A chemically defined system supports two distinct types of stem cell from a single blastocyst and their self‐assembly to generate blastoid

Abstract The pluripotent stem cells exist in a narrow window during early development and its derivation depends on intrinsic and extrinsic growth signalling in vitro. It has remained challenging to derive two or three distinct cell lines that are representative of blastocyst‐stage lineages from one preimplantation embryo simultaneously in a chemical defined condition. Therefore, it is desirable to establish a system by manipulating extrinsic signalling in culture to derive multiple types of stem cells from a single blastocyst. This study used a defined medium containing Activin A, WNT activator and LIF (ACL medium), enabling establishment of ACL‐ESCs and ACL‐XEN cells from one blastocyst. ACL‐blastoids were generated by suspending ACL‐ESCs and ACL‐XEN cells with ACL‐blastoid medium in three‐dimensional culture system. Lineage markers expression of ACL‐blastoids were performed by immunofluorescence. Our results indicate that ACL‐ESCs and ACL‐XEN cells derived from one blastocyst represent ICM and PrE lineages. Importantly, we obtained ACL‐blastoid from ACL‐ESCs and ACL‐XEN cells self‐aggregation, partially recapitulating early development and initiation of early implantation events. This study would not only provide ACL culture system for derivation and maintenance of two types of cell lines corresponding to ICM as well as PrE, but also reconstruct blastoids with them to deepen our understanding of early embryogenesis and widen insights into translational application of stem cells.


| INTRODUCTION
During the early stage of embryogenesis in mammals, the developmental potential of cells in the embryo is gradually restricted by a series of cleavages and early differentiation. During morula compaction, it is an initial cell fate commitment that outer cells segregate from the blastomeres as trophectoderm (TE), which surrounds the inner cell mass (ICM). Accordingly, a blastocyst is formed following Baojiang Wu and Zhiqing Yang have contributed equally to this study. cavitation. Prior to implantation, the inner cell mass generates epiblast progenitor cells and the bipotent extra-embryonic primitive endoderm (PrE), which differentiates into the endodermal lineages, parietal endoderm (PE) and visceral endoderm (VE). [1][2][3] Three representative stem cells derived from blastocysts can selfrenew, maintain and proliferate in vitro as ESCs, [4][5][6] TSCs 7,8 and XEN cells, 9 respectively. ESCs derive and maintain self-renewal and pluripotency properties via the addition of two small-molecule inhibitors of glycogen synthase kinase 3 and mitogen-activated protein kinase (2i), and leukaemia inhibitory factor (LIF). 6 Only ESCs have been confirmed through the gold-standard germline transmission test, and can result in the generation all body tissues. 10 TSCs are derived from trophoblasts and maintained in vitro in the presence of FGF4 and Heparin, while retaining their ability to differentiate into multiple cell types of the placenta. 7,11,12 XEN cells can be established and continuously passaged using the same TSC culture conditions or serum-containing medium, contributing to the extraembryonic endoderm cell types. 13,14 All three cell lineages are maintained indefinitely in their particular cell culture system as stable cell lines with essential signal requirements. The trophectoderm is specified due to compaction during morula and is restricted in its developmental potential with the expression of genes related to trophectoderm, which is distinct from pluripotent ESCs, the corresponding cell lineages of ICM in vitro. However, ICM 15 and ESCs 16 still can partially differentiate into trophoblast lineages.
The trophectoderm can be induced via the ectopic expression of the caudal-type homeobox transcription factor 2 (Cdx2) in ESCs. The overexpression of Gata4 or Gata6 in ESCs is sufficient to induce the establishment of self-renewing XEN cells, which are the in vitro counterparts of the PrE lineage of the mouse embryo. 17,18 In fact, XEN-like cells have been found to exist within ESCs cultured on XEN protocol. 4,19 Human or mouse XEN cells can be derived from ESCs via the addition of the endoderm agonist, Activin and WNT, and expand in the presence of LIF and low insulin without the requirement for gene manipulation. 20,21 Similarly, retinoic acid (RA) and Activin A applied progressively to ESCs can promote the differentiation of XEN cells in mice. 22 Although TSCs colonies are present during XEN cell line derivation from ICM or single blastocysts under TSCs derivation conditions, XEN cells remain as the dominant group. 7,9 Altogether, all three blastocyst lineages are not completely separated, but communicate with each other via several conserved signalling pathways. As representative cell lines of blastocyst lineages, ESCs, TSCs and XEN cells were recently combined with each other to model the in vivo interactions between embryonic and extra-embryonic lineages were used exclusively to reconstruct embryo-like structures. Early attempts to generate embryo-like structures via the spontaneous differentiation of ESCs in suspension culture resulted in disorganized cell aggregates termed as embryoid bodies. 19,23 The reconstruction of embryo-like structures in vitro offers new opportunities for understanding embryogenesis. Based on previous studies, ESCs and TSCs cooperate in vitro to form structures that resemble embryonic day 3.5 (E3. 5) blastocysts, termed blastoids. 24 Furthermore, three types of stem cells, ESCs, XEN cells and TSCs aggregate together, forming gastrulating embryo-like structures (or gastruloids). 25 27 As the three types of stem cells were derived from different culture media, identifying the factors that play an important role in blastoids proved difficult. The EPSCs could generate blastoids; however, the culture medium of EPS-blastoids was complicated, comprising FGF4, Heparin and serum. 28 Several studies have shown that human blastocyst-like structures can be generated from human pluripotent stem cells 29,30 or induced pluripotent stem cells (iPSC) from the reprogramming of fibroblasts. 28 Although embryo-like structures recapitulate the key features of early embryonic development, they do not support the development of bona fide embryos. In addition, ESCs can develop into organoids, including gastrulating embryo-like structures, which present an invaluable system to recapitulate early development in vitro. [31][32][33][34][35][36][37][38][39] Based on previous studies, stem cell cultures can self-assemble in vitro to generate gastruloids in human. 31 Most morphological characteristics and gene expression levels of gastruloids are very similar to those of natural embryos, with differences, such as the lack of gene expression associated with extra-embryonic cell types. 36 The developmental defect of stem cell-derived blastoids or gastruloids is mainly associated with the in vitro culture conditions; compared with in vivo development, it is difficult to provide the full requirements for stem cell-derived blastoids or gastruloids under in vitro culture conditions. Different signalling pathways play an important role in development and control differentiation into particular germ layers in vitro, such as Nodal/Activin, [40][41][42][43][44][45][46] WNT, 47-54 BMP 55-57 and LIF-STAT 58,59 signalling pathways, which are not only critical for cell lineage derivation and maintenance, but also for the generation of embryo-like structures in vitro. In mice, the activation of TGF-β signalling via the Activin A pathway was previously found to support ESCs proliferation, 60 but is also essential for PrE promotion when combined with CHIR99021 (CHIR), the stimulator of Wnt signalling, similar to that for human ESCs differentiation. 20,21 CHIR is not only a Wnt/ β-catenin agonist, in fact, CHIR represses GSK-3 to promote naive pluripotency and reduced variability within cohorts of cells. 6 During pre-implantation development, the LIF-STAT pathway plays a critical role in blocking Epi differentiation, supporting PrE expansion, the maintenance of XEN cells in vitro. Accordingly, XEN cells derived and proliferated in the condition of EMFIs or EMFI-CM, providing an adequate source of LIF. 9 FGF/ERK signalling is required for TE differentiation through FGFR1 and is important for PrE specification. Thus, FGF/ERK signalling plays a key role in maintaining appropriate proportions of cell types in the blastocyst. 61 Recently, studies have been conducted on the induction of embryo-like structures by modulating several signalling pathways. Bone morphogenetic protein (BMP4), Nodal, STAT, MAPK and WNT signalling are critical for blastoid formation and trophoblast epithelial morphogenesis. 24 Although the WNT signalling pathway is important for blastocyst development, 54 the combination of the WNT and FGF4 signalling pathways efficiently reconstruct EPSC-blastoids. 28 Nodal/Activin signalling can also support the development of the extra-embryonic compartment in gastruloid and natural embryos in early post-implantation stages. 62 To date, the derivation and maintenance of these three types of stem cell lines, which can be employed as the origins of embryo-like structures under the same culture conditions, have not been reported. As all the necessary requirements are available, three types of stem cell lines can be derived from one blastocyst using the same culture conditions. Due to restricted accessibility to human embryonic specimens donated for research and ethical limitations, mouse blastocysts were used in this study. 63 By focusing on Activin A, WNT activator (CHIR99021) and the LIF signalling pathway, we tested the following hypothesis: there is a defined culture condition sufficient to govern two or three stem cell induction and maintenance. Through medium test experiments, we found that the addition of Activin A, CHIR99021 and LIF (ACL medium) supported the establishment of two types of stem cells, ESCs (ACL-ESCs) and XEN-like (ACL-XEN) cells, from one blastocyst. The transcriptional characteristics and developmental competence indicated that ACL-ESCs and ACL-XEN cells were equivalent to the inner cell mass and primitive endodermal lineages of the preimplantation embryo. Notably, ACL-ESCs and ACL-XEN cells were demonstrated to self-organize into blastocyst-like ACL-blastoids.

| Derivation of ACL-XEN cells
The "flat" and GOF/GFP negative outgrowths that spread out on day 8-10 was disaggregated into single cells with TrypLE (Gibco) and cultured in ACL medium equally. When the colonies reached 80-90% confluence, cells were passaged every 2-3 days in a 24-well fibronectin-coated plate. These cells, referred to as ACL-XEN cells, could perform self-renewal for over 35 passages.

| Derivation of 2i/L-ESCs
E3.5 blastocysts with GOF/GFP collected from the uterus and zona pellucidae were removed using Acidic Tyrode's Solution (Sigma-Aldrich) and placed in a 24-well plate coated with fibronectin (16.7 μg/ml, Millipore) at least 0.5 h before use. Serum-free ESC culture medium (2i/L) was prepared as previously described. 6 Briefly, N2B27 basic medium was supplemented with PD0325901 (PD, 1 μM, Miltenyi Biotech), CHIR99021 (CH, 3 μM, Miltenyi Biotech), and leukaemia inhibitory factor (LIF, 1000 IU/ml, Millipore); the medium was named 2i/L-medium. Within 5-6 days, the ICM of the blastocyst cultures grew efficiently, and outgrowth occurred. When colonies grew to around 200 μm in diameter, they were picked and minced into pieces using glass needles and cultured in 2i/L medium. Colonies were passaged manually for 2-3 passages as described above, and then treated with Accutase (Gibco) every 2 days. Finally, these cells were named 2i/L-ESCs.

| Derivation of XEN cell lines
To derive the XEN cell lines, a previous protocol 13

| Production of chimeras from ACL-ESCs/ ACL-XEN cells
Using a piezo-assisted micromanipulator attached to an inverted microscope, approximately 8-12 ACL-ESCs were injected into eight-cell embryos collected from E2.5 CD1 (ICR) mice recipient, which were then cultured in KSOM medium (Millipore) overnight at 37 C in a 5% CO 2 atmosphere to obtain chimeric blastocysts.
After re-expansion of the blastocoel cavity, some chimeric blastocysts were transferred to the uteri of pseudopregnant CD1 (ICR) mice at 2.5 days post coitus (dpc) to generate chimeras, and the remaining chimeric blastocysts were cultured for another 24 h and fixed in 4% paraformaldehyde for immunofluorescence. Full-term chimeras were confirmed by the coat colour pattern of the pups at birth.
To generate chimeric embryos of ACL-XEN cells, 8-12 cells were gently injected into the perivitelline space of eight-cell embryos. After 48 h of culture in KSOM medium at 37 C in a 5% CO 2 atmosphere, chimeric embryos were hatched and then fixed for IF. Embryonic day 6.5 (E6.5) CD1 (ICR) wild-type gastrulae were also collected, and ACL-XEN cells were microinjected into the cavity of gastrulae, which were cultured in ACL medium for 48 h to trace ACL-XEN cells.

| Generation of ACL-blastoids
Following incubation with Accutase and TrypLE, ACL-ESCs with GOF/GFP and ACL-XEN cells with tdTomato were dissociated into single cells, and mixed at a ratio of 1:5 with a total concentration of 1 Â 10 5 cells/ml. After centrifugation for 3 min at 1300 rpm, the cell pellet was resuspended in 800 μl ACL-blastoid medium and transferred into a well of a 24-well plate coated with Anti-Adherence Rinsing Solution (StemCell Technologies) at least 0.5 h before use, which was day 0 of the entire aggregation process. The ACL-blastoid medium contained 50% N2B27 medium and 50% KSOM medium as a basic medium supplemented with Activin A (20 ng/ml, R&D Systems), CHIR9902 (3 μM, Miltenyi Biotech) and LIF (1000 IU/ml, Millipore).
From day 2, 400 μl of the supernatant was carefully replaced with fresh medium every 2 days. In the first 2 days, no cavity formation occurred; however, small aggregates were observed. The emergence of ACL-blastoids was observed at day 3 after cell seeding, and ACLblastoids continued to increase, reaching a normal size, resembling E3.5 blastocyst around day 5. The ACL-blastoids were retrieved with a mouth pipette and fixed for IF or RNA extraction. XEN cell lines were established according to a previous protocol, 13 with modifications. ACL-blastoids were plated individually in a 24-well plate pre-coated with fibronectin in XEN derivation medium (30% TS medium and 70% feeder-conditioned medium from mouse embryonic fibroblasts) and supplemented with 25 ng/ml rhFGF4 and 1 mg/ml Heparin. An outgrowth was observed around day 3. In the following days, the medium was changed every 3 days.

| Derivation of three types of stem cells from ACL-blastoids
At around day 10, the XEN cells were dissociated into single cells using TrypLE for 7 min at 37 C. Dissociation was stopped with release medium, and the cells were collected via centrifugation at 1300 rpm for 3 min. Thereafter, the pellets were resuspended and plated into a 24-well plate pre-coated with fibronectin. FGF4 and Heparin were removed from the XEN derivation medium after 2-3 stable passages, once the XEN cells were established and grew well.

| Embryos and ACL-blastoids transfer
The 2.5 dpc recipient was anaesthetised with Avertin (Aibei), and the uterine horn was exposed via surgery. Chimeric embryos or ACLblastoids on day 5 were picked and transferred into KSOM droplets using a mouth pipette and washed three times. Subsequently, approximately 10-15 chimeric embryos or ACL-blastoids were transferred to each side of the uterine horn and pre-punctured with a needle. At 6.5 or 7.5 dpc, post-implantation embryo-like structures were collected from deciduae at implantation sites in the dissected uterus.

| Karyotype
Both ACL-ESCs and ACL-XEN cells were prepared for cytogenetic analysis via treatment with colchicine (Sigma) at a final concentration of 0.2 μg/ml for 2.5 h to accumulate cells in metaphase. Following cell harvesting, the cell pellets were exposed to 8 ml 0.075 M KCl (Sigma) for 10 min at 37 C for hypotonic treatment, and cold fixative solution prepared with 1 ml of methanol: acetic acid (3:1) was gently added and mixed. After discarding the supernatant, the cells were fixed three times at 37 C with 8 ml fixative solution for 30 min each. Thereafter, the cells were suspended in 0.5 ml cold fixative solution and dropped onto pre-cold clean slides, which were dried for 1 h at 70 C in an incubator. After cooling to 25 C approximately, the slides were stained with Giemsa (Sigma) for 10 min, and the unfixed dyes were removed via washing with distilled water. The slides were photographed using a microscope (Nikon), and the preparations were analysed using LUCIA Cytogenetics (Lucia).

| RT-qPCR
Total RNA was extracted from cultured cells using RNeasy Mini Kit  Table S1.

| RNA extraction and sequencing
Total RNA was extracted from approximately 2 Â 10 6 cells using Trizol reagent kit (Invitrogen) according to the manufacturer's recommendations. mRNA was enriched by Oligo(dT) Beads from total RNA, which was then fragmented into short fragments using fragmentation buffer, and transcripts were reversed into cDNA with random primers.
Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. (Guangzhou, China).

| RNA-seq and analysis
Before alignment, raw data were trimmed to remove reads with more than 10% low-quality bases and trimmed adaptors. Thereafter, the clean reads were mapped to the mouse reference genome (10 mm) using TopHat (2.0.12) with default settings. 65 HTSeq (0.6.1) was used for reads counting, and the RefSeq gene expression level was esti- Trend analysis of DEGs was performed using Short Time-series Expression Miner software. 69

| Statistical analyses
Statistical analyses were performed using the GraphPad Prism software (v8.0.2). Data are presented as mean ± SD. Significance between each group was measured using unpaired two-tailed Student's t test, and a value of p < 0.05 was considered statistically significant.

| Activin A replaces the MEK inhibitor for the derivation of ESCs from blastocyst
Early embryonic development is affected by different signalling pathways at different stages of development; however, the development of all embryonic cell lineages occurs in the same environment of the oviducts or uterus. Therefore, under certain conditions, different stem cell lines may be generated from a single blastocyst. In ESCs culture medium, 2i/L (MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021 and LIF), the MEK inhibitor, PD0325901, prevents ESCs differentiation in long-term culture. 6 Nodal/Activin signalling is critical to embryonic development, particularly for extra-embryonic development.
Here, Activin A was used to replace the MEK inhibitor (PD0325901) in 2i/L medium (Activin A, CHIR99021 and LIF), which resulted in the ACL medium, to establish new embryonic stem cells from blastocysts.
Oct4-4PE-GFP (GOF/GFP, mixed background of MF1, 129/sv and C57BL/6J strains) 129/sv F1 mouse blastocysts were directly placed in chemically defined ACL medium on fibronectin-coated cell culture plates ( Figure 1A). Two types of cells were discovered in ACL medium: GOF/GFP-positive ESC-like cells and GOF/GFP-negative flat cells after 3 days of culture ( Figures 1B and S1A). GOF/GFP-positive cells were picked and digested with Accutase in the subsequent passages; these GOF/GFP-positive cells were defined as ACL-ESCs. ACL-ESCs were morphologically similar to ESCs and maintained self-renewal capability over passage 19 (p19) ( Figure 1B).
The key features of ACL-ESCs relative to those of ESCs were determined. ACL-ESCs exhibited high alkaline phosphatase  Figures 1D and S1D). However, ACL-ESCs at the TE position were negative for the trophectoderm marker, CDX2 ( Figures 1E and S1D). We proceeded to determine their ability to contribute to chimeric embryos at E6.5. Based on our findings, ACL-ESCs successfully developed into epiblasts (19/19, 100%) ( Figures 1F and   S1E). Notably, full-term chimeras (17/26, 65%) were obtained from ACL-ESCs and their germline transmission abilities were verified ( Figures 1G, H and S1F). To further determine the differentiation ability of ACL-ESCs in vitro, 2i/L-ESCs and ACL-ESCs were cultured in N2B27 basic medium without any components. After 3 days of in vitro differentiation, RT-qPCR analysis was performed. Compared with 2i/L-ESCs, the relative expression of all three germ layer markers was significantly increased in ACL-ESCs, except Hand1 ( Figure S1G).
These results indicate that ACL-ESCs have a strong differentiation ability that depends on environmental changes. Taken together, these data suggest that Activin A replaces the MEK inhibitor to support derivation of ACL-ESCs from blastocysts, and ACL-ESCs present similar pluripotency features to ESCs and have stronger differentiation ability than ESCs in vitro.

| ACL culture condition supports XEN-like cells derivation from blastocyst
When blastocysts were placed in ACL medium for 3-5 days, GOF/GFP-positive cells were picked, and GOF/GFP-negative flat cells were observed (Figure 2A). GOF/GFP-negative flat cells propagated rapidly, were highly refractile, and appeared epithelial-like, with features coinciding with the typical morphology of XEN cells or TSCs 7,9,13,14 ( Figure 2B). These cells displayed genome stability after more than 35 passages (Figures 2B and S2A) and highly expressed primitive endoderm cell lineage-related genes, such as Gata4, Gata6 and Sox17 ( Figure S2B). The IF staining results also showed that high levels of GATA4 and SOX17 proteins were observed in ACL cultured epithelial-like cells ( Figure 2C), and not detected CDX2 positive trophectoderm-like cells ( Figure S2C). Therefore, GOF/GFP-negative flat cells were designated as ACL-XEN cells. Previous work suggests that XEN cells become dominant over trophoblast from blastocyst outgrowths. 9 Therefore, we investigated whether TS-like cells existed in early stage of ACL cells derivation. We cultured blastocysts in ACL medium do not pick up the GOF/GFP-positive cells, and mixed with GOF/GFP-negative flat cells. We performed immunofluorescence staining, and found ACL cultured cells in early stage (passage 6) expressed pluripotent protein maker OCT4, SOX2 and endoderm protein maker GATA4, however, no trophectoderm marker CDX2 was detected ( Figure S2D, E). We proved that the majority cells are ES-like cells and XEN-like cells in ACL culture system. The above results are consistent with the previous report.

XEN cells can self-renew in in vitro culture and differentiate into
PrE derivatives, such as VE and PE, and contribute to chimeras (in vivo) in a lineage-appropriate manner, highlighting the developmental potential of their origin. 9 To determine the developmental potential of ACL-XEN cells in vivo, ACL-XEN cells with H2B tdTomato-labelled were injected into eight-cell embryos, which were then   Figure 3F). Taken together, these data strongly suggest that ACL conditions can establish and maintain two types of stem cell lines derived from a single blastocyst.

| Blastocyst-like structures reconstructed from ACL-ESCs and ACL-XEN cells
After fertilization, cleavage stage embryonic cells communicate with each other and develop into blastocysts containing three different cell types (ICM, PrE and TE). 2 We proceeded to determine whether the two types of stem cells derived from one blastocyst (ACL-ESCs and ACL-XEN cells) were cultured together under ACL conditions and could aggregate into blastocyst-like structures (blastoids) ( Figure 4A). To confirm this hypothesis, ACL-ESCs and ACL-XEN cell lines with reporters (GOF/GFP and H2B tdTomato, respectively) were used to trace their spatial locations for aggregation. When ACL-ESCs and ACL-XEN cells were cocultured on FN-coated plates with ACL medium, there were some small clones in which GOF/GFP-positive ACL-ESCs were surrounded by epithelial-like cells developed from ACL-XEN cells ( Figure S4A). We examined the expression of the key proteins, OCT4 in ACL-ESCs and SOX17 in ACL-XEN cells, and found that both proteins could be detected in co-cultured cells ( Figure S4B).
Interestingly, some cells co-expressed OCT4 and SOX17 proteins ( Figure S4B), indicating that the co-culture system could drive  Figures 4B and S4C).
Using the above optimized conditions, we consistently observed the development of ACL-blastoids ( Figure 4C) and found that they continued to enlarge and reach an early blastocyst-like size at around day 4. We also counted the total number of ACL-blastoids, solid and vacuole structures, respectively, and found that formation efficiency of ACL-blastoids with typical blastocyst-like morphology were 68.42% at day 4 ( Figure S4D). Notably, the average diameter and total cell number of ACL-blastoids were comparable to those of E3.5 blastocysts, and the ICM cell number of ACL-blastoids was higher than that of E3.5 blastocysts ( Figure 4D, E). We examined whether ACL-ESCs and ACL-XEN cell have the capacity to produce blastoids or gastruloids in ETX medium. 25 Most of the cells in ETX medium were found to be differentiated or apoptotic, and only few cells could induce smaller aggregations, but failed to form cavities of blastoids or gastruloids in morphology ( Figure S4E). Collectively, our results demonstrate that ACL-blastoids were reconstructed from ACL-ESCs and ACL-XEN cell aggregates in ACL medium.  Finally, we determined whether key cellular and molecular events that feature early pre-implantation development could be recapitulated during ACL-blastoid formation. We traced the dynamics of the two types of cell aggregation during the first 3 days. On days 1 and 2, the cells started to form compact aggregates, and the cell adhesion protein, E-cadherin, began to accumulate at the cell-cell junctions, similar to compacted embryos ( Figure 5F). Importantly, blastocyst cavity-like structures started to form on day 3 ( Figure 5F). In mouse early embryogenesis, TE and ICM lineages are specified from the early blastocyst stage, and YAP signalling is critical for this process. 8,[71][72][73][74] On day 2, YAP was found in some outside cells of ACL-ESCs and ACL-XEN cell aggregates; on day 3, the nucleus of most outside cells expressed the active YAP protein ( Figure 5G). The molecular characteristics of ACL-blastoids (day 5) were verified via qPCR using a single ACL-blastoid compared with a single blastocyst. Some markers of blastocyst lineage expression levels were found to be similar to those of natural blastocysts, such as Rex1, Sox17 and Eomes ( Figure 5H); however, some of the lineage markers, Oct4, Nanog, Gata6, Cdx2, Gata2 and Gata3, were lower in the ACL-blastoids than the natural blastocysts ( Figure S5E). Together, these findings indicate that ACL-blastoids resemble blastocysts, with inner cells expressing pluripotency and outer cells expressing trophectoderm lineage markers.

| In vitro and in vivo developmental potential of ACL-blastoids
During embryonic development, early blastomeres specialize in the ICM and TE, and the ICM results in the PrE and epiblast of the blastocysts. Moreover, ESCs, TSCs and XEN cells can be directly derived from blastocysts. 2, 75 We determined whether ACL-blastoids could also lead to these three stem cell lines. Here, we successfully generated ESCs lines from ACL-blastoids under 2i/L culture condition.
The morphologies of ACL-blastoid-derived ESCs were similar to those of natural blastocysts derived from ESCs and expressed the pluripotency protein, SOX2 ( Figure S5F). TSCs lines were also derived from ACL-blastoids using FGF4-and Heparin-containing conditioned medium on feeder cells and expressed the TSCs key factor CDX2 ( Figure S5F). XEN cell lines were also successfully established from ACL-blastoids and expressed the PrE transcription factor, GATA4 ( Figure S5F).
We assessed the in vivo developmental potential by transferring ACL-blastoids into pseudopregnant mice at 2.5 and 3.5 dpc ( Figure 6A). At 6.5-7.5 dpc, we collected developmental ACLblastoids and identified the formation of decidua in the uteri of surrogate ACL-blastoids ( Figure 6A). Overall, approximately 17% of the transferred ACL-blastoids were implanted and induced decidualization ( Figure 6A, B). However, only approximately 2% of the transferred ACL-blastoids formed gastrulation-like structures ( Figure 6C).
Although ACL-blastoids can successfully form gastrulation-like structures, the size of gastrulation-like structures varied and was markedly smaller than that of the control ( Figure 6B). Importantly, IF results indicated that deciduae induced by gastrulation-like structures contained SOX2, GATA6 and CDX2 positive cells ( Figure 6D). Meanwhile, lumenogenesis in post-implantation embryos is regulated by podocalyxin (PCX). 76 The levels of PCX and cell adhesion proteins, E-cadherin and N-cadherin, were also detected in the deciduae derived from ACL-blastoids ( Figure 6E cooperates with Activin A. 20 A previous study revealed that prolonged Mek1/2 suppression impairs the chromosomal stability of ESCs. 79 The cytokine, LIF, which potently promotes mouse ESC identity, is a key component of ESCs culture conditions, 80,81 while the LIF-STAT pathway may play a role in XEN cell maintenance with the expression of some components, such as LIFR, gp130, JAK1, JAK2, STAT1 and STAT3. 9 Our findings revealed that LIF alone is sufficient to maintain ESCs pluripotency in a hypermethylated state. 82 Considering the above results, we concluded that Activin/Nodal, CHIR and LIF (ACL medium) are sufficient to derive two distinct stem cell lines from blastocysts. However, how cell lineages of blastocysts develop in ACL medium and precisely regulate mechanisms for stem cell self-renewal remain largely elusive. Recent studies have shown that blastoids represent an accessible, scalable, and tractable model system that will be valuable for many applications in basic research and translational approaches. 24,[27][28][29][30]83,84 The blastoids were generated from three different stem cells (ESCs, TSCs and XEN cells) with high efficiency and developed into a gastrula-like stage. 27 Here, we demonstrate that ACL-ESCs and ACL-XEN cells can be reconstituted to blastocyst-like structures, which recapitulate early embryonic developmental processes and initiate early implantation events. Although we successfully generated ACL-blastoids from ACL-ESCs and ACL-XEN cell aggregation, the levels of most of the three lineage markers were lower than those in natural blastocysts, especially TE lineages, and consistent with previous reports that TE markers are lower in stem cell-derived embryonic models. 85 Nodal/Activin signalling is required for TSCs renewal in culture and blastoid induction in previous reports. 78,86,87 Our culture conditions comprise Activin A, and we hypothesized ACL-ESCs aggregating with ACL-XEN cells might initiate TSCs differentiation and form blastocyst-like structures. We attempted to generate blastoids by aggregating 2i/L-ESCs and ACL-XEN cells in ACL medium, but failed to obtain the TE marker, CDX2 positive cells. Overall, ACL-ESCs were demonstrated to be necessary for ACL-blastoid formation. Further, the development of CDX2 positive cells was found to rely on the interaction between the ACL-ESCs and ACL-XEN cells; however, whether ACL-ESCs directly differentiate into TE cells, or push ACL-XEN cells to reverse to TE cells remains unclear and should be further explored.
Here, we described a modified cell culture system that generates two types of stem cell lines from one blastocyst. These cells were used to reconstruct ACL-blastoids, which had many similarities to blastocysts at the morphological, developmental, molecular and functional levels; however, these processes were imperfect and perhaps less neatly regulated than those in natural embryos. Overall, a simple approach to derive two types of stem cells from one blastocyst and generate ACL-blastoids is to capture the mechanism of embryo lineage segregation.