Revealing cell populations catching the early stages of the human embryo development in naïve pluripotent stem cells

Moya-Jódar, Marta; Ullate-Agote, Asier; Barlabé, Paula; Rodríguez-Madoz, Juan R.; Abizanda, Gloria; Barreda, Carolina; Carvajal‐Vergara, Xonia; Vilas–Zornoza, Amaia; Romero, Juan Pablo; Gárate, Leire; Agirre, Xabier; Coppiello, Giulia; Prósper, Felipe; Aranguren, Xabier L.

doi:10.1101/2022.03.06.482812

Cited by 3 publications

(5 citation statements)

References 70 publications

(172 reference statements)

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“…In addition, similar to mouse 2CLCs, DUX overexpression increased the abundance of 8CLCs in culture by 20-fold. A separate study corroborates the finding of 8CLCs within naïve hPSC cultures [ 9 ]. Interestingly, this group also identified trophectoderm-like cells in the same cultures, further confirming the presence of multiple distinct cell types within naïve hPSC cultures.…”

supporting

confidence: 83%

Hidden totipotency in naïve human pluripotent stem cell cultures

Schmitz

2022

Life Medicine

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supporting

confidence: 83%

Hidden totipotency in naïve human pluripotent stem cell cultures

Schmitz

2022

Life Medicine

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“…The bulk RNA-seq and proteomics analyses of TIRN-SC (+/-iDUX4) revealed acquisition of pre-lineage embryonic transcriptional programs. However, rare subsets of cells expressing 8C-specific genes were reported in heterogenous naïve epiblast-like hESC populations (Moya-Jodar et al, 2023).…”

Section: Resultsmentioning

confidence: 99%

Proteogenomic Reprogramming to a Functional Human Totipotent Stem Cell State via a PARP-DUX4 Regulatory Axis

Zimmerlin,

Angarita,

Park

et al. 2024

Preprint

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PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome and proteome during zygotic genome activation (ZGA), totipotent, and pluripotent embryonic stages. Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naive reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.g., TPRXL, HOX clusters), eight-cell (8C)-specific (e.g., DUXA, GSC, GATA6), primitive endoderm-specific (e.g., GATA4, SOX17), trophectoderm-specific (e.g., CDX2, TFAP2C), and naive epiblast-specific (e.g., DNMT3L, NANOG, POU5F1(OCT4)) factors; all in a hybrid, combinatorial single-cell manner. Mapping of proteomic and single-cell expressions of TIRN cells against human preimplantation embryo references identified them as relatively homogenous 4C-8C stage populations. Injection of TIRN cells into murine 8C-16C-staged embryos resulted in efficient totipotent-like single cell contributions of human cells to both extra-embryonic (trophectoderm, placenta) and embryonic (neural, fetal liver, hematopoietic) lineages in human-murine blastocyst and fetal chimeras. Pairing of proteome with ubiquitinome analyses of TIRN cells revealed a global shutdown of ADP-ribosylation, and a perturbed TNKS/PARP1 equilibrium which not only impacted the protein levels of hundreds of TNKS/PARP1 substrates via a rewiring of the ubiquitin-proteosome system (UPS), but also de-repressed expression of hundreds of developmental genes associated with PARP1 suppression. ChIP-Seq analysis of core NANOG-SOX2-OCT4 (NSO) pluripotency factors in TIRN cells identified reprogrammed DUX4-accessible distal and cis-regulatory enhancer regions that were co-bound by PARP1 (NSOP). These NSOP enhancer regions possessed co-binding motifs for hundreds of the same ZGA-associated, embryonic, and extraembryonic lineage-specifying pioneer factors (e.g., HOX, FOX, GATA, SOX, TBX, CDX families) that were concurrently co-expressed in TIRN cells; suggesting that PARP1 and DUX4 cooperate with NSO pluripotency core factors to regulate the epigenetic plasticity of a human totipotency program. These findings provide the first demonstration that global, proteome-wide perturbations of post-translational modifications (i.e., ADP-ribosylation, ubiquitination) can regulate epigenetic reprogramming during human embryogenesis. Totipotent TIRN stem cells will provide a valuable cell culture model for studying the proteogenomic regulation of lineage specification from human blastomere stages and may facilitate the efficient generation of human organs in interspecies chimeras.

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“…During the conception and majority of the execution of this project, the presence of a totipotent state in PSC culture was considered a mouse-specific phenomenon. As of writing, several papers have been published on the existence of 8-cell-like cells (8CLC) within human naïve PSC cultures (Taubenschmid-Stowers et al, 2022; Mazid et al, 2022; Moya-Jódar et al, 2022; Yoshihara et al, 2021). These 8CLC have similar transcriptomes to the 2CLC of mouse cultures, expressing totipotency genes such as Zscan family genes and endogenous retroviruses (Mazid et al, 2022; Moya-Jódar et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…As of writing, several papers have been published on the existence of 8-cell-like cells (8CLC) within human naïve PSC cultures (Taubenschmid-Stowers et al, 2022; Mazid et al, 2022; Moya-Jódar et al, 2022; Yoshihara et al, 2021). These 8CLC have similar transcriptomes to the 2CLC of mouse cultures, expressing totipotency genes such as Zscan family genes and endogenous retroviruses (Mazid et al, 2022; Moya-Jódar et al, 2022). These subpopulations appear in small quantities – similar to the 2CLC in mouse – when using culture media designed to promote naïve PSC characteristics, such as PXGL, 5iLA, 5iLAF, t2iLGö, RSeT, but interestingly they were not seen in expanded potential stem cells (Mazid et al, 2022; Moya-Jódar et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

“…These 8CLC have similar transcriptomes to the 2CLC of mouse cultures, expressing totipotency genes such as Zscan family genes and endogenous retroviruses (Mazid et al, 2022; Moya-Jódar et al, 2022). These subpopulations appear in small quantities – similar to the 2CLC in mouse – when using culture media designed to promote naïve PSC characteristics, such as PXGL, 5iLA, 5iLAF, t2iLGö, RSeT, but interestingly they were not seen in expanded potential stem cells (Mazid et al, 2022; Moya-Jódar et al, 2022). It is not known whether naïve PSCs bearing two X Chromosomes would be more stable genetically, or if they would show a higher proportion of 8C-like cells (Fig 6E).…”

Section: Discussionmentioning

confidence: 99%

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Chromosome X Dosage Modulates Development of Aneuploidy in Genetically Diverse Mouse Embryonic Stem Cells

Stanton,

Aydin,

Skelly

et al. 2024

Preprint

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SUMMARYThe genetic integrity of pluripotent stem cells (PSC) is integral to their applications in research and therapy, but it is compromised by frequent development of copy number variations. Little is known about the basis of the variable genomic integrity among different PSC lines. Here we identify aneuploidies using RNA-seq and proteomics data from a panel of mouse embryonic stem cell (mESC) lines derived from 170 Diversity Outbred mice. We identified 62 lines with detectable aneuploid subpopulations and a subset of originally XX lines that lost one Chromosome X (XO). Strikingly, a much lower proportion of XX lines were aneuploid, compared to XY or XO lines. Two single-cell RNA-seq data sets demonstrated that aneuploid XY DO mESC also show lower Chromosome X gene expression and that XY mESC accumulate higher aneuploid proportions in culture than isogenic XX lines. Possible mechanisms for this protective effect of X chromosome dosage include our discovery that the lines with two active X Chromosomes have a higher expression of 2-cell-like state genes, and differential expression of X-linked tumor suppressor genes associated with DNA damage response.HighlightsFirst genetic analysis of predisposition to aneuploidy in pluripotent stem cell culturesChromosomal regions duplicated in aneuploid mouse embryonic stem cells are syntenic with regions overrepresented in human pluripotent stem cell lines bearing recurrent genetic abnormalitiesX-Chromosome dosage strongly influences susceptibility to aneuploidy in mouse embryonic stem cells and to a lesser degree in human pluripotent stem cellsXX mouse embryonic stem cell lines show a higher proportion of cells in 2 cell-like state and higher expression of tumor suppressor genes associated with DNA damage response

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Revealing cell populations catching the early stages of the human embryo development in naïve pluripotent stem cells

Cited by 3 publications

References 70 publications

Hidden totipotency in naïve human pluripotent stem cell cultures

Hidden totipotency in naïve human pluripotent stem cell cultures

Proteogenomic Reprogramming to a Functional Human Totipotent Stem Cell State via a PARP-DUX4 Regulatory Axis

Chromosome X Dosage Modulates Development of Aneuploidy in Genetically Diverse Mouse Embryonic Stem Cells

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