distance in PCA space and Jaccard similarities. Cluster1 Cluster4 Cluster2 Cluster3 Butler et al. 2018

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