EXTRAEMBRYO NIC MESODERM CELLS

FIELD OF THE INVENTION

The invention relates to methods for cultivating and isolating extraembryonic mesoderm stem cells.

INTRODUCTION

The extraembryonic mesoderm (EXM) is an important tissue with essential roles in development. EXM is implicated in primitive erythropoiesis and extracellular matrix formation, becomes an integral part of the amnion, yolk sac, allantois and chorion, and forms the primitive umbilical cord. Intriguingly, the mechanisms of EXM specification differ dramatically between species of mammals. In rodents, the EXM is specified only after gastrulation from the primitive streak. In primates however, the EXM starts to emerge earlier, before gastrulation, around Carnegie Stage 5 in humans. The EXM arises in close proximity to the epiblast, primitive endoderm (PrE) and trophoblast (TB) then spreads to line the inner surface of the cytotrophoblast (CTB) and the outer surface of the primitive yolk sac and amnion. It forms a connecting stalk between the CTB and the amnion, epiblast disc and PrE by day 13, which forms the primitive umbilical cord. Later on, extraembryonic mesoderm cells (EXCMs) fill chorionic villi. However, despite the importance of the EXM, our understanding of its cellular and molecular regulation in humans remains limited. Moreover, there are no in vitro models for primate EXM development [Ross & Boroviak (2020) Nat. Commun. 11, 3760]. An exciting prospect is the derivation of human in vitro extraembryonic mesoderm cells (extraembryonic mesoderm cells (EXCMs)) to model EXM development.

The lineage origin of the EXM in humans and other primates is unknown, and subject to considerable uncertainty, with multiple sources proposed [Pera and Rossant (2021) Cell Stem Cell 28, 1896-1906; Rossant & Tam (2022) Dev. Cell 57, 152- 165]. Early studies suggested that the EXM is derived from the trophoblast due to its location and emergence prior to formation of the primitive streak. Others suggested that the EXM originates from the early primitive streak, due to its appearance in a similar region of the epiblast. The EXM in mice and other species originates from the primitive streak at gastrulation. However, in primates, the EXM is found prior to primitive streak formation, therefore the primitive streak cannot be the only source of extraembryonic mesoderm cells (EXCMs). An epiblast origin has been suggested as cells expressing mesoderm genes align closely to epiblast cells in a monkey embryo scRNA-seq dataset, prior to the emergence of the primitive streak. EXM was also suggested to originate from PrE based on electron microscopy images, their shared gene expression with PrE and lineage tracing using mutations A combination of origins is also possible. The regulatory elements underlying EXM identity in humans are also unknown.

Human embryo development is difficult to study because it occurs in utero. Advances have enabled culturing human embryos ex utero up to 14 days [Deglincerti et al. (2016) Nature 533, 251-254; Shahbazi & Zernicka-Goetz (2018) Nat. Cell Biol. 20, 878-887; Shahbazi et al. (2016) Nat Cell Biol 18(6), 700-708; Xiang et al. (2020) Nature 577, 537-542; Zhou etal. (2019) Nature 572, 660-664]. However, obtaining human embryos for research remains a challenge due to extensive ethical and legal restrictions. As a result, our understanding of early human development remains limited. To fill this gap, a number of human stem cell-based embryo models have been developed to recapitulate specific stages of human embryogenesis. Naive human pluripotent stem cells (hPSCs) represent the preimplantation naive pluripotent epiblast and have the ability to differentiate into embryonic lineages as well as extraembryonic PrE and TB lineages, including human trophoblast stem cells (hTSCs) and amnion. [Castel et al. (2020) Stem Cells. Cell Rep. 33, 108419; Cinkornpumin et al. (2020) Stem Cell Reports 15, 198-213; Dong et al. (2020) Elife 9 e52504; Guo et al. (2021) Cell Stem Cell 28, 1040-1056; Io et al. (2021) Cell Stem Cell 28, 1023-1039; Karvas et al. (2022) Cell Stem Cell 29, 810-825; Linneberg-Agerholm et al. (2019) Development 146, devl80620; Rostovskaya et al. (2022) Cell Stem Cell 29, 744-759] However, whether naive hPSCs have the ability to form additional extraembryonic lineages such as the EXM is unknown.

Attempts to form human blastoids recapitulating aspects of blastocyst development and cellular composition have been made [Fan (2021) Cell Discov 7, 81; Kagawa et al. (2022) Nature 601, 600-605; Liu et al. (2021) Nature 591, 627-632; Yanagida et al. (2021) Cell Stem Cell 28, 1016-1022. e4; Yu et al. (2021) Nature 591, 620- 626: Zhao et al. (2021) Biorxiv May 7]. The power of such models to predict development depends on their ability to form cells reflecting the blastocyst stage. Blastoids generate different extents of 'off-target' cells depending on the initial cell state and molecules used to stimulate their formation. The lineage identity and developmental stages of the cells generated remain heavily debated and were proposed to correspond to postimplantation epiblast, primitive streak, amnion, mesoderm-like cells and extraembryonic mesoderm cells (EXCMs) in humans [Yanagida et al. (2021) cited above; Kagawa et al. (2022) Nature 601, 600-605; Zhao et al. (2021) cited above and to embryonic mesoderm or EXM in mouse [Posfai et a/. (2021) Cell Biol. 23, 49-60].

SUMMARY OF THE INVENTION

Here, we report the specification of extraembryonic mesoderm cells (EXMCs) from naive human pluripotent stem cells (hPSC) cultures. We surmise that modeling EXMC specification could help understand cell fate specification events in human periimplantation embryogenesis, defects of which may cause developmental failure. Our work demonstrates that naive hPSC cultures can differentiate into extraembryonic mesoderm cells (EXCMs), and establish a model that allows the study and manipulation of early human postimplantation development in vitro.

A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. extraembryonic mesoderm cells (EXCMs) are mesenchymal, can arise from an epiblast intermediate and are capable of self-renewal. Thus, extraembryonic mesoderm cells (EXCMs) arising via primatespecific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition [Kagawa et al. (2022) Nature 601, 600-605] correspond to extraembryonic mesoderm cells (EXCMs). Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects.

The invention relates to in vitro methods of differentiating human naive pluripotent stem cells into extraembryonic mesodermal cells (EXCM) comprising the steps of a) providing naive pluripotent stem cells (PSC), b) cultivating said naive PSC in a medium for the differentiation of PSC into trophoblast stem cells (TSC) ), or in a medium for the cultivation of TSC„ c) isolating from the cultivated cells in step b), those cells with a mesenchymal morphology.

Apart from the specific medium mentioned in the examples, other media used in the art for the conversion of cells into PSC or maintaining PSC in culture are equally considered. In these methods cells with a mesenchymal morphology, representing extraembryonic mesodermal cells (EXCM) are CDH1 (Cadherin 1) negative cells.

Herein CDH1 negative EXCM are separated from CHD1 positive trophoblast stem cells.

Further markers specific for extraembryonic mesodermal cells (EXCM) are mentioned in the detailed description and can be used to further characterize and/or purify EXCM.

Typically cell separation and isolation is performed by FACS.

As cells with EXCM properties appear from day 8 onwards, the cultivation in step b) can be performed for at least 8, at least 18 or at least 30 days.

Generally the medium is supplemented with EGF (Epidermal Growth Factor) and ITS- X (Insulin-Transferrin-Selenium-Ethanolamine). Omission of these supplements had the most impact on the generation of EXCM Typically the medium is supplemented with: -Bovine Serum Albumin (BSA), -Fetal Bovine Serum (FBS), -Penicillin-streptomycin, -Insulin-transferrin-selenium-ethanolamine, -L-ascorbic acid,

-an activin receptor-like kinase inhibitor, such as A83-01,

-a Transforming Growth Factor beta (TGF-beta) inhibitor, such as SB431542,

-human Epidermal Growth Factor (hEGF),

-a Glycogen Synthase Kinase-3 (GSK-3) inhibitor such as CHIR99021

-Valproic acid,

-p-mercapto-Ethanol,

-a Rock inhibitor such as Y-27632.

More specifically the medium is supplemented with:

-0.3% BSA,

-0.2% FBS,

-1% Penicillin-streptomycin,

-1% insulin-transferrin-selenium-ethanolamine- X 100 supplement,

-1.5 pg/ml L-ascorbic acid,

-0.5 pM A83-01,

-IpM SB431542,

-50ng/ml hEGF,

-2uM CHIR99021, -0.8 mM Valproic acid,

-0.1 mM [3-mercapto-EtOH, and

-5 pM Y-27632.

For alternative embodiment of the medium each of these supplements by differ from the above values by 5 or 10 % (e.g. BSA may range between 0.285 and 3.15 % or between 0.27 % and 3.3 %)

Typically the medium is DMEM/F12.

Alternatives described in the art for DMEM/F12 medium are RPMI 1640, MEM, IMDM, Ham's F-10 Medium, McCoy's 5A Medium.

After isolation of the extraembryonic mesodermal cells these cells can be further cocultivated with another cell type such as human trophoblast stem cells.

Another aspect relates to the use of the hereabove mentioned media for the cultivation of cell cultures comprising or consisting of human extraembryonic mesodermal cells.

The invention in another aspect relates to methods of isolating extraembryonic mesodermal cells (EXCM) from cell cultures comprising trophoblast stem cells (TSC), comprising the step of separating CHD-1 negative EXCM cells from CHD-1 positive TSC.

FIGURE LEGENDS

Figure 1. Derivation of extraembryonic mesoderm cells (EXCMs) from naive hPSCs.

A. Experimental strategy. Created with Biorender.

B. Bright-field microscopy images showing ICSIG-1 naive hPSCs and converted cells under ASECRiAV. Scale bar 500 pm.

C. IF for the indicated marker in PXGL and ASECRiAV. Scale bar 100 pm.

D. Flow cytometry contour plot of day 30 ASECRiAV cells analyzed for CDH1.

Microscopy images of naive hPSCs converted under ASECRiAV for 30 days and cells sorted for lack of CDH1.

Figure 2. Characterization of extraembryonic mesoderm cells (EXCMs).

A. Marker gene expression violin plots.

B. IF for the indicated markers in day 30 ASECRiAV cells. Scale bar 200 pm.

C. As in 2C for the indicated cell types. Scale bar 200 pm. Bottom: quantification. Nc=total nuclei count. D. IF for the indicated markers and cell types. Scale bar 100 pm.

E. IF for the indicated markers in a day 10 human embryo. Scale bar 100 pm.

F. BST2 flow cytometry of sorted CDH1- extraembryonic mesoderm cells (EXCMs).

G. IF for the indicated markers in day 30 ASECRiAV cells. Scale bar 200 pm.

Figure 3. Gene regulatory networks and scATAC-seq profiles of extraembryonic mesoderm cells (EXCMs).

A. Regulon activity for indicated TFs. Significant difference between regulon activity, Wilcoxon rank-sum test, *adjusted p<2*10' ¹⁶ , ** adjusted p=3.9*10' ⁸ ).

B. IF for the indicated markers in the indicated cell types. Scale bar 100 pm.

C. Dot plot of marker genes chromatin accessibility (scATAC-seq).

D. scATAC-seq motif enrichment.

Figure 4. Signaling pathways in extraembryonic mesoderm cells (EXCMs).

A. IF for the indicated markers in the indicated cell types. Scale bar 200 pm.

B. Number of extraembryonic mesoderm cells (EXCMs) grown for 5 or 10 days under either BMP4 inhibition (1 pM LDN-193189) or mTOR inhibition (1 pM GSK1059615). n = 2 experiments. Biological replicates are shown as individual data points.

Figure 5. Single-cell time course analysis

A. Experimental strategy. Image created with Biorender.

B. IF for the indicated markers during naive hPSC to ASECRiAV conversion. Scale bar 100 pm. Please note feeders have background staining for NR2F2 in D0-D4 images.

C. Quantification of B. n = two rounds of differentiation.

Figure 6: Origins of extraembryonic mesoderm cells (EXCMs).

A. Flow cytometry analysis of naive hPSCs (ICSIG-1).

B. Bright-field microscopy images of SUSD2+ and - ICSIG-1 naive hPSCs 24 hours after sorting and cultured back in PXGL (Top) and 8 days after switching to ASECRiAV (Bottom). Scale bar 500 pm.

C. IF for the indicated markers in day 12 ASECRiAV cells converted from SUSD2 sorted naive cells. Scale bar 100 pm.

D. Quantification of C. Nuclei counted from 5 random fields.

E. IF for the indicated markers in TROP2+/BST2- sorted ICSIG-1 hiPSCs, BST2+/TROP2- sorted ICSIG-1 hiPSCs and TROP2-/BST2- sorted cells at day 12 of ASECRiAV conversion. Scale bar 100 pm.

F. Quantification of E. Nuclei counted from 5 random fields.

G. As in E. in TROP2+/BST2- sorted ICSIG-1 hiPSCs, BST2+/TROP2- sorted ICSIG-1 hiPSCs and TROP2-/BST2- sorted cells at day 12 of ASECRiAV conversion. Scale bar, 100 pm. H. As in F but for G.

Figure 7. Blastoids contain EXM-like cells.

A-B. IF for the indicated markers after 96 hours of blastoid generation.

5 VI M+ structures indicated with white box (top left), enlarged at the bottom. Scale bar

200 pm. Non-cavitated (E, box) and cavitated structure (F, box).

C. Quantification of 1-B.

DETAILED DESCRIPTION

To study and manipulate molecular processes underlying cell fate specification after implantation, an in vitro model of human extraembryonic mesoderm development is needed. This model would not only enable us to study postimplantation functionally, but would also improve our understanding of human reproduction and diseases.

Here we report that naive hPSC cultures can differentiate into extraembryonic mesoderm cells (EXCMs) which transcriptionally resemble human and monkey embryo EXM. We submit that naive hPSCs could provide a useful model for EXMC specification, enabling the study of a major cell type of the early postimplantation primate embryo that arises in early postimplantation human embryogenesis.

Here we separated extraembryonic mesoderm cells (EXCMs) and hTSCs by sorting. Purified extraembryonic mesoderm cells (EXCMs) provide an ideal model for future studies investigating the function of extraembryonic mesoderm cells (EXCMs) in vitro. For example, extraembryonic mesoderm cells (EXCMs) could be used to investigate the ability of human EXM to contribute to the first wave of haematopoiesis in the human embryo. Co-culture of extraembryonic mesoderm cells (EXCMs) with other embryonic cell types will help understand the role that EXM plays during human embryogenesis.

The lineage origin of extraembryonic mesoderm cells (EXCMs) in human embryos is unknown. An epiblast origin of extraembryonic mesoderm cells (EXCMs) in human embryos remains to be tested but is supported by several lines of evidence. Human naive epiblast stem cells can differentiate into extraembryonic mesoderm cells (EXCMs) in vitro at least via an intermediate epiblast state. Primate extraembryonic mesoderm cells (EXCMs) first arise in close proximity to the epiblast at a time when the epiblast is in an intermediate state between pre-implantation naive epiblast and post-implantation primed epiblast. Recent monkey embryo scRNA-seq analyses suggest that extraembryonic mesoderm cells (EXCMs) arise from the epiblast [Yang et al. (2021) Nat. Commun. 12, 5126] and extraembryonic mesoderm cells (EXCMs) are specified from the epiblast in mice, although later in development [Saykali et al. (2019) Elife 8].

Recent work linked the extended epiblast peri-implantation period between the naive and primed epiblast states, which lasts 10 days in humans and 2 days in mice, to multiple distinct waves of amniogenesis [Rostovskaya et al. (2022) Cell Stem Cell 29, 744-759]. Similarly, it is possible that different waves of extraembryo mesogenesis (the development of EXM) exist in primates. Indeed, several human epiblast stem cell states appear to be competent for EXMC induction. Human extended pluripotent stem cells in monkey chimeras can give rise to extraembryonic mesoderm cells (EXCMs) [Tan et al. (2021) Cell 184, 3589]. Additionally, several studies have suggested that primed hPSCs may be competent to generate extraembryonic mesoderm cells (EXCMs) under certain conditions, although this remains to be strictly demonstrated [Tietze et a/. (2020) BioRxiv August 29;. Io et a/. (2021) Cell Stem Cell 28, 1023-1039; Markouli et al. (2021) Sci. Rep. 11, 8242; Simunovic et al. (2022) Cell Stem Cell 29, 962-972].

While the primate epiblast may have the capacity to activate the EXM developmental program, a TB origin has been proposed. Since extraembryonic mesoderm cells (EXCMs) were also obtained from TROP2+ cells in our experiments, a TB origin of extraembryonic mesoderm cells (EXCMs), although deemed unlikely in the embryo [Ross & Boroviak (2020) Nat. Commun. 11, 3760], cannot be excluded. On the other hand, there are several lines of evidence suggesting an origin of extraembryonic mesoderm cells (EXCMs) from the PrE [Bianchi et al. (1993) Am. J. Med. Gen. 46, 542-550]. Moreover, PrE conversion generated extraembryonic mesoderm cells (EXCMs). Although the intermediate epiblast route is also possible in these experiments, a PrE origin of extraembryonic mesoderm cells (EXCMs) cannot be excluded. The absence of PrE cells in ASECRiAV cultures suggest it is unlikely that PrE is the sole origin of extraembryonic mesoderm cells (EXCMs) in vitro. A combination of origins remains possible and understanding the origin and window of competence for EXMC differentiation in human development remains an exciting prospect for future studies.

We showed that VIM+ extraembryonic mesoderm cells (EXCMs) are present at low frequency (<0.02%) during the generation of human blastoids generated from triply- inhibited PXGL hPSCs. Others have shown that the starting cells and medium used to conduct blastoid specification strongly impacts the faithfulness of the model. Inadequacies in these parameters cause cells to follow an abnormal sequence of events, specify slowly and inefficiently and generate abnormal lineages and stages [Zhao et al. (2021) Biorxiv May 7]. Here we show the formation of putative EXM progenitors during the early steps of blastoid organization. A suboptimal blastoid medium or defects during blastoid formation could lead to the precocious activation or acceleration of EXMC specification. However, human blastoids may still be competent to initiate a bona fide postimplantation EXMC specification program later, when grown to postimplantation stages, which warrants future investigations.

Altogether, we have discovered that naive hPSC cultures can specify the EXMC fate, which provides a model to molecularly and functionally characterize EXM specification in vitro. This system is of particular interest given that in humans, EXM specification takes place after implantation and starts before gastrulation and is therefore inaccessible for experimentation. The induction and maintenance of extraembryonic mesoderm cells (EXCMs) from multiple naive hPSC lines will enable the study of EXM in culture and allow molecular, genetic and epigenetic manipulations, extraembryonic mesoderm cells (EXCMs) may also allow the development of improved integrated stem cell embryo models in combination with TB, epiblast and PrE lineage-derived cell types.

Derivation of extraembryonic mesoderm cells (EXCMs) from naive hPSCs.

We sought to derive hTSCs from naive hPSCs (PXGL) exposed to hTSC media ASECRiAV (Figure 1A) [Castel et al. (2020) Stem Cells. Cell Rep. 33, 108419; Dong et al. (2020) Elife 9 e52504; Okae et al. (2018) Cell Stem Cell 22, 50-63]. By day 30 of conversion, we observed colonies with hTSC morphology and GATA3 expression (Figure IB, 1C). These results suggested induction of hTSCs, as expected. Unexpectedly, another cell type with mesenchymal morphology and mostly lacking GATA3 expression was present in day 30 ASECRiAV cultures (Figure IB). We consistently obtained both hTSCs and the other cell type in all conversion attempts (>35 experiments) and the ratio of hTSCs to the other cell type varied. hTSCs, which express the epithelial marker CDH1, could be separated from CDH1- cells by fluorescence-activated cell sorting (FACS) (Figure ID). The CDH1- cells appeared to self-renew and expand for over 14 passages. Similar results were obtained with two other naive hPSC lines (H9 and WIBR2-MGT) grown in two different naive media. In summary, we found that differentiation of naive hPSCs using hTSC conditions gives rise to an unexpected, CDH1- mesenchymal cell type.

To establish the identity of all cell types obtained by ASECRiAV conversion and compare them to the human embryo, we applied single-cell RNA sequencing (scRNA- seq) on day 30 of conversion with naive and primed hPSCs as controls. We obtained the transcriptome of 629 single cells. Uniform manifold approximation and projection (UMAP) revealed 4 clusters, each corresponding to a specific cell type. To determine if cells correspond to cells of the human embryo, we integrated the scRNA-seq data with a reference human embryo atlas, which included datasets of preimplantation, postimplantation and a Carnegie Stage 7 human embryo ) as well as reference hPSCs. UMAP showed that cells arranged according to developmental progression; hPSCs and hTSCs overlapped with their embryo counterparts, namely epiblast and early postimplantation trophoblast, respectively. Unexpectedly, the undefined mesenchymal cells aligned mostly to the EXM of Carnegie Stage 7 human embryo, suggesting they reflect EXM. A few unidentified mesenchymal cells aligned to the PrE. We verified this analysis by integrating the scRNA-seq data with monkey embryo data which contains day 14 monkey EXM. Naive and primed hPSCs aligned with monkey epiblast, as expected. The unidentified mesenchymal cells aligned to the EXM which were annotated as extraembryonic mesenchyme cells by [Yang et al. (2021) Nat. Commun. 12, 5126]. Extraembryonic mesenchyme and extraembryonic mesoderm have been used interchangeably in the literature. Correlation analysis confirmed a high correlation of the mesenchymal cells with human and monkey embryo EXM. These results suggest that the unexpected, mesenchymal cell type obtained by differentiation of naive hPSC cultures are extraembryonic mesoderm cells (EXCMs).

In vitro extraembryonic mesoderm cells (EXCMs) recapitulate the gene expression profile of postimplantation human and monkey embryo EXM.

To determine if the gene expression profile of primate EXM in the embryo is recapitulated in vitro, we analyzed the expression of known marker genes for each cell type. Primed hPSCs expressed high levels of ZIC2 and CD24; naive hPSCs expressed KLF17, KLF4, DNMT3L and DPPA5; hTSCs expressed GATA2, GATA3 and KRT7. Importantly, in vitro extraembryonic mesoderm cells (EXCMs) expressed several primate embryo EXM marker genes, including POSTN, VIM and NID2. Most EXM marker genes were not expressed in primed and naive hPSCs nor hTSCs. Comparing in vitro extraembryonic mesoderm cells (EXCMs) to other in vitro and embryo data revealed that in vitro and embryo EXM are highly similar to each other and differ from other cell types. Although several EXMC genes such as VIM, LUM and POSTN mark the amnion, we found these genes to be more highly expressed in the EXM compared to the amnion (Figure 2A). Immunofluorescence (IF) imaging of day 30 ASECRiAV cultures and of extraembryonic mesoderm cells (EXCMs) confirmed the presence of VIM, LUM, POSTN, DCN, GATA4, GATA6 and absence of CDH1 in extraembryonic mesoderm cells (EXCMs), whereas hTSCs lacked VIM and expressed CDH1 (Figure 2B-2D). These results confirm that extraembryonic mesoderm cells (EXCMs) obtained by conversion from naive hPSC cultures transcriptionally match human and monkey embryo EXM, and express specific key proteins.

Extraembryonic mesoderm cells (EXCMs) did not represent another cell type, although there was partial overlap in gene expression with other cell types, extraembryonic mesoderm cells (EXCMs) lacked PrE marker FOXA2 and hTSC markers GATA2, TP63 and KRT7, but expressed SOX17, GATA4, GATA6 and PDGFRA (PrE), KRT18, HAND1 and NR2F2 (TSC). Extraembryonic mesoderm cells (EXCMs) did not express key mesoderm gene T/TBXT/BRACHYURY, in line with their pregastrulation origin and the lack of T expression in monkey embryo extraembryonic mesoderm cells (EXCMs), but unlike mouse extraembryonic mesoderm cells (EXCMs) which do express T/Brachyury due to their gastrulation origin. Multiple mesoderm markers including MESP1, GSC and EOMES were absent from extraembryonic mesoderm cells (EXCMs), indicating that extraembryonic mesoderm cells (EXCMs) do not represent embryonic mesoderm. Extraembryonic mesoderm cells (EXCMs) also did not express amnion marker genes such as ISL1, HEY1, CDH10 or CTSV.

Differential gene expression analysis between naive hPSCs and day 30 extraembryonic mesoderm cells (EXCMs) revealed 38 genes that were significantly increased in extraembryonic mesoderm cells (EXCMs) (>5.6 fold, p-value<0.05) including key human and monkey EXM genes such as VIM, POSTN, DCN, GATA4, LUM and H19. Gene ontology (GO) analysis of extraembryonic mesoderm cells (EXCMs) identified an enrichment of genes in the mesodermal commitment pathway.

In summary, these results suggest that exposing naive hPSC cultures to human hTSC culture conditions results in the induction of hTSCs as well as extraembryonic mesoderm cells (EXCMs), and that extraembryonic mesoderm cells (EXCMs) in vitro are similar to extraembryonic mesoderm cells (EXCMs) in early postimplantation primate embryos and distinct from other cell types. Altogether, we captured in vitro a primate-specific postimplantation human embryo cell type, making it accessible for experimentation.

Conversion of naive hPSCs to extraembryonic mesoderm cells (EXCMs) models an epithelial-to-mesenchymal transition

It has been proposed that in monkey embryos, extraembryonic mesoderm cells (EXCMs) are the first cells to undergo EMT during embryogenesis [Enders & King (1988) Am. J. Anat. 181, 327-340]. Thus, we sought to characterize epithelial-to- mesenchymal (EMT) transitions in extraembryonic mesoderm cells (EXCMs). extraembryonic mesoderm cells (EXCMs) showed mesenchymal morphology (Figure IB, 1C). As extraembryonic mesoderm cells (EXCMs) lost CDH1 expression, they gained expression of the mesenchymal marker CDH2. Extraembryonic mesoderm cells (EXCMs) expressed mesenchymal marker VIM (Figures 2A-2C) along with TWIST1, SNAI2 and ZEB2, which promote EMT. Trophectoderm (TE) maturation marker NR.2F2, which is expressed in extraembryonic mesoderm cells (EXCMs), also regulates expression of multiple key transcription factors (TFs) that promote EMT, such as ZEB1/2 and PRRX1. Enriched GO terms in extraembryonic mesoderm cells (EXCMs) included signaling for EMT, suggesting signaling might be implicated in EMT in extraembryonic mesoderm cells (EXCMs) We conclude that extraembryonic mesoderm cells (EXCMs) acquire features of mesenchymal cells consistent with an EMT.

Extracellular matrix gene expression in extraembryonic mesoderm cells (EXCMs).

A hallmark of extraembryonic mesoderm cells (EXCMs) in the early primate embryo and of stromal cells in general is production of extracellular matrix. We therefore analyzed the expression of key monkey and human embryo EXM extracellular matrix genes such as FN1, specific collagens (COL1A1, COL1A2, COL3A1, COL4A1, COL6A1 and COL6A3) and Laminins (LAMB1, LAMC1), which were increased in extraembryonic mesoderm cells (EXCMs) relative to naive hPSCs and to a large extent also to hTSCs. In addition, GO analysis identified 'Focal adhesion' and 'extracellular matrix receptor interactions' as top enriched pathways in extraembryonic mesoderm cells (EXCMs). Therefore, extraembryonic mesoderm cells (EXCMs) derived from naive hPSC cultures possess another hallmark of the EXM : the expression of a specific set of extracellular matrix genes.

BST2 as a cell surface marker of extraembryonic mesoderm cells (EXCMs) We aimed to identify EXMC cell surface markers by examining genes with differential gene expression between extraembryonic mesoderm cells (EXCMs) and hTSCs (adjusted p-value <0.01 and expression fold change >2). This identified 11 candidate cell surface genes, of which BST2 was chosen as it was also expressed in human embryo EXM, but not in most other embryonic cell types with the exception of epiblast and mesoderm. Flow cytometry for BST2 on extraembryonic mesoderm cells (EXCMs) revealed that 90.7% of extraembryonic mesoderm cells (EXCMs) were positive for BST2 (Figure 2F) which was further confirmed by IF (Figure 2G). Therefore, we identified BST2 as a cell surface marker of extraembryonic mesoderm cells (EXCMs).

Gene regulatory networks in extraembryonic mesoderm cells (EXCMs)

TFs control cell fate specification by binding to cis-regulatory regions, thus forming GRNs, yet the GRNs of human extraembryonic mesoderm cells (EXCMs) remain undefined. Therefore, we used single-cell regulatory network inference and clustering (SCENIC) analysis [Aibar et al. (2017) Nat. Methods 14, 1083-1086] to reconstruct GRNs and predict TFs with regulatory activity in extraembryonic mesoderm cells (EXCMs). SCENIC measures TF regulatory activity by combining expression of TFs and their candidate target genes which are co-expressed and have TF binding motifs. The expected regulons for KLF17, SOX11 and GATA3 were found active in naive, primed hPSCs and hTSCs, respectively. TFs recently shown to be essential regulators of human hTSC identity including ARID3A, GATA2 and ZNF407 had high regulatory activity in hTSCs, supporting the notion that the approach can identify critical regulators of cell identity. We detected high regulatory activity in extraembryonic mesoderm cells (EXCMs) for TFs including ARID5B, PLAGL1, CREB3L1, GATA4 and FOXF1 , in line with their reported expression or regulatory activity in monkey EXM. TWIST1, an important mesoderm regulator, also showed regulatory activity in extraembryonic mesoderm cells (EXCMs). Several HOX genes including HOXAIO, HOXA11, HOXA9, and HOXA13 had high activity in extraembryonic mesoderm cells (EXCMs) (Figure 3A), in line with the recently reported regulatory activity of HOXA11 in the monkey EXM. CREB3L1, which was previously reported as a human-specific epiblast factor , and has reported roles in extracellular matrix formation, was also active in extraembryonic mesoderm cells (EXCMs) (Figure 3A). We detected high activity of TFs shared between hTSCs and extraembryonic mesoderm cells (EXCMs) including HAND1 and NR2F2 (Figure 3A). The regulatory activity of NR2F2 was unexpectedly higher (adjusted p-value 3.9*10' ⁸ ) in extraembryonic mesoderm cells (EXCMs) than in hTSCs and naive hPSCs (Figure 3A, ). Surprisingly, although we could confirm the expression of NR2F2 in extraembryonic mesoderm cells (EXCMs) by IF, NR2F2 appeared more highly expressed at the protein level in hTSCs than in extraembryonic mesoderm cells (EXCMs) (Figure 3B). In summary, extraembryonic mesoderm cells (EXCMs) possess regulatory activity for a unique combination of TFs typically associated with TB, PrE and mesoderm as well as TFs expressed in human and monkey embryo EXM, providing a valuable resource for future studies. Single-cell chromatin accessibility profiling of extraembryonic mesoderm cells (EXCMs)

To further characterize extraembryonic mesoderm cells (EXCMs) and identify regulatory elements that may underlie EXMC identity, we performed single-cell ATAC sequencing (scATAC-seq). We obtained the chromatin accessibility landscape of 1133 cells comprising naive hPSCs, primed hPSCs, hTSCs and extraembryonic mesoderm cells (EXCMs) which clustered into 4 populations. 40314 peaks were called for the EXMC population, with 22444 being unique. scATAC-seq revealed that extraembryonic mesoderm cells (EXCMs) had high chromatin accessibility in the vicinity of most EXM marker genes (NID2, FOXF1, POSTN) and lacked chromatin accessibility at regions associated with several genes of alternative cell fates. TF motifs including HOXAIO, HOXA13, TWIST1, CREB3L1 and NR2F2 were found to be highly enriched in accessible chromatin of extraembryonic mesoderm cells (EXCMs), corroborating SCENIC analyses. Our results identify candidate cis-regulatory elements and TFs underlying the human EXM gene regulatory program. Of note, we also report as a resource single-cell epigenomic data for primed and naive hPSCs as well as hTSCs.

Contribution of ASECRiAV components to EXMC induction

We aimed to define the ASECRiAV components that are most important for EXMC induction. We removed ASECRiAV components individually or in combinations (SB431542/A83-01; ITS-X/hEGF), then induced EXMC conversion, and assessed induction by immunostaining for GATA4 and VIM at day 12. Despite high variability, all conditions contained GATA4/VIM double positive cells, suggesting that no single ASECRiAV component is strictly required for EXMC induction. However, ITS-X and hEGF removal decreased EXMC induction, especially when removed together. Insulin and the insulin-like growth factor activate the PI3/AKT and mTOR pathway, suggesting insulin might activate the mTOR pathway during EXMC induction, in addition to the recognized role of this pathway in human pluripotency. Other factors may also contribute to EXMC induction, although to a smaller extent. Collectively, these results suggest that ITS-X and hEGF are the most important factors in ASECRiAV for EXMC induction.

BMP4 and mTOR signaling in extraembryonic mesoderm cells (EXCMs)

We aimed to identify signaling pathways that maintain extraembryonic mesoderm cells (EXCMs). GO analysis revealed that the TGF-p superfamily and mTOR signaling pathways are enriched in extraembryonic mesoderm cells (EXCMs). TGF-p is surprising since ASECRiAV contains two TGF-p inhibitors, A83-01 and SB431542. However, these only target ALK4, 5 and 7 which are receptors activated by ligands of one branch of the TGF-p superfamily, namely ACTIVIN/NODAL/TGF-p. The other branch of the TGF-p superfamily is activated by the BMP signaling pathway, including BMP4, which acts through ALK1, 2, 3 or 6 and is not targeted by inhibitors in ASECRiAV. Accordingly, higher expression of BMP4 and of its downstream target genes ID2 and ID3 in extraembryonic mesoderm cells (EXCMs), both in vitro and in vivo as compared to naive hPSCs, suggest that this pathway is active in extraembryonic mesoderm cells (EXCMs). Several genes related to the mTOR pathway were also highly expressed in extraembryonic mesoderm cells (EXCMs), suggesting that the mTOR pathway is active.

Increased SMAD1/5/9 phosphorylation was validated by immunostaining in extraembryonic mesoderm cells (EXCMs) (Figure 4A). Collectively, these results show that the BMP4 and mTOR signaling pathways, but not the ACTIVIN/NODAL/TGF- p pathway, are active in extraembryonic mesoderm cells (EXCMs).

To determine whether the BMP4 and mTOR pathways are required for EXMC maintenance, we inhibited the BMP4 pathway using the inhibitor LDN-193189 (LDN), targeting receptors ALK1, 2, 3 and 6. extraembryonic mesoderm cells (EXCMs) were treated daily for 10 days. Western blot analysis confirmed a decrease of phosphorylated SMAD1/5/9 upon inhibition by LDN , which correlated with reduced EXMC growth (Figure 4A). These results show that the BMP4 pathway is needed for EXMC growth. To determine the effect of the mTOR pathway on EXMC maintenance, we inhibited the mTOR pathway using inhibitor GSK1059615 targeting PI3Ko/P/6/y and mTOR. GSK1059615 inhibited cell growth and induced cell death (Figure 4A). Thus, the mTOR pathway is required for EXMC growth and survival. Altogether, we conclude that the BMP4 and mTOR pathways are implicated in EXMC maintenance with the monkey EXM.

Single-cell transcriptome analysis of differentiation kinetics

To create a differentiation trajectory, we used single-cell RNA-seq time course analysis. We collected samples at different timepoints during ASECRiAV conversion (Figure 5A). Cells were passaged on day 5, 10 and 15. We included sorted day 70 extraembryonic mesoderm cells (EXCMs). After quality control, we obtained data for 12977 cells with an average of 1622 cells per sample. UMAP analysis showed the presence of distinct populations at different times during conversion. At day 0, the majority of cells were naive hPSCs and expressed pluripotency genes, with a small fraction of cells corresponding to 8-cell-l ike-cells (8CLCs) marked by expression of 8- cell stage embryo genes. Naive cells were only detected at day 0. By day 1, most cells appeared to progress into an epiblast intermediate , marked by decreased expression of naive pluripotency genes DNMT3L and KLF4 and continued expression of several formative pluripotency genes such as DPPA2, GDF3, ZNF728 and ZNF729. This intermediate epiblast population formed 3 clusters, which we termed epiblast intermediate 1, 2 and 3, reflecting their progression during differentiation. hTSCs represented a low fraction of the cells up to day 4 but became the most abundant cell type at day 8. Their abundance declined at day 13 and was minimal by day 18. The first extraembryonic mesoderm cells (EXCMs) appeared on day 8 and clustered with extraembryonic mesoderm cells (EXCMs) from day 13 and day 18; we termed this cluster "early extraembryonic mesoderm cells (EXCMs)". In early extraembryonic mesoderm cells (EXCMs), pluripotency genes were silenced and EXMC genes LUM, NID2, FOXF1, VIM, and POSTN were activated. At day 18, the majority of cells were extraembryonic mesoderm cells (EXCMs). Intriguingly, day 70 extraembryonic mesoderm cells (EXCMs) isolated by FACS formed a distinct population which we termed "late extraembryonic mesoderm cells (EXCMs)”. These cells clearly expressed multiple EXMC genes such as LUM, NID2, VIM, and POSTN. Several genes including NID2, POSTN and NR.2F2 were more highly expressed in late extraembryonic mesoderm cells (EXCMs) compared with early extraembryonic mesoderm cells (EXCMs). Together, these results show that extraembryonic mesoderm cells (EXCMs) can be derived in vitro by day 8, and that their identity can be maintained for at least 70 days, with expression of EXMC marker genes increasing over time.

To relate these changes in cell identity to embryo development, we integrated the time course scRNA-seq data with our initial day 30 data and human embryo atlas. The integration UMAP showed good correspondence between in vitro cell types and the embryo. In particular, naive hPSCs reflected the naive epiblast, and intermediate epiblast cells aligned between the naive and primed epiblast. hTSCs reflected early TB, while early extraembryonic mesoderm cells (EXCMs) reflected embryo EXM. Embryo EXM generally showed high correlation with in vitro extraembryonic mesoderm cells (EXCMs) at all time points. Day 14 embryo EXM correlated best to day 8 and 13 in vitro extraembryonic mesoderm cells (EXCMs), while Carnegie Stage 7 embryo EXM (embryonic day 16-19) correlated best to day 18 in vitro extraembryonic mesoderm cells (EXCMs). These results suggest that in vitro EXMC differentiation follows a progression that resembles that of the embryo EXM, with earlier in vitro cells correlating better with the earlier ex vivo embryo time point and later in vitro cells correlating better with later embryo stages. As the day 70 extraembryonic mesoderm cells (EXCMs) clustered separately from the other in vitro extraembryonic mesoderm cells (EXCMs) and embryo EXM, they may represent an as yet unstudied later stage of EXM development.

The scRNA-seq experiments above suggested that acquisition of the EXMC fate is a sequential process, marked by gradual acquisition of gene expression. While early extraembryonic mesoderm cells (EXCMs) activated the expression of GATA4, VIM, ANXA1, COL4A1, COL4A2, and BMP4, late extraembryonic mesoderm cells (EXCMs) showed higher expression of POSTN and PTX3 compared to early extraembryonic mesoderm cells (EXCMs) and appeared to show increased NR2F2 expression. Time course IF analysis confirmed the sequential activation of GATA4, then VIM and finally NR2F2 and the absence of VIM+/GATA4- cells during naive to ASECRiAV conversion (Figure 5A, 5B). Collectively, these results provide insights into the sequence and progression of EXMC differentiation starting from naive hPSCs.

Origin of extraembryonic mesoderm cells (EXCMs)

To determine the origin of extraembryonic mesoderm cells (EXCMs), we first investigated whether extraembryonic mesoderm cells (EXCMs) arise from predifferentiated cells in naive hPSC cultures by sorting SUSD2+ cells, which enriches for naive hPSCs, and SUSD2- cells, to enrich pre-differentiated cells from naive hPSC cultures (Figure 6A, 6B). After replating in PXGL for 24h then switching to ASECRiAV for 12 days, both populations gave rise to extraembryonic mesoderm cells (EXCMs) (Figure 6C, 6D). Hence, extraembryonic mesoderm cells (EXCMs) do not appear to preferentially arise from SUSD2- pre-differentiated cells in naive hPSC cultures.

PrE cells were absent in the ASECRiAV time course so in vitro extraembryonic mesoderm cells (EXCMs) are unlikely to be specified via a PrE intermediate. In the embryo, the EXM appears several days after the 8-cell stage. Thus, a direct 8CLC to EXMC origin is unlikely and would be devoid of developmental logic, although it cannot be excluded.

In the scRNA-seq time course, naive hPSCs were seen only at day 0, while extraembryonic mesoderm cells (EXCMs) were detected starting at day 8. Hence, extraembryonic mesoderm cells (EXCMs) likely do not originate directly from naive hPSCs. Instead, extraembryonic mesoderm cells (EXCMs) may arise from an intermediate cell state between naive and EXMC states. Intriguingly, at day 4 of the time course, the majority of cells possessed an intermediate epiblast state. These results suggest that intermediate epiblast cells might be a source of extraembryonic mesoderm cells (EXCMs).

To test the ability of intermediate epiblast cells to give rise to extraembryonic mesoderm cells (EXCMs), we used cell surface marker BST2 (Figure 2F, 2G), which is also expressed in intermediate epiblast cells prior to EXMC specification. We also used the TB lineage marker TROP2. We sorted cells at day 6 of conversion and grew these for another 6 days in ASECRiAV. BST2+/TROP2- epiblast intermediate cells gave rise to extraembryonic mesoderm cells (EXCMs), indicating that epiblast intermediate cells are competent to differentiate into extraembryonic mesoderm cells (EXCMs) (Figure 6E, 6F). hTSCs were also obtained (Figure 6G, 6H). These results suggest that epiblast intermediate cells are not irreversibly committed to the embryonic epiblast lineage but instead are competent to differentiate into extraembryonic cell types including extraembryonic mesoderm cells (EXCMs) and hTSCs.

BST2-/TROP2+ cells isolated at day 6 of differentiation and grown further into ASECRiAV were enriched for cells that form hTSCs, as expected (Figure 6G, 6H). However, extraembryonic mesoderm cells (EXCMs) were also obtained (Figure 6E, 6F). These results suggest that BST2-/TROP2+ cells at day 6 of differentiation may not yet be irreversibly committed to the TB lineage. However, in scRNA-seq data, we detected a small population of intermediate epiblast cells expressing TROP2, hence extraembryonic mesoderm cells (EXCMs) may arise from a few BST2-/TROP2+ epiblast intermediate cells. BST2/TROP2 double negative cells also gave rise to both extraembryonic mesoderm cells (EXCMs) and hTSCs (Figure 6E-6H). Intriguingly, BST2/TROP2 double positive cells died and did not give rise to extraembryonic mesoderm cells (EXCMs) or hTSCs (Figure 6E-6H). Collectively, these results suggest that epiblast intermediate cells marked by TROP2+ or BST2+ are both competent to differentiate into extraembryonic mesoderm cells (EXCMs) and hTSCs. We propose a model in which naive hPSCs give rise to epiblast intermediate cells from which extraembryonic mesoderm cells (EXCMs) originate.

Human naive pluripotent stem cell to PrE conversion

Several reports suggested a PrE origin of extraembryonic mesoderm cells (EXCMs). Thus, we tested if differentiation of naive hPSCs to the PrE fate induces extraembryonic mesoderm cells (EXCMs). We subjected naive hPSCs (PXGL) to RACL medium for 6 days to induce a PrE fate followed by an additional 24 days in NACL medium to induce a naive extraembryonic endoderm (nXEN) fate. To examine cell identity, we reconstructed single-cell transcriptomes at day 8 of RACL conversion and day 24 of NACL conversion. 593 and 75 cells passed quality control for RACL and NACL samples, respectively. The cells organized into 6 clusters. In addition to PrE cells, RACL conversion unexpectedly induced extraembryonic mesoderm cells (EXCMs). 'RACL extraembryonic mesoderm cells (EXCMs)' had low pluripotency gene expression and expressed EXMC genes including NID2, FOXF1, VIM, ANXA1 but not FOXA2 and LUM. By integration with human embryo and ASECRiAV conversion data they resemble embryo early extraembryonic mesoderm cells (EXCMs) and correspond to 'early extraembryonic mesoderm cells (EXCMs)' obtained by ASECRiAV conversion. Thus, unexpectedly, RACL conversion also induces the EXMC fate.

Interestingly, RACL also induced 3 'intermediate' populations, each of which were similar to the three corresponding epiblast intermediate populations obtained by ASECRiAV conversion, and expressed pluripotency genes P0U5F1, NANOG and SOX2, as well as formative genes DPPA2, GDF3, ZNF208 and ZNF729. These results raise the possibility that, in RACL media, extraembryonic mesoderm cells (EXCMs) also arise via an epiblast intermediate state, a PrE state, or both. In summary, we found that in addition to PrE-like cells, and extraembryonic mesoderm cells (EXCMs), RACL induces epiblast intermediate cells.

We next sought to test the cell types obtained after culture of RACL cells in NACL media reported to induce a nXEN state. Unexpectedly, most NACL cells comprised extraembryonic mesoderm cells (EXCMs), but this time the cells corresponded to 'late extraembryonic mesoderm cells (EXCMs)' with similar gene expression profile as obtained in the ASECRiAV experiments above. The extraembryonic mesoderm cells (EXCMs) found in both RACL and NACL media had correlated gene expression with both early and late extraembryonic mesoderm cells (EXCMs) found in ASECRiAV media and embryo EXM. These transcriptome analyses show that naive hPSCs grown in RACL and NACL generate several cell types, including extraembryonic mesoderm cells (EXCMs). As hTSCs were not present in RACL culture and PrE was not present in ASECRiAV culture, this further supports that extraembryonic mesoderm cells (EXCMs) can be generated from an epiblast intermediate.

Extraembryonic mesoderm cells (EXCMs) and human blastoids

Attempts to form human blastoids have generated different extents of nonblastocyst-stage cell types, which would impair the potency of embryo models to predict development. We wondered whether non-blastocyst-stage cell types might correspond to extraembryonic mesoderm cells (EXCMs), and re-analyzed data from human blastoids. Clustering of fully developed blastoids (96 hours post-induction) with primed and naive hPSCs revealed 6 distinct populations of cells. We then integrated datasets of fully developed blastoids and human embryos, along with in vitro extraembryonic mesoderm cells (EXCMs). We found that 1.6% (15/920) of the cells in fully developed blastoids did not align with the blastocyst-stage but rather with embryo EXM and in vitro extraembryonic mesoderm cells (EXCMs). These nonblastocyst-stage cells expressed key EXM marker genes and had reduced expression of marker genes of alternative lineages including epiblast, PrE, TB, and mesoderm. The majority of these non-blastocyst-stage cells corresponded to extraembryonic mesoderm cells (EXCMs) (93%, 14/15 cells), while 7%, (1/15 cells) also expressed amnion markers and may reflect amion cells. These results suggested that human blastoids form <2% non-blastocyst-stage cells that, in majority, are extraembryonic mesoderm cells (EXCMs). Analysis of blastoids generated through another method [Yu et al. (2021) Nature 591, 620-626] identified 3% of cells as EXMC-like cells, while we identified 9% of cells as EXMC-like cells in iBIastoids. Both of these other datasets included an additional 45% of cells not aligning to any of the four cell types examined here (blastocyst-stage epiblast, PrE, TB and EXM).

To investigate the origin of extraembryonic mesoderm cells (EXCMs) in blastoids, we analyzed earlier time points in blastoids. We found that 1.4% (2/139) of cells in the initial naive hPSC culture clustered with extraembryonic mesoderm cells (EXCMs) , either expressing PITX1 or PITX1 and NID2, but importantly lacking expression of most other EXMC marker genes, including VIM, with both cells expressing amnion markers, including ISL1. These pre-differentiated cells are therefore not mature extraembryonic mesoderm cells (EXCMs), but still may contribute to the population of extraembryonic mesoderm cells (EXCMs) we found in fully formed (96 hours) blastoids.

We then examined the presence of EXMC-like cells during the course of blastoid formation. We observed cells which aligned with extraembryonic mesoderm cells (EXCMs) at 24 hours (2%, 4/200 cells), and 60 hours (7.2%, 30/418 cells) of blastoid formation. Thus, EXMC-like cells became progressively more abundant during early blastoid formation but remained rare in fully developed blastoids (1.6%). Importantly, contrary to cells harvested from fully formed blastoids (the latter accounting for ~75% of the total number of structures), cells harvested at 24 and 60 hours also included the 25% of structures that will not form blastoids. Given the increased proportion of EXMC-like cells at 60 hours over 96 hours, this raised the possibility that EXMC-like cells might appear preferentially in the ~25% of structures that do not form blastoids. Because the EXM arises after implantation, we conclude that the presence of rare VIM+ EXMC-like cells in a blastocyst model is inappropriate. Altogether, we propose that understanding human EXMC specification will help to improve stem cell-based embryo models and enable us to gain insights into mechanisms of early human embryogenesis.

Examples

Example 1. Human primed pluripotent stem cell culture

Human primed pluripotent stem cells (H9 hESCs (WiCell#WA09), Sigma hiPSCs (Sigma#iPSC EPITHELIAL-1-IPSC0028, ICSIG-1) and WIBR2 29M-GP26-TN9 hESCs [Theunissen et al. (2016) Cell Stem Cell 19, 502-515] were grown with or without feeder conditions as per the cell lines, at normoxia conditions (5% CO2) and under humidified conditions at 37°C. In feeder-free conditions cells were cultured in precoated geltrex tissue culture treated plates in complete E8Flex medium (Stem cell technology). Cells were dissociated into smaller clumps every 5-6 days by incubating 5 min at room temperature in Versene. In feeder-dependent conditions primed hPSCs were grown on gelatin coated mitomycin -treated mouse embryonic fibroblast (MMC- MEF) feeders in human knockout serum replacement (KSR) primed medium containing 77.5% of DMEM/F12 (Gibco, 31330-038), 15% FBS (Gibco, 10270106), 5% KSR (Gibco, 10828028), non-essential amino acid (Gibco, 11140050), 2mM L- glutamine (Gibco, 25030081), 1% Penicillin-streptomycin (Gibco, 15140-122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), [3-mercapto-EtOH (Gibco, 31350- 010) and adding lOng/ml FGF2 (Peprotech) freshly everyday. Cells were passaged every 6-7 days using a 20 min incubation in Collagenase, (ThermoFisher 17104019). Media was changed every day.

Example 2. Human naive pluripotent stem cell culture

Naive hPSCs (H9 hESCs, WIBR2-MGT (converted from WIBR2 29M-GP26-TN9 hESCs) and Sigma hiPSCs) were cultured on MMC-MEF feeders in 5% 02 and 5% CO2 incubator under humidified conditions at 37°C. All naive hPSCs were cultured in PXGL medium [Bredenkamp et al. (2019) Stem Cell Reports 13, 1083-1098] which consists of 1 : 1 DMEM/F12 and Neurobasal, 0.5% N2-supplement, 1% B27- Supplement, 2 mM L-Glutamine, 0.1 mM [3-mercaptoethanol, 1% penicillinstreptomycin (Gibco, 15140-122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), 1 pM PD0325901 (Axon Medchem), 2 pM XAV939 (Sigma-Aldrich, X3004), 2 pM G66983 (Tocris, 2285), 20 pg/mL human LIF (PeproTech, 300-05) and 10 |jM Y-27632 (Tocris, 1254). Naive hPSCs were passaged every 4-5 days in a ratio 1 :2 or 1 :3 by single-cell dissociation with Accutase (Sigma-Aldrich, A6964-100ML) followed by filtering through a 40 pm cell strainer (Corning, 352340).

Example 3. Human primed to naive conversions

KLF4 mRNA Conversion

Starting from day 1 or day 2 after seeding primed hPSCs in E8 onto Geltrex, cells were lipofected daily with KLF4 mRNA (Miltenyi, 130-101-115) for 9 days [Liu et al. (2017) Nat. Methods 14, 1055-1062]. Per well of a 6-well plate, 2 pl KLF4 mRNA were diluted in 250 pL Opti-MEM (Gibco, 31985- 047) and 6 pl of lipofectamine RNAimax (Invitrogen, 13778075) in another 250 pL of Opti-MEM. Then the diluted mRNA was added to the diluted lipofectamine and incubated for 20 minutes at room temperature. After medium was exchanged to 1.5 mL fresh E8 flex, the mixture was added dropwise. After 10 days of lipofection, cells were passaged with Versene onto MMC-MEF feeders in E8 flex and transfected again. Starting from the following day, the medium was switched to t2iLG6 supplemented with Y-27632 (Tocris, 1254) and cells were transferred to hypoxia, while the lipofection was repeated every day for another 5 days. The naive t2iLG6 medium contains a 50: 50 mixture of DMEM/F12 (Gibco, 31330-038) and Neurobasal medium (Gibco, 21103-049), supplemented with 2 mM L-glutamine (Gibco), 0.1 mM [3-mercaptoethanol (Gibco, 31350-010), 0.5% N2 supplement (Gibco, 17502-048), 1% B27 supplement (Gibco, 17504-044), 1% Penicillin-streptomycin (Gibco, 15140-122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), 10 pg/ml human LIF (PeproTech, 300-05), 250 M L-ascorbic acid (Sigma-Aldrich, A4544-100G), 10 jg/ml recombinant human insulin (Sigma, 19278- 5ML), 1 M PD0325901 (Axon Medchem, 1408), 1 M CHIR99021 (Axon Medchem, 1386), 2.5 IJM G66983 (Tocris, 2285) [Liu et al. (2017) Nat. Methods 14, 1055- 1062]. After a total number of 15 days of transfection, cells were passaged with Accutase (Sigma-Aldrich, A6964-100ML) on the following day onto fresh feeders and continued to be cultivated in PXGL.

5iLA conversion

To convert primed hPSCs to naive hPSCs, trypsinized primed hPSCs were seeded onto a gelatin coated MMC-MEF feeders tissue culture treated 6 well plates and cultured with human KSR primed medium along with 10 pM of Y-27632 (Tocris, 1254) in a humidified normoxia (5% CO2) for 2 days. On the 3rd day and after giving a wash with Phosphate-buffered Saline (PBS (Gibco, 10010-015)) the medium was changed to 5iLA medium composed of 1 : 1 DMEM/F12 (Gibco, 31330-038) and Neurobasal (Gibco, 21103-049), 1% N2-supplement (Gibco, 17502-048), 2% B27 supplement (Gibco, 17504-044), 20 g/ml recombinant human LIF (PeproTech, 300-05), 2mM L- glutamine (Gibco, 25030-081), 1% non-essential amino acid, 0.1 mM [3- mercaptoethanol (Gibco, 31350-010), 1% Penicillin-streptomycin (Gibco, 15140- 122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), 50 pg/ml BSA (Sigma- Aldrich, A3059) and supplemented with 5 inhibitors: PD0325901 (Stemgent, 1 pM), IM-12 (Enzo, 1 pM), SB590885 (R&D systems, 0.5 pM), WH-4-023 (A Chemtek, 1 pM), Y-27632 (Tocris, 10 pM), and Activin A (Peprotech, 20 ng/ml) and grown in a humidified incubator in hypoxia condition (5% CO2 and 5% 02) at 37°C. After an initial wave of cell death around day 10-13, dome-shaped naive colonies started appearing. These cells were passaged into single cells every 4-5 days using 5 min incubation in Accutase (Sigma-Aldrich, A6964-100ML) at 37°C.

For some experiments, H9 and WIBR2-MGT naive hPSCs which were derived and cultured in 5iLA conditions were switched at passage 12 into PXGL naive medium for stable maintenance and expansion.

Example 4. Mouse feeders

MEFs were isolated from E14.5 pregnant WT C57/Black 6 mice. Male embryos were selected based on sex genotyping PCR and immortalized with Mitomycin C (Bioconnect). MEFs were cultured and harvested in a humidified incubator at 37°C and in 5% CO2 by using filter sterilized MEF medium consisting of 90% of DMEM supplemented with 10% FBS, 1% Glutamax, 1% Penicillin-streptomycin (Gibco, 15140-122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), lx non- essential amino acid and 0.1 mM p-mercaptoethanol and on 0.1% gelatin-coated tissue culture treated plates.

Example 5. Naive human pluripotent stem cells to trophoblast and EXMC fate conversion

Human naive to trophoblast and EXMC conversions were done using the following previously described protocols for hTSCs [Cinkornpumin et al. (2020) Stem Cell Reports 15, 198-213; Dong et al. (2020) Elife 9, e52504; Guo et al. (2021) Cell Stem Cell 28, 1040-1056; Io et al. (2021) Cell Stem Cell 28, 1023-1039]. Naive hPSCs were seeded in such a way that expecting at least 90% confluency by Day 2 on MMC-MEFs after dissociating in single-cell using TryplE (for 15 min at 37°C) in their respective naive culture conditions supplemented with lOpM Y-27632 (Tocris, 1254). The very next day, after a wash with PBS (Gibco, 10010-015), the media was switched from naive to ASECRiAV medium [Okae et al. (2018) Cell Stem Cell 22, SO- 63] consisting of DMEM/F12 (Gibco, 31330038) supplemented with 0.3% BSA (Sigma, A3059), 0.2% FBS (Gibco, 10270-106), 1% Penicillin-streptomycin (Gibco, 15140-122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), 1% insulin- transferrin-selenium-ethanolamine-X 100 supplement (Gibco, 51500056), 1.5 pg/ml L-ascorbic acid (Sigma, A8960), 0.5 pM A83-01 (Peprotech, 9094360), IpM SB431542 (Axon Medchem, 1661), 50ng/ml hEGF (Miltenyi Biotec, 130-097-750), 2uM CHIR99021 (Axon Medchem, 1386), 0.8 mM Valproic acid (Sigma, V0033000), 0.1 mM [3-mercapto-EtOH (Gibco, 31350-010) and 5 pM Y-27632 (Tocris, 1254). The medium was changed every two days and supplemented with 5 pM Y-27632. From passage 1 onwards both hTSCs and extraembryonic mesoderm cells (EXCMs) were cultured and maintained on 5 pg/ml Collagen IV coated tissue culture treated plates in hypoxia conditions (5% 02 and 5% CO2) and passaged every 5 days at 1 :3 or 1:6 splitting ratios. Collagen IV coated cell culture plates were coated overnight at 37°C. extraembryonic mesoderm cells (EXCMs) and hTSCs used in all experiments were always cultured and maintained in ASECRiAV medium unless otherwise specified.

Example 6. Naive human pluripotent stem cells to PrE and nXEN fate conversion

Human naive to PrE and nXEN conversions were done using a previously described protocol with minor adaptations [Linneberg-Agerholm et al. (2019) Development 146, devl80620]. Naive human PSCs grown in PXGL on feeders were seeded at a high density (split ratio of 1 : 1 or 2: 1) directly in RACL medium, which is made of Roswell Park Memorial Institute 1640 medium (RPMI; Gibco, 218750-34) supplemented with 1 X GlutaMAX, 1 X B27 minus insulin (Gibco, A18956- 01), 1 % (v/v) Pen/Strep (Gibco, 15140-122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), 100 ng/mL Activin A (PeproTech, 120-14E), 3 pM CHIR99021 and 10 pg/ml recombinant human LIF onto fresh MMC-MEF feeders in hypoxia. Cells were kept in RACL medium for at least 7-8 days and the medium was refreshed every day. After PrE cells reached confluency, they were dissociated with TrypLE or Accutase and re-plated onto fresh MMC-MEF feeders at a ratio of 1:2 to 1 :4 in NACL medium, which is composed of 1: 1 DMEM/F12 and Neurobasal, 0.5% N2-supplement, 1% B27- Supplement, 2 mM L-Glutamine, 0.1 mM [3-mercaptoethanol, lx penicillinstreptomycin (Gibco, 15140-122, 10,000 units Penicillin/mL, 10,000 pg Streptomycin/mL), supplemented with 100 ng/mL Activin A, 3 pM CHIR99021, 10 pg/ml recombinant human LIF and lOpM Y-27632 [Linneberg-Agerholm et al. (2019) Development 146, devl80620]. These nXEN cells were subcultured every 4-7 days with TrypLE or Accutase.

Example 7. Method details

Immunofluorescence and microscopy

Immunofluorescence staining was performed as described previously [Pasque et al. (2014) Cell 159, 1681-1697]. Cells were grown on 0.1% gelatinized 18 mm round coverslips with or without feeders. The next day cells were fixed in 4% paraformaldehyde-PBS for 10 min at room temperature in the dark and permeabilized with 0.5% Triton X-100 in PBS for 10 min and washed twice with 0.2% Tween20 in PBS (PBST) for 5 min each before proceeding to the staining. After this step, cells were either stored at 4°C or directly subjected to staining. Primary and secondary antibodies were diluted in a blocking buffer containing mainly PBST with 5% normal donkey serum and 0.2% fish skin gelatin. Cells on coverslips were incubated at 4°C with the specific primary antibodies in blocking solutions (1: 100 dilution for most antibodies, 1 :50 dilution for NANOG, 1 :40 dilution for FOXA2), after that washed three times with PBST each 5 min. After that it was incubated with the appropriate corresponding fluorophore conjugated secondary antibodies in blocking buffer (1:500 dilution) for Ih in the dark, washed again 3 times with PBST 5 min each, washed with 0.002% DAPI (Sigma-Aldrich, D9542) solution in PBST. The coverslips were mounted in Prolong Gold reagent with DAPI after a final wash in PBST. Mounted coverslips were kept at room temperature in the dark overnight before imaging. All immunofluorescence images were taken in a Zeiss Axioimager Al inverted microscope with an AxioCam MRc5 camera and processed in ImageJ. Bright field images were taken using a Nikon Eclipse Ti2 microscope and analyzed using ImageJ software.

Flow cytometry hPSCs were dissociated using Accutase (Sigma-Aldrich, A6964-100ML) into single cells by incubating 5 min at 37°C. Before proceeding the antibody staining, cells were washed 2 times with FACS buffer containing 1% BSA in PBS (Gibco, 10010-015). Fluorophore conjugated antibodies were diluted at a ratio of 1 :50 which is 1 pL of antibody in 50 pL of FACS buffer for around 50000 to 100000 cells, and incubated at 4°C in the dark at least for 30 min. Cells were washed again with FACS buffer and passed through a 40 pM cell strainer (Corning, 352340) and analyzed using a BD influx. Single stained controls were used for compensation and setting up the precise and stringent gate in the flow cytometer. Single-cell RNA sequencing

Cell Preparation

Day 30 cells were washed with PBS (Gibco, 10010-015), dissociated from culture dishes using Accutase (Sigma-Aldrich, A6964-100ML) (7 min. at 37°C) in hypoxic condition for naive hPSCs and hTSCs and in normoxic condition for primed hPSCs, and finally diluted with DMEM/F12 (Gibco, 31330-038). Single-cell suspensions were filtered through a 40pm cell strainer (Corning, 352340), centrifuged at 200 ref for 5 min., resuspended in PBS with 0.04% Bovine Serum Albumin (BSA) (Sigma Aldrich, A9418-50G) (1000 cells/pl).

Cells were collected at day 0, 1, 2, 4, 8, 13 and 18 during the time course ASECRiAV conversion. During the conversion cells were passaged at day 5, 10 and 15. Day 70 extraembryonic mesoderm cells (EXCMs) which were generated with an independent conversion and isolated by FACS were also included. Cells were washed with PBS, treated with Accutase (10 min. At 37°C, hypoxia), and diluted with DMEM/F12. Single cell suspensions were filtered through a 40pm cell strainer, and centrifuged (200 ref, 500min.). Cells before the first passage (day 0, 1, 2 and 4) were depleted from feeders, while cells after the first passage (day 8, 13 and 18) and at day 70 were immediately resuspended in resuspension buffer (PBS, 1% BSA). For feeder depletion, cells were resuspended and plated on a gelatine-coated plate for 35 min. in PXGL medium for cells at day 0 or in ASECRiAV medium for cells at day 1, 2 and 4, collected and centrifuged (200 ref, 500min.), and resuspended in resuspension buffer. Single cells in resuspension buffer were centrifuged (200 ref, 5min.) and resuspended again in resuspension buffer (1000 cells/pl). Finally, cells were filtered with the Flowmi 40pm tip strainer (Bel-Art, H13680-0040).

Cells collected at day 6 of the RACL conversion and at day 24 of the NACL conversion, were washed with PBS, treated with Accutase (15 min. at 37°, hypoxia), and diluted with DMEM/F12. Single cell suspensions were filtered through a 40pm cell strainer, centrifuged at 200 ref for 5 min., resuspended in resuspension buffer (PBS, 1%BSA), centrifuged (200rcf, 500min.), and finally resuspended in resuspension buffer (1000 cells/pl) and filtered with a Flowmi 40pm tip strainer. All cells were counted with the Luna-FL automated Fluorescence Cell Counter (Logos Biosystems).

Library preparation and sequencing

Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) targeting 2000, 4000 or 5000 cells (Next GEM Single Cell 3' library and Gel Bead Kit v3.1) according to the manufacturer's protocol (lOx User Guide; CG000204, Revision D). Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT cleanup, complementary DNA amplification and library construction were all performed according to the manufacturer's protocol. Individual sample quality was assessed using a Tapestation (Agilent). Qubit 2.0 (ThermoFisher Scientific) and KAPA Library Quantification Kit for Illumina Platform (KAPA Biosystems) were used for library quantification before pooling. The final library pool was sequenced on a NovaSeq6000 (Illumina) or NextSeq2000 (Illumina) instrument using NovaSeq SP reagent kit vl.5 (Illumina, 20028401) or NextSeq 1000/2000 P3 kit v3 for 2 lanes of 100-base-pair paired-end reads, or NextSeq 1000/2000 P2 kit v3 for 1 lane of 100-base-pair paired end reads.

Single-cell RNA-seq analysis

Raw sequence reads were quality-checked using the FastQC software. The CellRanger version 4.0.0 was used to process, align and summarize unique molecular identifier (UMI) counts against the 10X Genomics pre-built human GRCh38 and mouse mmlO reference genome datasets (2020-A, July 7, 2020). Downstream analyses were performed in R using Seurat (v4.0.1). Human cells were retained and mouse cells were filtered out by adjusting the number of counts per cell (nCount_RNA) and the number of mapped genes per cell (nFeature_RNA) to only keep cells that were mostly mapped to the human GRCh38 (hg38) genome (nFeature_RNA > 100 and nFeature_RNA < 15000). Cells with more than 25% of mitochondrial counts were filtered out. The count matrix was normalized with Seurat global-scaling normalization method "LogNormalize" that normalizes the feature expression measurements for each cell by the total expression, multiplies this by a 10.000 scale factor, and log-transforms the result. Differential expression testing was performed with the FindMarkers function in Seurat based on the non-parametric Wilcoxon rank sum test applying the logFC threshold of averaged Iog2 FC > 0.25. A graph-based cell clustering approach was used to cluster cells with FindClusters function in Seurat.

Single-cell gene expression analysis of merged datasets

Single-cell RNA-seq datasets [Tyser et al. (2021) Nature 600, 285-289; Messmer et a/. (2019) Cell Rep. 26, 815-824; Petropoulos et al. (2016) Cell 165, 1012-1026; Zhou et al. (2019) Nature 572, 660-664] were integrated with the data generated within this study performed using Seurat v3 integration standard workflow [Stuart et al. (2019) Cell 177, 1888-1902; Butler et a/. (2018) Nat. Biotechnol. 36, 411-420]. Datasets were normalized and scaled before selecting the 2000 most variable genes. The FeaturePlot function was used to project individual gene expression on UMAP. Differential expression analysis was performed with the FindMarkers function based on the non-parametric Wilcoxon rank sum test applying the logFC threshold of averaged Iog2 FC > 0.25. Similar integrations were performed with blastoid data [Kagawa et al. (2022) Nature 601, 600-605] and with monkey data [Tan et al. (2021) Cell 184, 3589; Yang et al. (2021) Nat. Commun. 12, 5126].

Correlation coefficients were calculated on the basis of the top 2000 most highly variable genes across all data sets using corrplot v.0.92.

Gene regulatory network interference

GRNs were inferred using pySCENIC. First raw expression data were normalized by dividing feature counts of each cell by the total counts for that cell and multiplying by a factor of 10.000, followed by logi _p transformation. The normalized counts were used to generate the co-expression modules using GRNboost. Next, the RcisTarget package was used to assess target binding motif enrichment and create regulons with only genes containing a binding motif, where a regulon is a transcription factor and its target genes. Subsequently, AUCell was used to measure regulon activity. Here, AUCell used the area under the curve to calculate the enrichment of the regulon across the ranking of all genes in a particular cell, resulting in a matrix of the activity of each regulon in each cell. Downstream analyses were done using the Seurat package.

Single-cell ATAC-seq cell preparation and sequencing Cell lines samples collection and nuclei isolation

Cells were washed with PBS (Gibco, 10010-015), dissociated from culture dishes by Accutase (Sigma-Aldrich, A6964-100ML) (7 min. at 37°C) in hypoxic condition for naive hPSCs and hTSCs and in normoxic condition for primed hPSCs, and finally diluted with DMEM/F12. Single-cell suspensions were filtered through a 40pm cell strainer (Corning, 352340), centrifuged at 200 ref for 5 min., resuspended in PBS (Gibco, 10010-015) with 0.04% Bovine Serum Albumin (BSA), and counted with a NucleoCounter NC-100 (Chemometec). 100.000 to 1.000.000 cells were added to a 2-ml microcentrifuge tube and were centrifuged (300 ref, 5min at 4°C). The supernatant was removed without disrupting the cell pellet and lOOpI chilled lysis buffer (10 mM Tris-HCI (pH 7.4), 10 mM NaCI, 3 mM MgCI2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% digitonin and 1% BSA) was added and mixed by pipetting 10 times. The microcentrifuge tube was incubated on ice for 4 min and then 1ml chilled wash buffer (10 mM Tris-HCI (pH 7.4), 10 mM NaCI, 3 mM MgCI2, 0.1% Tween-20 and 1% BSA) was added and mixed by pipetting 5 times. Nuclei were centrifuged (500 ref, 5 min at 4°C) and the supernatant was removed without disrupting the nuclei pellet. Nuclei were resuspended in a chilled Diluted Nuclei Buffer (lOx Genomics; 2000153) at 610-1540 nuclei per pl based on the starting number of cells. The nuclei concentration was determined using a NucleoCounter NC-100 (Chemometec). Nuclei were pooled before loading onto the 10X Chromium using the following ratio (H9 primed hPSCs: Sigma primed hPSCs: H9 naive hPSCs: Sigma naive hPSCs: Sigma day 30 conversion = 1 : 1: 1 : 1 :2) and were immediately used to generate scATAC-seq libraries as described in the methods below.

Library preparation and sequencing scATAC-seq libraries were prepared according to the Chromium Single Cell ATAC Reagent Kits User Guide (lOx user guide; CG000168, Revision D). Nuclei were loaded onto the lOx Chromium Single Cell Platform (lOx Genomics) at a concentration targeting 2000 nuclei, according to the manufacturer's protocol. Nuclei transposition, generation of gel beads in emulsion (GEMs), barcoding, GEM cleanup, and library construction were performed with the Chromium Single Cell ATAC Reagent Kits (vl Chemistry) according to the manufacturer's protocol. Library Quality control and quantification was assessed using the Tapestation (Agilent) and Qubit 2.0 (ThermoFisher Scientific). The library was sequenced on a Nextseq500 (Illumina) instrument using the MID output kit (Illumina) (20024904).

Single-cell ATAC-seq analysis

Preprocessing of single-cell chromatin accessibility data was performed using the cellranger-atac version 1.2.0 pipeline (10X Genomics). Read filtering, alignment, cell and peak calling, as well as cell-by-peak count matrix generation were performed using the "count" option (cellranger-atac count) with default parameters against the 10X Genomics pre-built human GRCh38 and mouse mmlO reference genome datasets (GRCh38_and_mmlO Reference-1.2.0, November 21, 2019). Downstream analysis was performed using Signac. The count matrix was filtered for cells where at least 15% of all fragments fell within peaks, with less than 5% of fragments falling within blacklist regions, with less than 4% nucleosome signal, with at least 2% enrichment for transcription start sites, and peaks with a minimum of 2500 fragments and a maximum of 20000 fragments. Dimensionality reduction was performed with the RunUMAP function from the Seurat package. Cell clusters were identified using the FindNeighbors function with parameters "reduction = 'Isi', dims = 2:30"' and FindClusters function with parameters "algorithm = 3, resolution = 0.5". Upon initial clustering, cluster-specific peaks were called with MACS2 using the function CallPeaks with parameters "group. by = 'idents'". These peaks were used to generate a final Signac object which was used for all downstream analyses. Fragment counts were mapped to peaks using FeatureMatrix with parameters "process_n = 2000". The new count matrix was filtered for cells where at least 20% of fragments fell within peaks, with less than 0.8% of fragments falling within blacklist regions, with less than 2% nucleosome signal, with at least 2% enrichment for transcription start sites, and for peaks with a minimum of 1500 and a maximum of 10000 fragments. Clustering was performed on the new object as described above. Peaks were then filtered for mean accessibility, keeping all peaks with greater than 0.1 mean accessibility across cells. The Upset plot was generated using the UpSetR package. Motifs were mapped using the motifmatchr package and the JASPAR2018 database. Motifs were added using the AddMotifs function with default parameters. Motifs were mapped to all peaks uniquely accessible per cluster. FindMotifs was used to test enriched motifs, and peaks were controlled for length and GC content.

Accessibility scores were calculated per cell type by averaging the log normalized number of fragments between 2kb upstream of the transcription start site and the transcription termination site for each gene across each cluster. Motifs were enriched in the cluster-specific peaks of each cluster.

Quantification and statistical analysis

Statistical tests and data processing were performed in R (v4.0.3). Information on each statistical test and multi-testing correction used can be found in the result section and figure legends. For statistical tests on single cell RNA-seq experiments, one replicate was included per time point. Cells were filtered to keep only cells with between 100 and 15000 human genes expressed, and cells with <25% mitochondrial reads were kept. Mouse cells were also excluded as described in the scRNA-seq analysis section. Differential gene expression analysis was performed using the Seurat function FindMarkers based on the non-parametric Wilcoxon rank sum test, using cutoffs of adjusted p-value<0.05 and Iog2 fold change>0.25. Regulon comparisons used a Wilcoxon rank sum test, with Bonferroni adjusted p-values.

For this work more than 35 naive to ASECRiAV conversions were performed. All experiments have been repeated at least three or two times, with exceptions. Experiments were repeated three times for Figure 2F, 2G, Figure 3B and two times for all other figures except for Figure 6E, 6G which were performed once. Experiments were not blinded. No data were excluded.