A single cell trajectory of human archetypal pluripotent stem cell differentiation to trophoblast stem cells reveals induction of endogenous BMP5/7 and GATA3 without transitioning through a naive state

  1. Ethan Tietze
  2. Andre Rocha Barbosa
  3. Bruno Henrique Silva Araujo
  4. Veronica Euclydes
  5. Hyeon Jin Cho
  6. Yong Kyu Lee
  7. Arthur Feltrin
  8. Bailey Spiegelberg
  9. Alan Lorenzetti
  10. Joyce van de Leemput
  11. Pasquale Di Carlo
  12. Tomoyo Sawada
  13. Gianluca Ursini
  14. Kynon J. Benjamin
  15. Helena Brentani
  16. Joel E. Kleinman
  17. Thomas M. Hyde
  18. Daniel R. Weinberger
  19. Ronald McKay
  20. Joo Heon Shin
  21. Apua C.M. Paquola
  22. Jennifer A. Erwin

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Abstract

The human placenta is increasingly a focus of research related to early child development and the impact of maternal hyperimmune states. Primary human trophoblast stem cells (hTSC) and human pluripotent stem cells (hPSC) differentiated to hTSC can potentially model placental processes in vitro . Yet, it remains controversial how the differentiation of human pluripotent stem cells to trophectoderm relates to in vivo development and the factors required for this differentiation. Here, we demonstrate that the primed pluripotent state retains potency to generate trophoblast stem cells by activating EGF and WNT and inhibiting TGFb, HDAC and ROCK signaling without exogenous BMP4 (named TS). We map this specification by temporal single cell RNAseq compared to activating BMP4 or activating BMP4 and inhibiting WNT. TS conditions generate a stable proliferating cell type that is highly similar to six-week placental cytotrophoblasts with activation of endogenous retroviral genes and without amnion expression. Multiple primed iPSC and ES lines differentiate to iPS-derived-TSCs that can be passaged for at least 30 passages and differentiate to pure populations of multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our findings establish that primed iPS cell specification to hTSC with TS conditions involves induction of TMSB4X , BMP5/7 , GATA3 and TFAP2A without transitioning through a naive state. Collectively, our results suggest that the primed state is on a continuum of potency and can differentiate to trophoblast stem cells via multiple paths.

Significance Statement

In the present study, we map the specification of primed induced pluripotent stem cells to trophoblast stem cells (TSC). Primed iPS-derived-TSC share transcriptional, morphological and functional characteristics with human ex vivo cytotrophoblasts including capacity of self-renewal and the ability to differentiate to pure extravillous and syncytiotrophoblasts. iPS-derived TSC display a uniquely active transcriptional network of human endogenous retroviruses similar to in vivo trophoblast. In addition, the fast conversion of primed iPSC to TSC allows for modeling placental diseases from large pluripotent stem cell cohorts which are traditionally banked at the primed state. Collectively, our results suggest that the primed state is on a continuum of potency which can differentiate to trophoblast stem cells via multiple paths.

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  1. Version published to 10.1101/2020.08.29.273425v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.08.29.273425: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Aliquots of 3,000 beads in 24.6 µL of H2O were amplified with 0.8 µM SMART PCR primer (IDT, 5’-AAG CAG TGG TAT CAA CGC AGA GT-3’) and 2X KAPA HiFi Hotstart Ready Mix (Fisher Scientific,
    SMART
    suggested: (SMART, RRID:SCR_005026)
    Preprocessing of Drop-seq data: Raw sequencing data was preprocessed using the pipeline “Drop-seq Alignment Cookbook” v2.0.0 found at https://github.com/broadinstitute/Drop-seq/releases/ and described in (Macosko et al., 2015).
    Cookbook”
    suggested: (Wikibooks, RRID:SCR_008799)
    SMART adapters at 5’ end and polyA tails at 3’ end with 6 or more bp were removed from the second pair, and then aligned to the reference human (GRCh38) genome using HISAT2 v2.1.0 (Kim et al., 2015) with the default settings.
    HISAT2
    suggested: (HISAT2, RRID:SCR_015530)
    Graphic visualizations of clusters and gene expression were done using Seurat and ggplot2 in R.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    (Chen et al., 2019) Python package.
    Python
    suggested: (IPython, RRID:SCR_001658)
    Marker genes of each branch were defined by Leaf gene detection analysis implemented in the STREAM package.
    STREAM
    suggested: (HTStream, RRID:SCR_018354)
    Circus plot with GO terms was done using GOplot v1.0.2 R package.
    GOplot
    suggested: None
    To profile the expression patterns of the 19 clusters detected in this work, we applied the Cell-Specific Expression Analysis (CSEA) as implemented in the pSI R package (Xu et al., 2014).
    Cell-Specific Expression Analysis
    suggested: None
    PPI network of TFs was built using STRING database v11.0 (
    STRING
    suggested: (STRING, RRID:SCR_005223)
    (downloaded from https://string-db.org/) and score interactions were divided by 1000 to initiate the PANDA.
    PANDA
    suggested: (PANDA, RRID:SCR_002511)
    Graphic visualization, gene clusterization, and GO enrichment analysis of gene clusters within networks were performed using Cytoscape v3.7.2 (Shannon et al., 2003) and ClueGO v2.5.4.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    ClueGO
    suggested: (ClueGO, RRID:SCR_005748)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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  3. Version published to 10.1101/2020.08.29.273425v1 on bioRxiv