Bridging the gap in embryogenesis research: North Bornean orangutan-derived blastoids as models for human development

Bridging the gap in embryogenesis research: North Bornean orangutan-derived blastoids as models for human development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bridging the gap in embryogenesis research: North Bornean orangutan-derived blastoids as models for human development Nicole Tay, Qiuye Bao, Uma Kamaraj, Ying Ying Zeng, Ka Wai Wong, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8154543/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Understanding early primate embryogenesis remains limited by ethical and technical constraints. To address this, we established a stem cell-based embryo model from the North Bornean orangutan ( Pongo pygmaeus pygmaeus ). Using integration-free induced pluripotent stem cells (iPSCs), we generated blastoids that self-organized into structures resembling human blastocysts in morphology, lineage composition, and transcriptomic identity. Initial aggregation of primed iPSCs produced blastoids with organized lineage segregation. We enhanced this approach by combining isogenic iPSCs maintained under different culture conditions at optimized ratios, generating "bi-blastoids" with high formation efficiency, balanced lineage specification, and dimensions matching human blastocysts. Single-cell RNA sequencing revealed epiblast-, trophectoderm-, and hypoblast-like populations with transcriptomic profiles closely resembling human E6-7 blastocysts. Comparative analysis demonstrated that orangutan blastoids exhibited stronger transcriptional fidelity to human embryos than existing macaque models, establishing their unique value for developmental studies. Functional validation confirmed that orangutan blastoids could generate embryonic stem cell-like and trophectoderm stem cell-like lines, recapitulate trophoblast differentiation and secrete chorionic gonadotropin. However, primitive endoderm/hypoblast-like cells failed to form a distinct compartment, mirroring limitations observed across other primate models. This orangutan blastoid system provides a less ethically restrictive platform for studying human-relevant embryogenesis while maintaining high developmental fidelity. Beyond developmental insights into primate development, this model may inform future reproductive interventions for critically endangered great apes as conservation technologies advance. Biological sciences/Developmental biology/Embryogenesis/Cell lineage Biological sciences/Developmental biology/Pluripotency Biological sciences/Developmental biology/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Biological sciences/Stem cells/Reprogramming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Human embryo development is a complex and dynamic process, and understanding how cells assemble into functional embryos is fundamental for uncovering the origins of developmental disorders 1 . Yet, despite advances in imaging and molecular profiling, a comprehensive view of the mechanisms underlying peri-implantation human embryogenesis remains elusive due to limited access to samples and stringent ethical constraints 2 . Embryological studies have relied heavily on model organisms such as the mouse, which are inexpensive and reproduce efficiently. These studies have revealed essential principles of early development 3 – 5 . Yet, their applicability is limited by key interspecies differences 6 – 8 . Although human and mouse blastocysts appear morphologically similar at pre-implantation, they differ profoundly at the cellular and molecular levels, with distinct gene expression and signalling pathways 5 , 9 – 11 . These differences become even more pronounced during post-implantation development and placentation 12 . These differences restrict murine models from fully capturing human-specific biology. Progress in human in vitro culture systems has yielded valuable insights into blastocyst and peri-implantation stages 13 – 18 . However, embryo availability remains limited, manipulation is technically challenging, and ethical considerations limit experimental scope 19 – 23 . The peri-implantation stage, which is largely inaccessible to study, is particularly critical, as abnormalities during this stage underlie infertility, pregnancy loss, and developmental condition 24 – 29 . To overcome these barriers, stem cell-based embryo models have emerged. Initial efforts used embryonal carcinoma-derived embryoid bodies 30 , 31 , and embryonic stem cells (ESCs) which could complement natural embryos through blastocyst chimerism 32 or tetraploid complementation 33 . However, these approaches still relied on natural embryos. More recently, the ability of pluripotent stem cells (PSCs) to self-organize in response to mechanochemical cues has enabled the creation of “blastoids” – 3D structures that mimic key features of blastocyst development. These models mimic many aspects of blastocyst development and can be observed and manipulated in high throughput, offering complex spatiotemporal insights into lineage segregation, cellular mechanics, implantation, and epigenetic regulation 34 – 36 . While containing both embryonic and extra-embryonic cell types, these structures can provide complex spatiotemporal molecular information on the development of the blastocyst and peri-implantation embryo, including lineage segregation, cellular mechanics, implantation, the effect of epigenetic abnormalities, and many other processes 37 . In humans, blastoids have been generated from partially reprogrammed fibroblasts 38 , naïve induced pluripotent stem cells (iPSCs) 39 – 41 and expanded potential / extended pluripotent stem cells (EPSCs) 42 , 43 . Despite variations between protocols, these structures resemble human blastocysts in morphology, size, cell number, lineage allocation, markers, transcriptomic profiles, and functional potential, including stem cell derivation and implantation modelling. Importantly, they bypass some limitations of using human embryos for research, while enabling applications such as drug and therapeutic screening 41 , 44 . Despite their potential, human blastoid studies remain ethically and legally constrained. Although the International Society for Stem Cell Research (ISSCR) recently lifted the blanket “14-day rule” restricting embryo research 45 , in vitro culture beyond 14 days is still evaluated on a case-by-case basis, and in vivo transplantation into a uterus remains forbidden. Thus, both human and murine models remain limited in their ability to capture peri-implantation development in full biological context. NHPs offer a promising alternative. Compared to human models, they have less stringent ethical concerns and provide greater sample availability, while allowing studies to extend into gastrulation and later stages 46 . Progress in NHP embryology 21 , 47 and in vitro techniques 48 support their value for modelling human embryonic development, given the conservation of key developmental mechanisms amongst primates 11 , 49 . The recent creation of cynomolgus macaque blastoids 50 further demonstrates the utility of such systems. Among > 300 primate species, Great Apes (family Hominidae ) are the closest relatives of humans, diverging only 12–16 million years ago compared to ~ 25–33 million years for macaques ( Macaca sp.) 51–53 . Developmental and placental differences between the macaques and humans 11 , 12 underscore the need for a Great Ape model 54 . Yet, invasive studies in living Great Apes are prohibited, leaving a critical gap for cellular approaches. Research on early embryonic development in orangutans ( Pongo sp.) is limited by their critically threatened status, the scarcity of embryos, and ethical restrictions. All three orangutan species are listed as Critically Endangered (CR) under the International Union for Conservation of Nature (IUCN) Red List 55 and share a close phylogenetic relationship with humans 56 , 57 . Their embryogenesis and placentation are comparable to humans and other apes 58 – 61 , yet little is known about their reproductive physiology, and assisted/advanced reproductive technologies (ART) approaches have seen limited success 62 , although new information and techniques will emerge as zoological institutions progress in this field. Here, we report the first successful generation of blastoids from the North Bornean orangutan ( Pongo pygmaeus pygmaeus ). These blastoids closely recapitulated human counterparts in morphology, organization, and transcriptomic identity. Functional validation through stem cell derivation and in vitro culture confirmed their developmental potential. The creation of orangutan blastoids establishes a new comparative system for studying primate embryogenesis while offering a less ethically restrictive alternative to human models. Beyond advancing developmental biology, this system may hold future potential for conservation, should supporting technologies mature. Orangutan iPSC and blastoid platforms could one day inform reproductive interventions or assist in developing improved ART for critically endangered great apes, and in the long term, might provide last-resort options for species preservation. RESULTS Formation of orangutan blastoids To model preimplantation development, we employed the AggreWell platform to aggregate feeder-dependent footprint-free induced pluripotent stem cells (F + iPSCs) under limiting dilution (Suppl. Figure 1). Optimal blastoid formation was achieved at a seeding density of 25–30 cells per microwell (Suppl. Figure 2a). By one day post-aggregation (DPA), uniform spheroids had formed, which expanded progressively until 5 DPA (Fig. 1 b; Suppl. Figure 2b). At 6 DPA, aggregates acquired a cystic architecture resembling human blastocysts, including a fluid-filled cavity, an outer epithelial layer, and an inner cell mass-like cluster (Fig. 1 a, c). Immunostaining confirmed lineage segregation, with expression of epiblast (EPI; OCT4, SOX2), trophectoderm (TE; GATA2, CDX2), and primitive endoderm / hypoblast (PE/HYPO; SOX17, GATA4) markers in distinct regions (Fig. 1 d; Suppl. Figure 2c). The spatial organization mirrored that of human blastocysts 49 , 63 – 66 . By contrast, aggregates derived from feeder-free orangutan iPSCs (F – iPSC) formed irregular cysts with poorly defined patterning (Suppl. Figure 2b, c) and were excluded from downstream analyses. Consistent with observations in human and NHPs 67 , aggregation of F + iPSCs led to upregulation of pluripotency and early lineage-associated transcripts (Suppl. Figure 2d), supporting the notion that orangutan blastoids undergo self-organization through conserved developmental programs. Collectively, these findings establish orangutan blastoids as a tractable in vitro system to investigate early embryogenesis in this endangered primate. Transcriptome analysis To resolve cellular identities at single-cell resolution, we performed scRNA-seq on 210 orangutan blastoids, generating transcriptomes from 7,028 cells. Dimensionality reduction and clustering identified seven transcriptionally distinct populations (Fig. 1 e; Suppl. Figure 3a, b). Marker analysis revealed populations corresponding to EPI (POU5F1, SOX2, KLF4, DPPA2/3/5, DNMT3L, PRDM14, KLF17, FGF4, GDF3), TE (CDX2, GATA2/3, PLAC8, KRT7/8/18/19, CD24), and PE/HYPO (SOX17, GATA4/6, PDGFRA, COL4A1) lineages (Suppl. Figure 3c). Correlation analyses indicated that orangutan blastoids aligned more closely with human blastoids and E6 human blastocysts (Fig. 1 g). Comparison with cynomolgus macaque ( Macaca fascicularis ) blastocyst datasets 68 further showed strong transcriptional similarity between orangutan and human blastoids and blastocysts (Fig. 1 h), reinforcing the orangutan as a valuable model for human embryonic development. Notably, POU5F1 and SOX2 displayed heterogeneous expression across clusters, consistent with their roles in multiple lineages during preimplantation development 65 . Cluster identities were assigned by cross-referencing with transcriptional profiles from human blastocysts and blastoids 5 , 17 , 38 , 65 , 69 (Fig. 1 f). These results confirm that F + iPSC-derived blastoids encompass the three principal lineages of the preimplantation embryo and recapitulate early cell fate decisions observed in human and other primates. Formation of bi-line orangutan blastoids Given the divergent characteristics of F + blastoids and F – blastoids, where F + iPSCs supported organized lineage segregation, while F – iPSCs produced poorly structured cysts with a bias toward the TE-like identity, we hypothesized that combining both cell types could improve structural integrity and lineage balance. Serial dilutions of F + iPSCs and F – iPSCs revealed that aggregation at a 1:3 ratio markedly improved blastoid formation efficiency compared with either population alone (Suppl. Figure 4a). The resulting chimeric “bi-line” blastoids (bi-blastoids) were composed of fewer cells than F + blastoids and were intermediate in size between F + and F – blastoids, closely resembling the dimensions of human blastoids (Suppl. Figure 4b-d). Bi-blastoids expressed core pluripotency markers (NANOG, POU5F1, SOX2, KLF4) as well as naïve pluripotency markers (DPPA3, DPPA5, DNMT3L, KLF5, KLF17, PRDM14, SUSD2) at levels comparable to mono-blastoids (F + or F – blastoids) (Fig. 2 b). Importantly, TE (CDX2, GATA2/3, KRT8/18, PLAC8, TFAP2C, TF63, HAVCR1), PE/HYPO (SOX17, GATA6, PDGFRA), and post-implantation lineage markers including amnion (GABRP, BMP4) and primordial germ cells (PRDM1) were all upregulated relative to mono-blastoids (Fig. 2 b), indicating enhanced lineage diversification. As with mono-blastoids, bi-blastoids displayed spatial organization resembling human blastocysts, with peripheral GATA2 + and CDX2 + TE-like cells, centrally located SOX2 + EPI-like cells, as well as SOX17 PE/HYPO-like cells and a blastocoel-like cavity (Fig. 2 c; Suppl. Figure 4e). To track lineage contribution, F - iPSCs stably expressing GFP were aggregated with F + iPSCs at the 1:3 ratio to form bi-blastoids GFP (Fig. 2 a). GFP + cells localized predominantly to the outer TE layer, co-expressing CDX2 and the tight-junction marker ZO-1, and were rarely observed within NANOG + or SOX2 + EPI-like cells or GATA6+/SOX17 + PE/HYPO-like cells. This pattern indicates a bias of the GFP + F - iPSC fraction toward the TE lineage (Fig. 2 d; Suppl. Figure 4e). Single-cell transcriptomics of orangutan bi-blastoids To resolve lineage specification within bi-blastoids, we performed scRNA-seq on approximately 200 aggregates, generating > 8,000 single-cell transcriptomes. Dimensionality reduction and clustering revealed nine transcriptionally distinct clusters (Fig. 3 a, b). Marker analysis revealed populations corresponding to EPI (POU5F1, SOX2, KLF4, DPPA2/3/5, DNMT3L, PRDM14, KLF17, FGF4, GDF3), TE (CDX2, GATA2/3, PLAC8, KRT7/8/18/19, CD24), and PE/HYPO (SOX17, GATA6/4, PDGFRA, COL4A1) lineages (Suppl. Figure 5a). Benchmarking against transcriptional signatures of human blastocysts and blastoids 5 , 17 , 38 , 65 , 69 confirmed lineage identities for each cluster (Fig. 3 b, c). Differentially expressed genes across each cluster were examined (Fig. 3 e). Comparative analysis with orangutan blastoids, human blastoids, and human blastocysts 38 , 65 , revealed broad transcriptional overlap (Suppl. Figure 5b). Similar to blastocysts, and in contrast to mono-blastocysts, bi-blastoids lacked intermediate populations (Fig. 3 d; Suppl. Figure 5b). Furthermore, they exhibited lineage distributions and gene expression pattern closely resembling human blastocysts at E6-7 (Fig. 3 d, f). Derivation of ESC-like and trophectoderm stem cell-like cells Natural blastocysts provide pluripotent and trophectoderm stem cells (TSCs). To evaluate whether orangutan blastoids harbour expandable stem cell populations, we plated bi-blastoids GFP under lineage-specific culture conditions. In iPSC medium, alkaline phosphatase-positive ESC-like cells (ESCLCs) emerged, displaying the characteristic morphology of primed pluripotent colonies – compact and flat with sharp borders and a high nuclear-to-cytoplasmic ratio (Fig. 4 a). Transcriptomic analysis confirmed robust expression of primed pluripotency markers at levels comparable to parental iPSCs (Fig. 4 b; Suppl. Figure 6a). These ESCLC lines exhibited minimal expression of TE-associated transcripts (Suppl. Figure 6b). Immunostaining further validated expression of pluripotency markers (NANOG, OCT4, SOX2, SSEA-4) in the absence of GFP signal, consistent with their derivation from the F + iPSC fraction (Fig. 4 d). When cultured under TSC-supportive conditions, bi-blastoids GFP gave rise to trophectoderm stem cell–like cells (TSCLCs) exhibiting the cobblestone morphology typical of human TSCs (Fig. 4 a). Transcriptomic profiling revealed upregulation of trophoblast-associated markers relative to iPSCs and ESCLCs (Fig. 4 c; Suppl. Figure 6b), while immunostaining confirmed expression of TE markers (CDX2, GATA2, GATA3) (Fig. 4 d). Under these conditions, pluripotency markers (NANOG, OCT4, SOX2, SSEA-4) were not detected. Most TSCLC clones were GFP⁺, although a minority of GFP⁻ clones were also obtained, suggesting that F⁺iPSCs, when aggregated, can give rise to both TE- and EPI-like lineages, whereas F⁻iPSCs show limited potential to generate EPI-like cells. Collectively, these results confirms that orangutan blastoids can generate both ESCLC and TSCLCs, further validating their quality. In vitro implantation assay During the implantation process, trophoblasts of a blastocyst attach to the uterine wall, and after adhesion, the inner cell mass forms the pro-amniotic cavity before initiating gastrulation 5 . To evaluate the peri-implantation potential of the orangutan blastoids, we adapted a human blastoid attachment assay 13 , 14 , 38 . Bi-blastoids GFP adhered within 24–48 h, flattened, and produced outgrowths (Suppl. Figure 7a). Immunostaining identified OCT4 + and SOX2 + EPI-like cells at the centre and CDX2 + and GATA3 + TE-like cells at the periphery, mirroring spatial organisation in the 3D blastoids (Suppl. Figure 7b). A minor population co-expressed SOX2 and CDX2, suggestive of an amnion-like identity 41 . By day 5 post-attachment, subsets of TE-like cells showed positive staining for a HLA-G analogue and CGβ, markers associated with maternal-foetal tolerance and early pregnancy maintenance (Fig. 5 a-c). ELISA and commercial pregnancy tests confirmed CGβ secretion into conditioned media, indicating functional syncytiotrophoblast-like differentiation (Fig. 5 d). These findings were reproducible in mono-blastoids (Fig. 5 e). Together, these data demonstrate that orangutan blastoids can recapitulate key cellular and molecular events of blastocyst implantation. The identification of post-implantation markers indicates that the orangutan blastoids are capable of progressing beyond pre-implantation stages, exhibiting hallmarks of peri-implantation development in vitro . DISCUSSION This study represents the first successful generation of integration-free orangutan iPSCs and their subsequent use to generate blastoids. Orangutan blastoids consisted of lineage-representative populations with transcriptomic profiles resembling those of human blastocysts and blastoids, capturing several functional hallmarks of peri-implantation development, with transcriptomic profiles resembling those of human blastocysts and blastoids. Notably, orangutan blastoids exhibited greater transcriptional similarity to human blastocysts than those of macaque models, underscoring their distinctive value as a comparative system for studying human blastocyst development and implantation. Protocols were adapted from the pioneering human blastoid study 38 . The bi-blastoid protocol achieved a maximum efficiency of 13.1%, close to the reported median efficiency of published human blastoids (median = 14.4%, range 1.9–89%) 38–43,67 . While most prior studies predominantly used naïve PSCs or EPSCs to initiate blastoid formation, this study demonstrates that primed iPSCs can also support blastoid generation. This suggests that the blastoid formation conditions may have driven partial conversion of primed iPSCs toward a naïve-like state. Such primed-to-naïve transitions may represent a rate-limiting step, accounting for reduced efficiency, but also highlight a tradeoff: primed iPSCs are easier to maintain than naïve cultures, potentially broadening accessibility of blastoid approaches. The bi-blastoid protocol markedly outperformed mono-blastoid conditions. Interestingly, F + iPSCs predominantly contributed to EPI-like cells, while F − iPSCs favoured TE-like differentiation, despite being derived isogenically from the same parental clone. This suggests that culture conditions, rather than clonal genetics, shaped lineage bias. We propose that F − iPSCs transiently adopted a naïve-like state, expanding developmental potential but subsequently “downgraded” to a primed state predisposed toward TE. Rather than enhancing blastoid formation, this shift skewed lineage orientation, preventing proper blastoid architecture. These findings highlight plasticity of primate pluripotency states and suggest that lineage orientation can be deliberately programmed through culture conditions. Although average cell numbers were comparable across orangutan blastoids, human blastocysts, and human blastoids 38 , their physical dimensions differed. Orangutan mono-blastoids displayed significantly larger diameters than mature human blastocysts (p = 0.0084), while orangutan bi-blastoids closely matched human blastocyst dimensions 70 . Together with their higher formation efficiency, these findings indicate that the bi-blastoid represents a more accurate and reliable orangutan model of blastocyst development. A persistent limitation of blastoid models is incomplete lineage specification. Across species, blastoids frequently lack defined PE/HYPO compartments 38 – 40 , 42 , 43 , 71 and often include non-relevant cell types, including uncommitted PSCs 39 , 40 , lineage intermediates expressing multiple lineages markers 38 , 40 , 42 ), or cell types associated with later developmental stages 38 , 72 . In orangutan blastoids, PE/HYPO-like cells were detected by transcriptomics and immunostaining but failed to organize into a distinct compartment, and diverged from human profiles. This limitation mirrors other primate models and underscores the challenge of fully recapitulating extraembryonic lineages in vitro . Cell-type annotation in embryo models is strongly influenced by available reference datasets 73 , 74 . While extensive single-cell datasets exist for mouse and primate development, equivalent data for wildlife species remain scarce. Defining lineages in stem cell-based embryo models without species-specific reference remains contentious 15 . Interspecies comparisons, applied here, provide partial solutions 64 , 69 , 75 , and human embryo reference maps remain fragmented, limiting lineage resolution. Nevertheless, recent NHP blastoid models 50 , 76 and the orangutan model described here showed strong transcriptional resemblance to human blastoids and blastocysts, particularly at E7, underscoring their relevance for comparative studies and the urgent need to expand reference datasets across primates. Functionality evaluation of blastoid-derived cells extends beyond morphological comparisons. Orangutan blastoids generated ESCLCs and TSCLCs, paralleling ESCs and TSCs from blastocysts. Unlike mouse PSCs, which are largely incapable of TE differentiation, human and other great ape PSCs possess this ability 77 – 80 , facilitating the formation of TE-containing blastoids using only PSCs to start. However, establishing robust, long-term TSCLC cultures remains a barrier, limiting the model's broader applicability. Functional validation was further explored using an implantation assay. In vitro implantation assays revealed that orangutan blastoids adhered, differentiated, and expressed markers of post-implantation lineages, recapitulating key features of uterine attachment. Yet, the absence of maternal-foetal crosstalk limits the modelling of later events such as mural TE development, parietal endoderm formation, and ecoplacental cone establishment. Incorporating hormonally stimulated endometrial models, as has been applied to human blastoids, could improve fidelity 41 . This orangutan blastoid model bridges a crucial gap between human and non-primate systems. It offers an ethically viable and scientifically robust platform for studying early embryogenesis without embryo destruction. Although current human blastoid models cannot progress beyond early stages, further advances may eventually extend developmental capacity and raise ethical considerations. In this context, great ape blastoids represent a valuable compromise – scientifically informative while ethically viable. Perspective – Expanding the Conservation Toolkit The blastoid system – also referred to as integrated stem cell-based embryo models, synthetic embryos, or blastocyst-like structures – offers unique opportunities for conservation. For critically endangered species, where natural embryos are scarce or inaccessible, blastoids may provide an unprecedented window into developmental stages otherwise unobtainable. Here, we established the first integration-free iPSC lines from P. p. pygmaeus , creating a renewable biobanking resource. Importantly, our findings demonstrate that lineage bias can be programmed by culture conditions, raising the possibility of engineering interspecies blastoids in which ICM-like cells from endangered species are combined with TE-biased cells from surrogate species. This would form analogues of embryos derived from blastocyst-based ICM transplantation or tetraploid complementation, and such strategies may help overcome barriers to interspecies implantation. Hence, integrated stem cell-based embryo models may expand the conservation toolkit beyond artificial insemination, IVF, and somatic cell nuclear transfer, which are constrained by gamete and embryo availability. Stem cell-derived blastoids may therefore offer a scalable alternative, particularly when generated from cryopreserved somatic cells, enabling recovery of genetic diversity from deceased individuals. Beyond their potential to generate embryos, blastoids allow systematic exploration of interspecies compatibility, trophoblast biology, and host-surrogate interactions, which are crucial for advancing cross-species ART. Coupled with biobanking initiatives, iPSC-based embryo modelscan safeguard threatened taxa today while providing tools for future reproductive technologies. Amid the accelerating biodiversity crisis, stem cell-based embryo models should be viewed not only as experimental platforms for developmental biology but also as integral tools for conservation. When integrated with reproductive technologies and species recovery strategies, these models provide a forward-looking approach with tangible potential to mitigate biodiversity loss. MATERIALS AND METHODS Ethics statement This project was approved by the Mandai Wildlife Group (MWG) Research Panel under the project code MWG230104. No animals were harmed in the preparation of this manuscript. Access to skin samples The Institute of Molecular and Cell Biology – Endangered Species Conservation via Assisted Reproduction (IMCB-ESCAR) laboratory is part of the Mandai Wildlife Group, which is the steward of Mandai Wildlife Reserve, home to Singapore Zoo, Night Safari, River Wonders, Bird Paradise, and Rainforest Wild Asia. Skin samples were obtained from only one female North Bornean Orangutan ( Pongo pygmaeus pygmaeus ), who died of natural causes at 6 years of age. Derivation and culture of North Bornean orangutan primary fibroblasts At post-mortem, the animals’ skin from the ear was cleaned with 70% ethanol and shaved before aseptic surgical preparation of the area of interest. A sterile scalpel blade was used to harvest a 3cm x 3cm sample of full-thickness skin. In sterile conditions, any remaining fur, fat, and epidermis were removed from the skin sample, and the sample was cut into smaller pieces. After washing in PBS, the pieces were incubated in DMEM with 1X Antibiotic–Antimycotic (Gibco, 15240062), at room temperature for 30 min. After further washing in PBS, the pieces were placed in a 0.1% gelatin-coated sterile tissue culture dishes and cultured in P0 media; P0 media contained Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 11966025), 20% Fetal Bovine Serum (FBS) (Gibco, 26140079), 1X Minimum Essential Medium (MEM) non-essential amino acids (NEAA) (Gibco, 11140076), 1X Penicillin–Streptomycin (Gibco, 15140122) (equivalent to 100U/ml Penicillin and 100µg/ml Streptomycin), and 2mM L-glutamine (Gibco, A2916801). All cell cultures were incubated at 37° Celsius, 5% CO 2 , and 20% O 2 , unless otherwise described. When confluent, fibroblasts were passaged with 0.25% trypsin and maintained on 0.1% gelatin-coated sterile tissue culture dishes with fibroblast media (FM); FM contained DMEM, 10% FBS, 1X MEM NEAA, 100µM β-mercaptoethanol (Gibco, 21985022), 1X Penicillin–Streptomycin, and 2mM L-glutamine. For efficient reprogramming, fibroblasts were transduced or transfected at passage 8 or earlier. Episomal reprogramming for iPSCs Cell counting was performed with the Countess™3 Automated Cell Counter (Invitrogen, AMQAX2000) and verified manually. 600,000 fibroblasts were transfected with 2ug of each plasmid pCXLE-OCT3/4-p53shRNA, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA-1 (Addgene #27077, #27078, #27080, and #37624 respectively 81 ). Electroporation was performed via the Neon™ Transfection System (Invitrogen, MPK5000, MPK1025) at 1650V, 10ms, and 3 pulses. Transfected cells were seeded onto 0.1% gelatin-coated 6cm dishes and cultured in FM without Penicillin–Streptomycin (Day 0). 24h later (Day 1), the media was changed to FM containing 10ng/ml bFGF. On Day 2, the media was changed to FM containing 10ng/ml bFGF and 0.5mM sodium butyrate, and was refreshed daily. On Day 7, cells were passaged and split 1:4 into 6-well plates coated with irradiated CF1 mouse embryonic fibroblast (iMEF) (Gibco, A34180) feeder layers, and cultured in a 1:1 ratio of FM and PluriSTEM™ Human ES/iPS Cell Medium (MERCK, SCM130). On Day 9, the media was changed to PluriSTEM™ only, and was refreshed every other day. iPSC colonies started forming by Day 12. iPSC colonies were picked from Day 21, and expanded and subsequently maintained in 6-well plates coated with feeders in PluriSTEM™. iPSCs were passaged with ReLeSR™ (STEMCELL Technologies, #100–0484) every 4–6 days and split 1:10–20 into PluriSTEM™, with 10µM Y-27632 for the 1st 24h post-passaging; PluriSTEM™ was changed daily or every other day. iPSCs were stable and could be passaged for more than 20 passages. Six iPSC lines were generated and one was used for further analysis. Sendai viral reprogramming for iPSCs 250,000 fibroblasts were seeded onto 0.1% gelatin-coated 3 cm dishes and cultured in FM. 24h later (Day 0), fibroblasts were transduced with Sendai virus from the CytoTune™-iPS 2.0 kit (Invitrogen, A16517) containing expression cassettes for human KLF4 , OCT3/4 , SOX2 , and c-MYC , in 1 ml of FM. On Day 1, the media was changed to FM containing 10ng/ml bFGF and 0.5 mM sodium butyrate, and was refreshed daily. On Day 7, cells were passaged and split 1:4 into 6-well plates coated with iMEF feeder layers or Matrigel®, and cultured in FM with bFGF. On Day 8, the media was changed to homemade pluripotent stem cell media (bFGF-media) and refreshed daily. bFGF-media contained DMEM, 15% ESC FBS (Gibco, 16141002), 1X MEM NEAA, 100µM β-mercaptoethanol, 1X Penicillin–Streptomycin, 2mM L-glutamine, 10ng/ml bFGF (Gibco, PHG0264), and 10ng/ml hLIF (PeproTech, 3000525UG). iPSC colonies were picked from Day 14, and expanded and subsequently maintained in 6-well plates seeded with feeders in bFGF-media (F + iPSCs), or on Matrigel®-coated plates in StemFlex™ Medium (Gibco, A3349401) (F − iPSCs). iPSCs were passaged with ReLeSR™ (STEMCELL Technologies, #100–0484) every 4–6 days and split 1:10–20 into their respective media, with 10µM Y-27632 for the 1st 24h post-passaging; media was changed daily or every other day. iPSCs were stable and could be passaged for more than 20 passages. Ten iPSC lines were generated and one was used for further analysis. Detection of Sendai viral genome transgenes was performed following manufacturer’s instructions, with transgene-specific primers for the specific detection of transgenes carried by the Sendai reprogramming vectors. Constructing GFP + F − iPSCs F − iPSCs were passaged at 60–80% confluence. 24h (Day 0) later, F − iPSCs were transduced with 6ul Lentivirus-packed pSin-GFP-Fg (Addgene #174307). On Day 2, 0.8µg/ml puromycin was applied for selection. GFP + F − iPSC conlonies were picked from Day 14, and expanded and subsequently maintained in 6-well plates coated with Matrigel® in StemFlex™ Medium with 0.6ug/ml puromycin. Alkaline phosphatase staining Alkaline phosphatase staining was performed using Vector® Blue Substrate Kit (Alkaline Phosphatase) according to the manufacturer’s instructions (Vector Laboratories, SK-5300). Karyotyping Cells were treated overnight with 2.5mg/ml 5-bromo-2-deoxyuridine and 10ug/ml colcemid. Cells were then dissociated with 0.5% trypsin–EDTA for 10 min, collected into a centrifuge tube, and treated with 5 ml hypotonic solution containing 75mM KCl and 0.8% sodium citrate for 20min. Cells were then fixed with a series of 3:1 methanol and acetic acid fixative. G-banding and chromosome analyses were performed in accordance with standard procedures: cell suspensions were dropped onto clean wet slides and allowed to air dry, before being placed in a 90 degree Celsius oven prior to staining using GTG-banding techniques; 20 cells were analyzed and 5 full karyotypes were performed per sample. Reference karyograms were taken from the Atlas of Mammalian Chromosomes 82 . Chromosomes were counted and checked for numerical and/or structural abnormalities. Embryoid body formation and differentiation into three germ layers iPSCs were passaged and split 1:3 into ultralow attachment 24-well plates. Cells were cultured in bFGF-media without bFGF nor hLIF for 7 days. EBs formed were either maintained in suspension for another 7 days before being harvested for RNA extraction, or transferred to a 0.1% gelatin-coated plate for another 7 days before being fixed for immunostaining. Blastoid formation Blastoid formation using F + iPSCs, F − iPSCs, or a combination of both was performed in a similar manner to published iBlastoid protocol 38 . AggreWell™400 24-well or 6-well culture plates (STEMCELL Technologies, #34421) were prepared according to the manufacturer’s instructions as above. ReLeSR™ was added to wells containing iPSC colonies for 1 min before aspiration. The wells were then incubated for 2 mins at 37° Celsius. TrypLE™ was then used to flush out iPSCs sans iMEF feeder cells when present, and cells were incubated in TrypLE™ for 2 mins at 37° Celsius to promote singularization of iPSCs. iPSCs were resuspended in 1ml of iBlastoid media (iBM) with Y-27632; iBM contained 36.6% DMEM/F12 GlutaMAX (Gibco, 10565018), 38.8% Advanced DMEM/F12 (Gibco, 12634010), 12.1% Neurobasal medium, 10.1% conditioned FBS, 0.075% BSA, 1.25mM L-glutamine, 0.625X Penicillin-Streptomycin, 50µM β-mercaptoethanol, 0.75X Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (Gibco, 51500056), 0.125X N-2 Supplement, 0.125X B-27™ Supplement, 12.5µM N- acetyl-L-cysteine (Sigma-Aldrich, A7250), 4nM β-oestradiol (Sigma-Aldrich, E4389100MG), 100ng/ml Progesterone (Sigma-Aldrich, P7556100MG), 375.2ng/ml L-ascorbic Acid, 2µM CHIR99021, 0.5µM A83-01 (Sigma-Aldrich, SML0788), 1µM SB431542 (STEMCELL Technologies, 72234), 0.8mM Valproic Acid (VPA) (Sigma-Aldrich, P4543), 50ng/ml EGF (Sigma-Aldrich, E5036500UG), and 10ng/ml BMP4 (Miltenyi Biotec, 130111168). Cell numbers were quantified, and a minimum cell viability of 80% was ensured before proceeding for blastoid culture. iPSCs at defined ratios for 25 cells per microwell (1200 microwells in an AggreWell™400 24-well plate, 5900 microwells in an AggreWell™400 6-well plate), were collected and transferred into each AggreWell™ well. The plate was centrifuged at x300 g for 3 mins and transferred back into the incubator 37° Celsius, 5% CO 2 , and 5% O 2 . 24h later (Day 0), iBM without Y-27632 was carefully replaced. 156h after initial cell seeding (Day 6.5), aggregates were collected from each AggreWell™ well using wide-bore P1000 tips. Visual analysis and picking of cavitated blastoids were performed under a stereomicroscope. Efficiency of blastoid formation was calculated by dividing the number of cavitated structures collected from each AggreWell™ well by the number of microwells in each AggreWell™ well. Morphometric analyses of blastoids Diameters of blastoids were measured using ImageJ (NIH, version 1.54f) 83 with the x-axis determined as a line drawn between the widest section of the blastoid parallel to the edge of the inner cell mass-like structure adjacent to the blastocoel, and the y-axis determined as a line drawn between the widest section of the blastoid perpendicular to the x-axis. Number of cells per blastoid were calculated by first washing a number of collected blastoids in 0.04% BSA in PBS, followed by digestion in a 1:1 mix of Accumax™ (STEMCELL Technologies, #07921) and Versene Solution (Gibco, 15040066) at 37° Celsius in a thermoshaker for 30 mins or until blastoids have been dissociated into single cells. The cell suspension was then subjected to centrifugation and resuspension to achieve an appropriate dilution for cell counting. The total number of cells calculated to be collected was then divided by the number of blastoids dissociated to determine the number of cells per blastoid. Derivation of stem cells from blastoids To derive ESCLCs and TSCLCs, blastoids were collected and deposited onto iMEF feeders in bFGF-medium supplemented with 10µM Y-27632, or in trophectoderm stem cell (TSC) medium, and refreshed daily without Y-27632. TSC medium contained DMEM/F12 GlutaMAX, 0.2% characterized FBS (HyClone, SH30071.03), 0.3% BSA, 0.1mM β-mercaptoethanol, 0.5% Penicillin-Streptomycin, 1% ITS-X, 0.5mM VPA, 50ng/ml EGF, 2µM CHIR99021, 1.5µg/ml L-ascorbic Acid, 0.5µM A83-01, 1µM SB431542, 5µM Y-27632. ESCLCs and TSCLCs were passaged with ReLeSR™ or TrypLE™ respectively every 4–6 days and split 1:10–20 into their respective media, which was refreshed daily or every other day. 4–8 ESCLC and TSCLC lines were generated. In vitro attachment assay The extended in vitro attachment assay was adapted from previously published protocols 13 , 14 , 38 . Blastoids were collected and deposited onto 0.1% gelatin- or Matrigel®-coated optical-grade tissue culture plates, and cultured in in vitro culture medium 1 (IVC1); IVC1 contained Advanced DMEM/F12, 1% ITS-X, 2mM L-glutamine, 0.5% Penicillin-Streptomycin, 20% characterized FBS, 25µM N- acetyl-L-cysteine, 8nM β-oestradiol, and 200ng/ml Progesterone. 48h later, media was refreshed to in vitro culture medium 2 (IVC2); IVC2 was identical to IVC1 with the exception of FBS, which was replaced by 30% knockout serum replacement (Gibco, 10828028). Blastoids were cultured in this manner for 5 days. hCG-analogue assay To assess the presence and concentration of chorionic gonadotrophin (CG) produced by syncytiotrophoblast-like cells, IVC1 and IVC2 were collected from the in vitro attachment assay at Days 2 and 5 respectively, and subjected to commercially available human pregnancy test trips (Guardian, 622125) or a human CG (hCG) beta DuoSet solid phase sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems, DY9034-05), following manufacturer’s instructions. Quantitative RT-PCR Total RNA was extracted using Monarch® Total RNA Miniprep Kit (New England BioLabs Inc., T2010) and reverse transcribed into cDNA with iScript™ Reverse Transcription Supermix (Bio-Rad, 1,708,840). qPCR was performed using KAPA SYBR® FAST qPCR Master Mix (2X) Universal (Kapa Biosystems, KK4618) with 3.3ng cDNA with a primer concentration of 115nM. Primers are listed in Supplementary Table 1; these were designed to be specific only to predicted orangutan sequences. Samples were run in either duplicates or triplicates and GAPDH and ACTB were used as internal controls. CFX Maestro Software was used for qPCR analyses. Immunofluorescence staining Attached cells were washed in PBS, floating organoids were washed in 0.04% BSA in PBS. Fixing was performed with 4% PFA for 10-12min. Blocking was performed at room temperature for 2h; blocking buffer contained 2% BSA, 0.3% triton X-100, in PBS. Incubation with primary antibodies was performed at 4° Celsius overnight, before washing with 0.1% tween-20 in wash buffer. Incubation with secondary antibodies and DAPI was performed in the dark at room temperature for 1h. Images were acquired on a fluorescence microscope (Nikon ECLIPSE Ts2, 4X, 10X, 20X) or on an inverted confocal microscope (ZEISS LSM 700, 10X, 20X). ImageJ or ZEISS ZEN Microscopy Software (blue edition) (RRID:SCR_013672) were used for fluorescence or confocal microscopy images respectively, to overlay images and to improve visualisation of the scale bars when necessary. Antibodies are listed in Supplementary Table 2.For blastoids, each immunostaining condition was performed in at least 10 blastoids. Images are only presented if > 50% of the blastoids examined demonstrated spatial localization of embryonic and extraembryonic lineages that resembled those of blastocysts. Single cell RNA-sequencing library generation For single cell RNA-sequencing (scRNA-seq), 210 F + blastoids and 200 bi-blastoids were selected on the basis of morphological assessment. The selection criteria for blastoids were the presence of a blastocoel, the presence of an ICM-like structure, and similarity in size to human blastocysts. The selected blastoids were dissociated to obtain single-cell suspensions as described before, with the addition of 0.05X DNAse I (STEMCELL Technologies, #07900). Cell numbers were quantified, and a minimum cell viability of 70% was ensured before proceeding for library generation. For each type of blastoid, approximately 10,000 cells were loaded into a Chromium Next GEM Chip G (10X Genomics, 2000177) for isolation, encapsulation, and construction of gel beads in-emulsions with the Chromium controller (10X Genomics, 1000204) according to the manufacturer’s instructions; GEM was checked for homogenous emulsion and no clog formation, indicating successful GEM generation. Libraries were generated according to the manufacturer's instructions using the Chromium Next GEM Single Cell 3’ GEM and Library & Gel Bead Kit v3.1 (10X Genomics, PN-1000121). Libraries were pooled and sequencing was performed by Mayo Clinic, on the NovaSeq 6000 System (Illumina) at a read depth of approximately 700M pair-end reads of 150bp per sample/library. Single cell RNA-sequencing analyses With 10X Genomics data obtained, alignment and UMI counting were executed using the Cell Ranger (RRID:SCR_017344) program under default parameters and the ponAbe3 genome. The feature-barcode count matrix generated by Cellranger was utilized for downstream analyses with Seurat in R. During quality control, cells expressing fewer than 500 genes or having over 10% of mitochondrial gene content were considered abnormal and excluded. Genes expressed in fewer than three cells were excluded. Uniform manifold approximation and projection (UMAP) visualization was performed with 20 dimensions following dimensionality reduction using principal component analysis (PCA). Differentially expressed genes (DEGs) in each cluster or cell type were identified using the FindAllMarkers function. DEGs between samples were identified using the FindMarkers function with Wilcoxon’s rank sum tests, requiring a minimum upregulation of 0.1 log-fold and an adjusted p-value threshold of 0.01. Average expression values for each cell type in each sample were computed. These steps were performed in collaboration with a bioinformatician. Integrated single cell RNA-sequencing analyses For the integration of publicly available datasets from human blastocysts 65 and blastoids 38 , Seurat’s FindIntegrationAnchors and IntegrateData functions were employed. UMAP was used for dimensionality reduction. Transcriptome correlation was calculated using integrated gene expression values, and average expression values for each cell type in each sample were computed. Based on these average expression values, PCA and hierarchical clustering were conducted across samples. These steps were performed in collaboration with a bioinformatician. Bulk RNA-sequencing analyses Total RNA was extracted using Monarch® Total RNA Miniprep Kit. Qualitative control was performed to ensure that total RNA samples were ≥ 400ng in ≥ 20µl for a concentration of ≥ 20ng/µl, with an RNA Integrity Number of ≥ 6.8 (Agilent 2100™ Bioanalyzer) and a purity (OD260/280 and OD260/230) of ≥ 2.0 (NanoDrop™ ND-1000 Spectrophotometer, Thermo Scientific, RRID:SCR_016517). For bulk RNA-seq, sequencing was performed by Novogene, at 40M PE150 reads per sample. Reads were mapped to the ponAbe3 genome using STAR with default parameters. The GTF annotation file for genes was downloaded from the UCSC genome browser. Cufflinks (v2.2.1) was used for gene assembly and quantification to generate fragments per kilobase per million mapped reads (FPKM) tables based on mapped BAM files and the GTF annotation file. Transcript counts were computed using Htseq (v.0.12.4). DESeq2 was employed for differential gene analysis in samples with replicates, with a p-value cutoff of 0.05 and a fold change cutoff of 1.5. For samples without replicates, differential gene analysis was performed using EdgeR, with a p-value cutoff of 0.05. These steps were performed in collaboration with a bioinformatician. Principal component analysis Principal component analysis (PCA) was conducted on RNA-seq counts with at least five mapped reads across at least 10% of samples. Batch effects were corrected using the ComBat function from the sva Bioconductor package (v.3.34.0). PCA analysis was performed using the FactoMineR R package. A cluster dendrogram was plotted following hierarchical clustering with the hclust function. Statistics and reproducibility The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessments. Data and statistical analyses presented, if not otherwise stated, were plotted and performed using GraphPad Prism. For qPCR, relative normalized gene expression data between two genes were compared using a two sample t-test with a two-tailed p threshold of 0.05, with a Levene’s test for equality of variances with a p threshold of 0.05; relative normalized gene expression data between more than two genes were compared using a one-way ANOVA with a p threshold of 0.05, followed by a post-hoc Tukey’s multiple comparisons test with a p threshold of 0.05. For efficiency, morphology, and ELISA data, a Shapiro-Wilk test for normality was employed with a p threshold of 0.05; normally distributed data was compared using a one-way ANOVA with a p threshold of 0.05, followed by a post-hoc Tukey’s multiple comparisons test with a p threshold of 0.05; and non-normally distributed data compared using either a Mann-Whitney test or a Kruskal-Wallis test with a p threshold of 0.05, followed by a post-hoc Dunn’s multiple comparisons test with a p threshold of 0.05; means were compared with reference means using a one-sample two-tailed t-test with a p threshold of 0.05. Declarations AUTHORSHIP NLT: conceptualization, methodology, software, validation, formal analysis, investigation, data curation, writing (original draft), visualization. QB: methodology, validation, formal analysis, investigation. UMK, YZ: methodology, software, formal analysis, investigation, visualization. KWW: methodology, investigation. 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Nat Methods 9:671–675. https://doi.org/10.1038/nmeth.2089 Additional Declarations There is NO Competing Interest. Supplementary Files orangblastoidsupp251112nc.docx Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, Supplementary Figure 4, Supplementary Figure 5, Supplementary Figure 6, Supplementary Figure 7, Supplementary Tables Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":1913592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOrangutan iPSCs can form blastoids that encompass the three principal lineages of the preimplantation embryo and recapitulate early cell fate decisions observed in humans. (A)\u003c/strong\u003e Orangutan primary dermal fibroblasts were reprogrammed to form integration-free iPSCs, which could self-aggregate under specific conditions to form blastoids. \u003cstrong\u003e(B)\u003c/strong\u003e Day 0 and Day 6 of orangutan blastoid formation. \u003cstrong\u003e(C)\u003c/strong\u003e Orangutan blastoids resemble a hatched human blastocyst. \u003cstrong\u003e(D)\u003c/strong\u003e Immunostaining confirming localization of OCT4 (EPI-marker) to cells in the ICM-like compartment, and CDX2 (TE-marker) to cells in the TE-like compartment, of orangutan blastoids. \u003cstrong\u003e(E)\u003c/strong\u003e UMAP of the transcriptome of single cells originating from orangutan blastoids, demonstrating 7 cell clusters. \u003cstrong\u003e(F)\u003c/strong\u003e UMAP of the transcriptome of single cells originating from orangutan blastoids, annotated; intmd = intermediate. \u003cstrong\u003e(G)\u003c/strong\u003eCorrelation analysis and dendrogram demonstrating relative transcriptomic similarity of orangutan blastoids to other human blastocysts and blastoids. \u003cstrong\u003e(H)\u003c/strong\u003ePCA and correlation analysis demonstrating relative dissimilarity of macaque blastocysts to Great Ape blastocysts and blastoids.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8154543/v1/2443eeaf451f9f052468dc2d.jpg"},{"id":97706808,"identity":"4d1013b4-66a1-46c4-aa8c-2c758ef0ccf8","added_by":"auto","created_at":"2025-12-08 13:05:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1812871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAggregation of orangutan F\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eiPSCs and F\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eiPSCs at a 1:3 ratio formed blastoids with improved efficiency. (A)\u003c/strong\u003e Orangutan primary dermal fibroblasts were reprogrammed to form integration-free iPSCs, which were maintained in different conditions, and when aggregated at a specific ratio could self-aggregate under specific conditions to form bi-blastoids. \u003cstrong\u003e(B)\u003c/strong\u003e Quantitative RT-PCR (qRT-PCR) analysis for the expression of core pluripotency (NANOG, POU5F1, SOX2, KLF4), naïve pluripotency (DPPA3, DPPA5, DNMT3L, KLF5, KLF17, PRDM14, SUSD2), TE (CDX2, GATA2, GATA3, KRT8, KRT18, PLAC8, TFAP2C, TF63, HAVCR1), PE/HYPO (SOX17, GATA6, PDGFRA), amnion (GABRP, BMP4), and PGC (PRDM1) markers in aggregates harvested from bi-blastoid formation and their starting iPSCs, normalized to GAPDH and ACTB, and controlled to fibroblasts (for pluripotency markers) or bi-blastoids (for TE, PE/HYPO, amnion, and PGC markers). \u003cstrong\u003e(C) \u003c/strong\u003eImmunostaining confirming localization of OCT4 (EPI-marker) to cells in the ICM-like compartment, and CDX2 (TE-marker) to cells in the TE-like compartment, of orangutan bi-blastoids. \u003cstrong\u003e(D) \u003c/strong\u003eImmunostaining demonstrating localization of SOX2 to GFP\u003csup\u003e-\u003c/sup\u003e cells in the ICM-like compartment, surrounded by localization of CDX2 to GFP\u003csup\u003e+\u003c/sup\u003e cells in the TE-like compartment, in orangutan bi-blastoids.\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8154543/v1/d99783218de1fcd4f7f4469e.jpg"},{"id":97706812,"identity":"0f50c808-4fd2-4cac-84c9-500c175e725a","added_by":"auto","created_at":"2025-12-08 13:05:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1055342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOrangutan bi-blastoids exhibited lineage distributions closely resembling human E6-7 blastocysts. (A) \u003c/strong\u003eViolin plot demonstrating exclusion of cells containing less than 500 genes per cell in scRNA-seq analysis of orangutan bi-blastoids. \u003cstrong\u003e(B) \u003c/strong\u003eUMAP of the transcriptome of single cells originating from orangutan bi-blastoids, demonstrating 9 cell clusters. \u003cstrong\u003e(C)\u003c/strong\u003e UMAP of the transcriptome of single cells originating from orangutan bi-blastoids, annotated. \u003cstrong\u003e(D) \u003c/strong\u003eDifferent proportions of cell types in human blastocysts, macaque blastocysts, human blastoids, and orangutan bi-blastoids; inter = intermediate. \u003cstrong\u003e(E) \u003c/strong\u003eTop 9 DEGs in each cluster in bi-blastoids. \u003cstrong\u003e(F) \u003c/strong\u003eCorrelation analysis and dendrogram demonstrating relative transcriptomic similarity of orangutan mono- and bi-blastoids to other human blastocysts and blastoids.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8154543/v1/ad14d9f23ed86030a89c3e09.jpg"},{"id":97895016,"identity":"c9ccda8a-0a49-4032-830e-5af7c87fc8fe","added_by":"auto","created_at":"2025-12-10 15:33:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":370325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOrangutan blastoids can generate both ESCLCs and TSCLCs. (A) \u003c/strong\u003eUnder lineage-specific culture conditions, orangutan bi-blastoids were able to generate AP-positive ESCLCs, as well as TSCLCs. \u003cstrong\u003e(B) \u003c/strong\u003eqRT-PCR analysis for the expression of core pluripotency markers in ESCLCs, normalized to GAPDH and ACTB, and controlled to fibroblasts. \u003cstrong\u003e(C) \u003c/strong\u003eqRT-PCR analysis for the expression of TE-associated markers in TSCLCs, compared to F\u003csup\u003e+\u003c/sup\u003eiPSCs, F\u003csup\u003e-\u003c/sup\u003eiPSCs, and ESCLCs.\u003cstrong\u003e (D) \u003c/strong\u003eImmunostaining confirming positive expression of pluripotency and TE-associated markers in ESCLCs and TSCLCs respectively.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8154543/v1/bd48f2dfa9930dd93ccd4b21.jpg"},{"id":97706815,"identity":"d736e5e2-bb13-4b9d-8816-46de5d941aed","added_by":"auto","created_at":"2025-12-08 13:05:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1254903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOrangutan blastoids are capable of progressing beyond pre-implantation stages, exhibiting hallmarks of peri-implantation development \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A) \u003c/strong\u003eImmunostaining demonstrating cytotrophoblast-like cells expressing CK7, and extravillous trophoblast-like cells expressing an HLA-G analogue, in orangutan bi-blastoids after \u003cem\u003ein vitro\u003c/em\u003eattachment assay. \u003cstrong\u003e(B) \u003c/strong\u003eImmunostaining demonstrating polar trophectoderm-like cells expressing NR2F2, and CGβ secreted by syncytiotrophoblast-like cells, in orangutan bi-blastoids after \u003cem\u003ein vitro\u003c/em\u003eattachment assay. \u003cstrong\u003e(C) \u003c/strong\u003eThese are markers associated with maternal-foetal tolerance and early pregnancy maintenance. \u003cstrong\u003e(D) \u003c/strong\u003eELISA measurements of the concentration of the protein CGβ secreted into IVC medium of attached and unattached bi-blastoids at Day 2 and 5 of extended \u003cem\u003ein vitro\u003c/em\u003e attachment assay. \u003cstrong\u003e(E) \u003c/strong\u003eELISA measurements of the concentration of the protein CGβ secreted into IVC medium of attached and unattached mono-blastoids at Day 2 and 5 of extended \u003cem\u003ein vitro\u003c/em\u003e attachment assay.\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8154543/v1/491dc10d3eccf5646883e878.jpg"},{"id":101879105,"identity":"f82577cc-d734-4796-8d1a-4fde38ffd7af","added_by":"auto","created_at":"2026-02-04 14:47:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7784950,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8154543/v1/2891e8e9-a6f7-4477-942f-31059fb5b4cb.pdf"},{"id":97894800,"identity":"ae6a7680-1984-4c4b-ac91-2581bcff097e","added_by":"auto","created_at":"2025-12-10 15:33:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5039783,"visible":true,"origin":"","legend":"Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, Supplementary Figure 4, Supplementary Figure 5, Supplementary Figure 6, Supplementary Figure 7, Supplementary Tables","description":"","filename":"orangblastoidsupp251112nc.docx","url":"https://assets-eu.researchsquare.com/files/rs-8154543/v1/4519a39e792f1806b7abea59.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Bridging the gap in embryogenesis research: North Bornean orangutan-derived blastoids as models for human development","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eHuman embryo development is a complex and dynamic process, and understanding how cells assemble into functional embryos is fundamental for uncovering the origins of developmental disorders \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Yet, despite advances in imaging and molecular profiling, a comprehensive view of the mechanisms underlying peri-implantation human embryogenesis remains elusive due to limited access to samples and stringent ethical constraints \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEmbryological studies have relied heavily on model organisms such as the mouse, which are inexpensive and reproduce efficiently. These studies have revealed essential principles of early development \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Yet, their applicability is limited by key interspecies differences \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although human and mouse blastocysts appear morphologically similar at pre-implantation, they differ profoundly at the cellular and molecular levels, with distinct gene expression and signalling pathways \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These differences become even more pronounced during post-implantation development and placentation \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These differences restrict murine models from fully capturing human-specific biology.\u003c/p\u003e\u003cp\u003eProgress in human \u003cem\u003ein vitro\u003c/em\u003e culture systems has yielded valuable insights into blastocyst and peri-implantation stages \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, embryo availability remains limited, manipulation is technically challenging, and ethical considerations limit experimental scope \u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The peri-implantation stage, which is largely inaccessible to study, is particularly critical, as abnormalities during this stage underlie infertility, pregnancy loss, and developmental condition \u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo overcome these barriers, stem cell-based embryo models have emerged. Initial efforts used embryonal carcinoma-derived embryoid bodies \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and embryonic stem cells (ESCs) which could complement natural embryos through blastocyst chimerism \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e or tetraploid complementation \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. However, these approaches still relied on natural embryos.\u003c/p\u003e\u003cp\u003eMore recently, the ability of pluripotent stem cells (PSCs) to self-organize in response to mechanochemical cues has enabled the creation of \u0026ldquo;blastoids\u0026rdquo; \u0026ndash; 3D structures that mimic key features of blastocyst development. These models mimic many aspects of blastocyst development and can be observed and manipulated in high throughput, offering complex spatiotemporal insights into lineage segregation, cellular mechanics, implantation, and epigenetic regulation \u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. While containing both embryonic and extra-embryonic cell types, these structures can provide complex spatiotemporal molecular information on the development of the blastocyst and peri-implantation embryo, including lineage segregation, cellular mechanics, implantation, the effect of epigenetic abnormalities, and many other processes \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn humans, blastoids have been generated from partially reprogrammed fibroblasts \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, na\u0026iuml;ve induced pluripotent stem cells (iPSCs) \u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e and expanded potential / extended pluripotent stem cells (EPSCs) \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Despite variations between protocols, these structures resemble human blastocysts in morphology, size, cell number, lineage allocation, markers, transcriptomic profiles, and functional potential, including stem cell derivation and implantation modelling. Importantly, they bypass some limitations of using human embryos for research, while enabling applications such as drug and therapeutic screening \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite their potential, human blastoid studies remain ethically and legally constrained. Although the International Society for Stem Cell Research (ISSCR) recently lifted the blanket \u0026ldquo;14-day rule\u0026rdquo; restricting embryo research \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ein vitro\u003c/em\u003e culture beyond 14 days is still evaluated on a case-by-case basis, and \u003cem\u003ein vivo\u003c/em\u003e transplantation into a uterus remains forbidden. Thus, both human and murine models remain limited in their ability to capture peri-implantation development in full biological context.\u003c/p\u003e\u003cp\u003eNHPs offer a promising alternative. Compared to human models, they have less stringent ethical concerns and provide greater sample availability, while allowing studies to extend into gastrulation and later stages \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Progress in NHP embryology \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ein vitro\u003c/em\u003e techniques \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e support their value for modelling human embryonic development, given the conservation of key developmental mechanisms amongst primates \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The recent creation of cynomolgus macaque blastoids \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e further demonstrates the utility of such systems.\u003c/p\u003e\u003cp\u003eAmong \u0026gt;\u0026thinsp;300 primate species, Great Apes (family \u003cem\u003eHominidae\u003c/em\u003e) are the closest relatives of humans, diverging only 12\u0026ndash;16\u0026nbsp;million years ago compared to ~\u0026thinsp;25\u0026ndash;33\u0026nbsp;million years for macaques (\u003cem\u003eMacaca\u003c/em\u003e sp.) \u003csup\u003e51\u0026ndash;53\u003c/sup\u003e. Developmental and placental differences between the macaques and humans \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e underscore the need for a Great Ape model \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Yet, invasive studies in living Great Apes are prohibited, leaving a critical gap for cellular approaches.\u003c/p\u003e\u003cp\u003eResearch on early embryonic development in orangutans (\u003cem\u003ePongo\u003c/em\u003e sp.) is limited by their critically threatened status, the scarcity of embryos, and ethical restrictions. All three orangutan species are listed as Critically Endangered (CR) under the International Union for Conservation of Nature (IUCN) Red List \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e and share a close phylogenetic relationship with humans \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Their embryogenesis and placentation are comparable to humans and other apes \u003csup\u003e\u003cspan additionalcitationids=\"CR59 CR60\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, yet little is known about their reproductive physiology, and assisted/advanced reproductive technologies (ART) approaches have seen limited success \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, although new information and techniques will emerge as zoological institutions progress in this field.\u003c/p\u003e\u003cp\u003eHere, we report the first successful generation of blastoids from the North Bornean orangutan (\u003cem\u003ePongo pygmaeus pygmaeus\u003c/em\u003e). These blastoids closely recapitulated human counterparts in morphology, organization, and transcriptomic identity. Functional validation through stem cell derivation and \u003cem\u003ein vitro\u003c/em\u003e culture confirmed their developmental potential. The creation of orangutan blastoids establishes a new comparative system for studying primate embryogenesis while offering a less ethically restrictive alternative to human models. Beyond advancing developmental biology, this system may hold future potential for conservation, should supporting technologies mature. Orangutan iPSC and blastoid platforms could one day inform reproductive interventions or assist in developing improved ART for critically endangered great apes, and in the long term, might provide last-resort options for species preservation.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFormation of orangutan blastoids\u003c/h2\u003e\u003cp\u003eTo model preimplantation development, we employed the AggreWell platform to aggregate feeder-dependent footprint-free induced pluripotent stem cells (F\u003csup\u003e+\u003c/sup\u003eiPSCs) under limiting dilution (Suppl. Figure\u0026nbsp;1). Optimal blastoid formation was achieved at a seeding density of 25\u0026ndash;30 cells per microwell (Suppl. Figure\u0026nbsp;2a). By one day post-aggregation (DPA), uniform spheroids had formed, which expanded progressively until 5 DPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb; Suppl. Figure\u0026nbsp;2b). At 6 DPA, aggregates acquired a cystic architecture resembling human blastocysts, including a fluid-filled cavity, an outer epithelial layer, and an inner cell mass-like cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, c).\u003c/p\u003e\u003cp\u003eImmunostaining confirmed lineage segregation, with expression of epiblast (EPI; OCT4, SOX2), trophectoderm (TE; GATA2, CDX2), and primitive endoderm / hypoblast (PE/HYPO; SOX17, GATA4) markers in distinct regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed; Suppl. Figure\u0026nbsp;2c). The spatial organization mirrored that of human blastocysts \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan additionalcitationids=\"CR64 CR65\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. By contrast, aggregates derived from feeder-free orangutan iPSCs (F\u003csup\u003e\u0026ndash;\u003c/sup\u003eiPSC) formed irregular cysts with poorly defined patterning (Suppl. Figure\u0026nbsp;2b, c) and were excluded from downstream analyses. Consistent with observations in human and NHPs \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, aggregation of F\u003csup\u003e+\u003c/sup\u003eiPSCs led to upregulation of pluripotency and early lineage-associated transcripts (Suppl. Figure\u0026nbsp;2d), supporting the notion that orangutan blastoids undergo self-organization through conserved developmental programs. Collectively, these findings establish orangutan blastoids as a tractable \u003cem\u003ein vitro\u003c/em\u003e system to investigate early embryogenesis in this endangered primate.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTranscriptome analysis\u003c/h3\u003e\n\u003cp\u003eTo resolve cellular identities at single-cell resolution, we performed scRNA-seq on 210 orangutan blastoids, generating transcriptomes from 7,028 cells. Dimensionality reduction and clustering identified seven transcriptionally distinct populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee; Suppl. Figure\u0026nbsp;3a, b). Marker analysis revealed populations corresponding to EPI (POU5F1, SOX2, KLF4, DPPA2/3/5, DNMT3L, PRDM14, KLF17, FGF4, GDF3), TE (CDX2, GATA2/3, PLAC8, KRT7/8/18/19, CD24), and PE/HYPO (SOX17, GATA4/6, PDGFRA, COL4A1) lineages (Suppl. Figure\u0026nbsp;3c).\u003c/p\u003e\u003cp\u003eCorrelation analyses indicated that orangutan blastoids aligned more closely with human blastoids and E6 human blastocysts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Comparison with cynomolgus macaque (\u003cem\u003eMacaca fascicularis\u003c/em\u003e) blastocyst datasets \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e further showed strong transcriptional similarity between orangutan and human blastoids and blastocysts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh), reinforcing the orangutan as a valuable model for human embryonic development.\u003c/p\u003e\u003cp\u003eNotably, POU5F1 and SOX2 displayed heterogeneous expression across clusters, consistent with their roles in multiple lineages during preimplantation development \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Cluster identities were assigned by cross-referencing with transcriptional profiles from human blastocysts and blastoids \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). These results confirm that F\u003csup\u003e+\u003c/sup\u003eiPSC-derived blastoids encompass the three principal lineages of the preimplantation embryo and recapitulate early cell fate decisions observed in human and other primates.\u003c/p\u003e\n\u003ch3\u003eFormation of bi-line orangutan blastoids\u003c/h3\u003e\n\u003cp\u003eGiven the divergent characteristics of F\u003csup\u003e+\u003c/sup\u003eblastoids and F\u003csup\u003e\u0026ndash;\u003c/sup\u003eblastoids, where F\u003csup\u003e+\u003c/sup\u003eiPSCs supported organized lineage segregation, while F\u003csup\u003e\u0026ndash;\u003c/sup\u003eiPSCs produced poorly structured cysts with a bias toward the TE-like identity, we hypothesized that combining both cell types could improve structural integrity and lineage balance.\u003c/p\u003e\u003cp\u003eSerial dilutions of F\u003csup\u003e+\u003c/sup\u003eiPSCs and F\u003csup\u003e\u0026ndash;\u003c/sup\u003eiPSCs revealed that aggregation at a 1:3 ratio markedly improved blastoid formation efficiency compared with either population alone (Suppl. Figure\u0026nbsp;4a). The resulting chimeric \u0026ldquo;bi-line\u0026rdquo; blastoids (bi-blastoids) were composed of fewer cells than F\u003csup\u003e+\u003c/sup\u003eblastoids and were intermediate in size between F\u003csup\u003e+\u003c/sup\u003e and F\u003csup\u003e\u0026ndash;\u003c/sup\u003eblastoids, closely resembling the dimensions of human blastoids (Suppl. Figure\u0026nbsp;4b-d). Bi-blastoids expressed core pluripotency markers (NANOG, POU5F1, SOX2, KLF4) as well as na\u0026iuml;ve pluripotency markers (DPPA3, DPPA5, DNMT3L, KLF5, KLF17, PRDM14, SUSD2) at levels comparable to mono-blastoids (F\u003csup\u003e+\u003c/sup\u003e or F\u003csup\u003e\u0026ndash;\u003c/sup\u003eblastoids) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Importantly, TE (CDX2, GATA2/3, KRT8/18, PLAC8, TFAP2C, TF63, HAVCR1), PE/HYPO (SOX17, GATA6, PDGFRA), and post-implantation lineage markers including amnion (GABRP, BMP4) and primordial germ cells (PRDM1) were all upregulated relative to mono-blastoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), indicating enhanced lineage diversification.\u003c/p\u003e\u003cp\u003eAs with mono-blastoids, bi-blastoids displayed spatial organization resembling human blastocysts, with peripheral GATA2\u0026thinsp;+\u0026thinsp;and CDX2\u0026thinsp;+\u0026thinsp;TE-like cells, centrally located SOX2\u0026thinsp;+\u0026thinsp;EPI-like cells, as well as SOX17 PE/HYPO-like cells and a blastocoel-like cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec; Suppl. Figure\u0026nbsp;4e). To track lineage contribution, F\u003csup\u003e-\u003c/sup\u003eiPSCs stably expressing GFP were aggregated with F\u003csup\u003e+\u003c/sup\u003eiPSCs at the 1:3 ratio to form bi-blastoids\u003csup\u003eGFP\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). GFP\u0026thinsp;+\u0026thinsp;cells localized predominantly to the outer TE layer, co-expressing CDX2 and the tight-junction marker ZO-1, and were rarely observed within NANOG\u0026thinsp;+\u0026thinsp;or SOX2\u0026thinsp;+\u0026thinsp;EPI-like cells or GATA6+/SOX17\u0026thinsp;+\u0026thinsp;PE/HYPO-like cells. This pattern indicates a bias of the GFP\u0026thinsp;+\u0026thinsp;F\u003csup\u003e-\u003c/sup\u003eiPSC fraction toward the TE lineage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed; Suppl. Figure\u0026nbsp;4e).\u003c/p\u003e\n\u003ch3\u003eSingle-cell transcriptomics of orangutan bi-blastoids\u003c/h3\u003e\n\u003cp\u003eTo resolve lineage specification within bi-blastoids, we performed scRNA-seq on approximately 200 aggregates, generating\u0026thinsp;\u0026gt;\u0026thinsp;8,000 single-cell transcriptomes. Dimensionality reduction and clustering revealed nine transcriptionally distinct clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Marker analysis revealed populations corresponding to EPI (POU5F1, SOX2, KLF4, DPPA2/3/5, DNMT3L, PRDM14, KLF17, FGF4, GDF3), TE (CDX2, GATA2/3, PLAC8, KRT7/8/18/19, CD24), and PE/HYPO (SOX17, GATA6/4, PDGFRA, COL4A1) lineages (Suppl. Figure\u0026nbsp;5a). Benchmarking against transcriptional signatures of human blastocysts and blastoids \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e confirmed lineage identities for each cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c). Differentially expressed genes across each cluster were examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003eComparative analysis with orangutan blastoids, human blastoids, and human blastocysts \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, revealed broad transcriptional overlap (Suppl. Figure\u0026nbsp;5b). Similar to blastocysts, and in contrast to mono-blastocysts, bi-blastoids lacked intermediate populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed; Suppl. Figure\u0026nbsp;5b). Furthermore, they exhibited lineage distributions and gene expression pattern closely resembling human blastocysts at E6-7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, f).\u003c/p\u003e\n\u003ch3\u003eDerivation of ESC-like and trophectoderm stem cell-like cells\u003c/h3\u003e\n\u003cp\u003eNatural blastocysts provide pluripotent and trophectoderm stem cells (TSCs). To evaluate whether orangutan blastoids harbour expandable stem cell populations, we plated bi-blastoids\u003csup\u003eGFP\u003c/sup\u003e under lineage-specific culture conditions. In iPSC medium, alkaline phosphatase-positive ESC-like cells (ESCLCs) emerged, displaying the characteristic morphology of primed pluripotent colonies \u0026ndash; compact and flat with sharp borders and a high nuclear-to-cytoplasmic ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Transcriptomic analysis confirmed robust expression of primed pluripotency markers at levels comparable to parental iPSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; Suppl. Figure\u0026nbsp;6a). These ESCLC lines exhibited minimal expression of TE-associated transcripts (Suppl. Figure\u0026nbsp;6b). Immunostaining further validated expression of pluripotency markers (NANOG, OCT4, SOX2, SSEA-4) in the absence of GFP signal, consistent with their derivation from the F\u003csup\u003e+\u003c/sup\u003eiPSC fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eWhen cultured under TSC-supportive conditions, bi-blastoids\u003csup\u003eGFP\u003c/sup\u003e gave rise to trophectoderm stem cell\u0026ndash;like cells (TSCLCs) exhibiting the cobblestone morphology typical of human TSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Transcriptomic profiling revealed upregulation of trophoblast-associated markers relative to iPSCs and ESCLCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec; Suppl. Figure\u0026nbsp;6b), while immunostaining confirmed expression of TE markers (CDX2, GATA2, GATA3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Under these conditions, pluripotency markers (NANOG, OCT4, SOX2, SSEA-4) were not detected. Most TSCLC clones were GFP⁺, although a minority of GFP⁻ clones were also obtained, suggesting that F⁺iPSCs, when aggregated, can give rise to both TE- and EPI-like lineages, whereas F⁻iPSCs show limited potential to generate EPI-like cells.\u003c/p\u003e\u003cp\u003eCollectively, these results confirms that orangutan blastoids can generate both ESCLC and TSCLCs, further validating their quality.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eimplantation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring the implantation process, trophoblasts of a blastocyst attach to the uterine wall, and after adhesion, the inner cell mass forms the pro-amniotic cavity before initiating gastrulation \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To evaluate the peri-implantation potential of the orangutan blastoids, we adapted a human blastoid attachment assay \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Bi-blastoids\u003csup\u003eGFP\u003c/sup\u003e adhered within 24\u0026ndash;48 h, flattened, and produced outgrowths (Suppl. Figure\u0026nbsp;7a). Immunostaining identified OCT4\u0026thinsp;+\u0026thinsp;and SOX2\u0026thinsp;+\u0026thinsp;EPI-like cells at the centre and CDX2\u0026thinsp;+\u0026thinsp;and GATA3\u0026thinsp;+\u0026thinsp;TE-like cells at the periphery, mirroring spatial organisation in the 3D blastoids (Suppl. Figure\u0026nbsp;7b). A minor population co-expressed SOX2 and CDX2, suggestive of an amnion-like identity \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBy day 5 post-attachment, subsets of TE-like cells showed positive staining for a HLA-G analogue and CGβ, markers associated with maternal-foetal tolerance and early pregnancy maintenance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). ELISA and commercial pregnancy tests confirmed CGβ secretion into conditioned media, indicating functional syncytiotrophoblast-like differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These findings were reproducible in mono-blastoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Together, these data demonstrate that orangutan blastoids can recapitulate key cellular and molecular events of blastocyst implantation. The identification of post-implantation markers indicates that the orangutan blastoids are capable of progressing beyond pre-implantation stages, exhibiting hallmarks of peri-implantation development \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study represents the first successful generation of integration-free orangutan iPSCs and their subsequent use to generate blastoids. Orangutan blastoids consisted of lineage-representative populations with transcriptomic profiles resembling those of human blastocysts and blastoids, capturing several functional hallmarks of peri-implantation development, with transcriptomic profiles resembling those of human blastocysts and blastoids. Notably, orangutan blastoids exhibited greater transcriptional similarity to human blastocysts than those of macaque models, underscoring their distinctive value as a comparative system for studying human blastocyst development and implantation.\u003c/p\u003e\u003cp\u003eProtocols were adapted from the pioneering human blastoid study \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The bi-blastoid protocol achieved a maximum efficiency of 13.1%, close to the reported median efficiency of published human blastoids (median\u0026thinsp;=\u0026thinsp;14.4%, range 1.9\u0026ndash;89%) \u003csup\u003e38\u0026ndash;43,67\u003c/sup\u003e. While most prior studies predominantly used na\u0026iuml;ve PSCs or EPSCs to initiate blastoid formation, this study demonstrates that primed iPSCs can also support blastoid generation. This suggests that the blastoid formation conditions may have driven partial conversion of primed iPSCs toward a na\u0026iuml;ve-like state. Such primed-to-na\u0026iuml;ve transitions may represent a rate-limiting step, accounting for reduced efficiency, but also highlight a tradeoff: primed iPSCs are easier to maintain than na\u0026iuml;ve cultures, potentially broadening accessibility of blastoid approaches.\u003c/p\u003e\u003cp\u003eThe bi-blastoid protocol markedly outperformed mono-blastoid conditions. Interestingly, F\u003csup\u003e+\u003c/sup\u003eiPSCs predominantly contributed to EPI-like cells, while F\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSCs favoured TE-like differentiation, despite being derived isogenically from the same parental clone. This suggests that culture conditions, rather than clonal genetics, shaped lineage bias. We propose that F\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSCs transiently adopted a na\u0026iuml;ve-like state, expanding developmental potential but subsequently \u0026ldquo;downgraded\u0026rdquo; to a primed state predisposed toward TE. Rather than enhancing blastoid formation, this shift skewed lineage orientation, preventing proper blastoid architecture. These findings highlight plasticity of primate pluripotency states and suggest that lineage orientation can be deliberately programmed through culture conditions.\u003c/p\u003e\u003cp\u003eAlthough average cell numbers were comparable across orangutan blastoids, human blastocysts, and human blastoids \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, their physical dimensions differed. Orangutan mono-blastoids displayed significantly larger diameters than mature human blastocysts (p\u0026thinsp;=\u0026thinsp;0.0084), while orangutan bi-blastoids closely matched human blastocyst dimensions \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Together with their higher formation efficiency, these findings indicate that the bi-blastoid represents a more accurate and reliable orangutan model of blastocyst development.\u003c/p\u003e\u003cp\u003eA persistent limitation of blastoid models is incomplete lineage specification. Across species, blastoids frequently lack defined PE/HYPO compartments \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e and often include non-relevant cell types, including uncommitted PSCs \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, lineage intermediates expressing multiple lineages markers \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e), or cell types associated with later developmental stages \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. In orangutan blastoids, PE/HYPO-like cells were detected by transcriptomics and immunostaining but failed to organize into a distinct compartment, and diverged from human profiles. This limitation mirrors other primate models and underscores the challenge of fully recapitulating extraembryonic lineages \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eCell-type annotation in embryo models is strongly influenced by available reference datasets \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. While extensive single-cell datasets exist for mouse and primate development, equivalent data for wildlife species remain scarce. Defining lineages in stem cell-based embryo models without species-specific reference remains contentious \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Interspecies comparisons, applied here, provide partial solutions \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e, and human embryo reference maps remain fragmented, limiting lineage resolution. Nevertheless, recent NHP blastoid models \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e and the orangutan model described here showed strong transcriptional resemblance to human blastoids and blastocysts, particularly at E7, underscoring their relevance for comparative studies and the urgent need to expand reference datasets across primates.\u003c/p\u003e\u003cp\u003eFunctionality evaluation of blastoid-derived cells extends beyond morphological comparisons. Orangutan blastoids generated ESCLCs and TSCLCs, paralleling ESCs and TSCs from blastocysts. Unlike mouse PSCs, which are largely incapable of TE differentiation, human and other great ape PSCs possess this ability \u003csup\u003e\u003cspan additionalcitationids=\"CR78 CR79\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, facilitating the formation of TE-containing blastoids using only PSCs to start. However, establishing robust, long-term TSCLC cultures remains a barrier, limiting the model's broader applicability.\u003c/p\u003e\u003cp\u003eFunctional validation was further explored using an implantation assay. \u003cem\u003eIn vitro\u003c/em\u003e implantation assays revealed that orangutan blastoids adhered, differentiated, and expressed markers of post-implantation lineages, recapitulating key features of uterine attachment. Yet, the absence of maternal-foetal crosstalk limits the modelling of later events such as mural TE development, parietal endoderm formation, and ecoplacental cone establishment. Incorporating hormonally stimulated endometrial models, as has been applied to human blastoids, could improve fidelity \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis orangutan blastoid model bridges a crucial gap between human and non-primate systems. It offers an ethically viable and scientifically robust platform for studying early embryogenesis without embryo destruction. Although current human blastoid models cannot progress beyond early stages, further advances may eventually extend developmental capacity and raise ethical considerations. In this context, great ape blastoids represent a valuable compromise \u0026ndash; scientifically informative while ethically viable.\u003c/p\u003e\n\u003ch3\u003ePerspective – Expanding the Conservation Toolkit\u003c/h3\u003e\n\u003cp\u003eThe blastoid system \u0026ndash; also referred to as integrated stem cell-based embryo models, synthetic embryos, or blastocyst-like structures \u0026ndash; offers unique opportunities for conservation. For critically endangered species, where natural embryos are scarce or inaccessible, blastoids may provide an unprecedented window into developmental stages otherwise unobtainable.\u003c/p\u003e\u003cp\u003eHere, we established the first integration-free iPSC lines from \u003cem\u003eP. p. pygmaeus\u003c/em\u003e, creating a renewable biobanking resource. Importantly, our findings demonstrate that lineage bias can be programmed by culture conditions, raising the possibility of engineering interspecies blastoids in which ICM-like cells from endangered species are combined with TE-biased cells from surrogate species. This would form analogues of embryos derived from blastocyst-based ICM transplantation or tetraploid complementation, and such strategies may help overcome barriers to interspecies implantation.\u003c/p\u003e\u003cp\u003eHence, integrated stem cell-based embryo models may expand the conservation toolkit beyond artificial insemination, IVF, and somatic cell nuclear transfer, which are constrained by gamete and embryo availability. Stem cell-derived blastoids may therefore offer a scalable alternative, particularly when generated from cryopreserved somatic cells, enabling recovery of genetic diversity from deceased individuals.\u003c/p\u003e\u003cp\u003eBeyond their potential to generate embryos, blastoids allow systematic exploration of interspecies compatibility, trophoblast biology, and host-surrogate interactions, which are crucial for advancing cross-species ART. Coupled with biobanking initiatives, iPSC-based embryo modelscan safeguard threatened taxa today while providing tools for future reproductive technologies.\u003c/p\u003e\u003cp\u003eAmid the accelerating biodiversity crisis, stem cell-based embryo models should be viewed not only as experimental platforms for developmental biology but also as integral tools for conservation. When integrated with reproductive technologies and species recovery strategies, these models provide a forward-looking approach with tangible potential to mitigate biodiversity loss.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e This project was approved by the Mandai Wildlife Group (MWG) Research Panel under the project code MWG230104. No animals were harmed in the preparation of this manuscript.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAccess to skin samples\u003c/h2\u003e\u003cp\u003eThe Institute of Molecular and Cell Biology \u0026ndash; Endangered Species Conservation via Assisted Reproduction (IMCB-ESCAR) laboratory is part of the Mandai Wildlife Group, which is the steward of Mandai Wildlife Reserve, home to Singapore Zoo, Night Safari, River Wonders, Bird Paradise, and Rainforest Wild Asia. Skin samples were obtained from only one female North Bornean Orangutan (\u003cem\u003ePongo pygmaeus pygmaeus\u003c/em\u003e), who died of natural causes at 6 years of age.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDerivation and culture of North Bornean orangutan primary fibroblasts\u003c/h2\u003e\u003cp\u003eAt post-mortem, the animals\u0026rsquo; skin from the ear was cleaned with 70% ethanol and shaved before aseptic surgical preparation of the area of interest. A sterile scalpel blade was used to harvest a 3cm x 3cm sample of full-thickness skin. In sterile conditions, any remaining fur, fat, and epidermis were removed from the skin sample, and the sample was cut into smaller pieces. After washing in PBS, the pieces were incubated in DMEM with 1X Antibiotic\u0026ndash;Antimycotic (Gibco, 15240062), at room temperature for 30 min. After further washing in PBS, the pieces were placed in a 0.1% gelatin-coated sterile tissue culture dishes and cultured in P0 media; P0 media contained Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) (Gibco, 11966025), 20% Fetal Bovine Serum (FBS) (Gibco, 26140079), 1X Minimum Essential Medium (MEM) non-essential amino acids (NEAA) (Gibco, 11140076), 1X Penicillin\u0026ndash;Streptomycin (Gibco, 15140122) (equivalent to 100U/ml Penicillin and 100\u0026micro;g/ml Streptomycin), and 2mM L-glutamine (Gibco, A2916801). All cell cultures were incubated at 37\u0026deg; Celsius, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 20% O\u003csub\u003e2\u003c/sub\u003e, unless otherwise described. When confluent, fibroblasts were passaged with 0.25% trypsin and maintained on 0.1% gelatin-coated sterile tissue culture dishes with fibroblast media (FM); FM contained DMEM, 10% FBS, 1X MEM NEAA, 100\u0026micro;M β-mercaptoethanol (Gibco, 21985022), 1X Penicillin\u0026ndash;Streptomycin, and 2mM L-glutamine. For efficient reprogramming, fibroblasts were transduced or transfected at passage 8 or earlier.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eEpisomal reprogramming for iPSCs\u003c/h2\u003e\u003cp\u003eCell counting was performed with the Countess\u0026trade;3 Automated Cell Counter (Invitrogen, AMQAX2000) and verified manually. 600,000 fibroblasts were transfected with 2ug of each plasmid pCXLE-OCT3/4-p53shRNA, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA-1 (Addgene #27077, #27078, #27080, and #37624 respectively \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e). Electroporation was performed via the Neon\u0026trade; Transfection System (Invitrogen, MPK5000, MPK1025) at 1650V, 10ms, and 3 pulses. Transfected cells were seeded onto 0.1% gelatin-coated 6cm dishes and cultured in FM without Penicillin\u0026ndash;Streptomycin (Day 0). 24h later (Day 1), the media was changed to FM containing 10ng/ml bFGF. On Day 2, the media was changed to FM containing 10ng/ml bFGF and 0.5mM sodium butyrate, and was refreshed daily. On Day 7, cells were passaged and split 1:4 into 6-well plates coated with irradiated CF1 mouse embryonic fibroblast (iMEF) (Gibco, A34180) feeder layers, and cultured in a 1:1 ratio of FM and PluriSTEM\u0026trade; Human ES/iPS Cell Medium (MERCK, SCM130). On Day 9, the media was changed to PluriSTEM\u0026trade; only, and was refreshed every other day. iPSC colonies started forming by Day 12. iPSC colonies were picked from Day 21, and expanded and subsequently maintained in 6-well plates coated with feeders in PluriSTEM\u0026trade;. iPSCs were passaged with ReLeSR\u0026trade; (STEMCELL Technologies, #100\u0026ndash;0484) every 4\u0026ndash;6 days and split 1:10\u0026ndash;20 into PluriSTEM\u0026trade;, with 10\u0026micro;M Y-27632 for the 1st 24h post-passaging; PluriSTEM\u0026trade; was changed daily or every other day. iPSCs were stable and could be passaged for more than 20 passages. Six iPSC lines were generated and one was used for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSendai viral reprogramming for iPSCs\u003c/h2\u003e\u003cp\u003e250,000 fibroblasts were seeded onto 0.1% gelatin-coated 3 cm dishes and cultured in FM. 24h later (Day 0), fibroblasts were transduced with Sendai virus from the CytoTune\u0026trade;-iPS 2.0 kit (Invitrogen, A16517) containing expression cassettes for human \u003cem\u003eKLF4\u003c/em\u003e, \u003cem\u003eOCT3/4\u003c/em\u003e, \u003cem\u003eSOX2\u003c/em\u003e, and \u003cem\u003ec-MYC\u003c/em\u003e, in 1 ml of FM. On Day 1, the media was changed to FM containing 10ng/ml bFGF and 0.5 mM sodium butyrate, and was refreshed daily. On Day 7, cells were passaged and split 1:4 into 6-well plates coated with iMEF feeder layers or Matrigel\u0026reg;, and cultured in FM with bFGF. On Day 8, the media was changed to homemade pluripotent stem cell media (bFGF-media) and refreshed daily. bFGF-media contained DMEM, 15% ESC FBS (Gibco, 16141002), 1X MEM NEAA, 100\u0026micro;M β-mercaptoethanol, 1X Penicillin\u0026ndash;Streptomycin, 2mM L-glutamine, 10ng/ml bFGF (Gibco, PHG0264), and 10ng/ml hLIF (PeproTech, 3000525UG). iPSC colonies were picked from Day 14, and expanded and subsequently maintained in 6-well plates seeded with feeders in bFGF-media (F\u003csup\u003e+\u003c/sup\u003eiPSCs), or on Matrigel\u0026reg;-coated plates in StemFlex\u0026trade; Medium (Gibco, A3349401) (F\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSCs). iPSCs were passaged with ReLeSR\u0026trade; (STEMCELL Technologies, #100\u0026ndash;0484) every 4\u0026ndash;6 days and split 1:10\u0026ndash;20 into their respective media, with 10\u0026micro;M Y-27632 for the 1st 24h post-passaging; media was changed daily or every other day. iPSCs were stable and could be passaged for more than 20 passages. Ten iPSC lines were generated and one was used for further analysis. Detection of Sendai viral genome transgenes was performed following manufacturer\u0026rsquo;s instructions, with transgene-specific primers for the specific detection of transgenes carried by the Sendai reprogramming vectors.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eConstructing GFP\u003csup\u003e+\u003c/sup\u003e F\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSCs\u003c/h2\u003e\u003cp\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSCs were passaged at 60\u0026ndash;80% confluence. 24h (Day 0) later, F\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSCs were transduced with 6ul Lentivirus-packed pSin-GFP-Fg (Addgene #174307). On Day 2, 0.8\u0026micro;g/ml puromycin was applied for selection. GFP\u003csup\u003e+\u003c/sup\u003e F\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSC conlonies were picked from Day 14, and expanded and subsequently maintained in 6-well plates coated with Matrigel\u0026reg; in StemFlex\u0026trade; Medium with 0.6ug/ml puromycin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAlkaline phosphatase staining\u003c/h2\u003e\u003cp\u003eAlkaline phosphatase staining was performed using Vector\u0026reg; Blue Substrate Kit (Alkaline Phosphatase) according to the manufacturer\u0026rsquo;s instructions (Vector Laboratories, SK-5300).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eKaryotyping\u003c/h2\u003e\u003cp\u003eCells were treated overnight with 2.5mg/ml 5-bromo-2-deoxyuridine and 10ug/ml colcemid. Cells were then dissociated with 0.5% trypsin\u0026ndash;EDTA for 10 min, collected into a centrifuge tube, and treated with 5 ml hypotonic solution containing 75mM KCl and 0.8% sodium citrate for 20min. Cells were then fixed with a series of 3:1 methanol and acetic acid fixative. G-banding and chromosome analyses were performed in accordance with standard procedures: cell suspensions were dropped onto clean wet slides and allowed to air dry, before being placed in a 90 degree Celsius oven prior to staining using GTG-banding techniques; 20 cells were analyzed and 5 full karyotypes were performed per sample. Reference karyograms were taken from the Atlas of Mammalian Chromosomes \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Chromosomes were counted and checked for numerical and/or structural abnormalities.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eEmbryoid body formation and differentiation into three germ layers\u003c/h2\u003e\u003cp\u003eiPSCs were passaged and split 1:3 into ultralow attachment 24-well plates. Cells were cultured in bFGF-media without bFGF nor hLIF for 7 days. EBs formed were either maintained in suspension for another 7 days before being harvested for RNA extraction, or transferred to a 0.1% gelatin-coated plate for another 7 days before being fixed for immunostaining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eBlastoid formation\u003c/h2\u003e\u003cp\u003eBlastoid formation using F\u003csup\u003e+\u003c/sup\u003eiPSCs, F\u003csup\u003e\u0026minus;\u003c/sup\u003eiPSCs, or a combination of both was performed in a similar manner to published iBlastoid protocol \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. AggreWell\u0026trade;400 24-well or 6-well culture plates (STEMCELL Technologies, #34421) were prepared according to the manufacturer\u0026rsquo;s instructions as above. ReLeSR\u0026trade; was added to wells containing iPSC colonies for 1 min before aspiration. The wells were then incubated for 2 mins at 37\u0026deg; Celsius. TrypLE\u0026trade; was then used to flush out iPSCs sans iMEF feeder cells when present, and cells were incubated in TrypLE\u0026trade; for 2 mins at 37\u0026deg; Celsius to promote singularization of iPSCs. iPSCs were resuspended in 1ml of iBlastoid media (iBM) with Y-27632; iBM contained 36.6% DMEM/F12 GlutaMAX (Gibco, 10565018), 38.8% Advanced DMEM/F12 (Gibco, 12634010), 12.1% Neurobasal medium, 10.1% conditioned FBS, 0.075% BSA, 1.25mM L-glutamine, 0.625X Penicillin-Streptomycin, 50\u0026micro;M β-mercaptoethanol, 0.75X Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (Gibco, 51500056), 0.125X N-2 Supplement, 0.125X B-27\u0026trade; Supplement, 12.5\u0026micro;M \u003cem\u003eN-\u003c/em\u003eacetyl-L-cysteine (Sigma-Aldrich, A7250), 4nM β-oestradiol (Sigma-Aldrich, E4389100MG), 100ng/ml Progesterone (Sigma-Aldrich, P7556100MG), 375.2ng/ml L-ascorbic Acid, 2\u0026micro;M CHIR99021, 0.5\u0026micro;M A83-01 (Sigma-Aldrich, SML0788), 1\u0026micro;M SB431542 (STEMCELL Technologies, 72234), 0.8mM Valproic Acid (VPA) (Sigma-Aldrich, P4543), 50ng/ml EGF (Sigma-Aldrich, E5036500UG), and 10ng/ml BMP4 (Miltenyi Biotec, 130111168). Cell numbers were quantified, and a minimum cell viability of 80% was ensured before proceeding for blastoid culture. iPSCs at defined ratios for 25 cells per microwell (1200 microwells in an AggreWell\u0026trade;400 24-well plate, 5900 microwells in an AggreWell\u0026trade;400 6-well plate), were collected and transferred into each AggreWell\u0026trade; well. The plate was centrifuged at x300\u003cem\u003eg\u003c/em\u003e for 3 mins and transferred back into the incubator 37\u0026deg; Celsius, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 5% O\u003csub\u003e2\u003c/sub\u003e. 24h later (Day 0), iBM without Y-27632 was carefully replaced. 156h after initial cell seeding (Day 6.5), aggregates were collected from each AggreWell\u0026trade; well using wide-bore P1000 tips. Visual analysis and picking of cavitated blastoids were performed under a stereomicroscope. Efficiency of blastoid formation was calculated by dividing the number of cavitated structures collected from each AggreWell\u0026trade; well by the number of microwells in each AggreWell\u0026trade; well.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eMorphometric analyses of blastoids\u003c/h2\u003e\u003cp\u003eDiameters of blastoids were measured using ImageJ (NIH, version 1.54f) \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e with the x-axis determined as a line drawn between the widest section of the blastoid parallel to the edge of the inner cell mass-like structure adjacent to the blastocoel, and the y-axis determined as a line drawn between the widest section of the blastoid perpendicular to the x-axis. Number of cells per blastoid were calculated by first washing a number of collected blastoids in 0.04% BSA in PBS, followed by digestion in a 1:1 mix of Accumax\u0026trade; (STEMCELL Technologies, #07921) and Versene Solution (Gibco, 15040066) at 37\u0026deg; Celsius in a thermoshaker for 30 mins or until blastoids have been dissociated into single cells. The cell suspension was then subjected to centrifugation and resuspension to achieve an appropriate dilution for cell counting. The total number of cells calculated to be collected was then divided by the number of blastoids dissociated to determine the number of cells per blastoid.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eDerivation of stem cells from blastoids\u003c/h2\u003e\u003cp\u003eTo derive ESCLCs and TSCLCs, blastoids were collected and deposited onto iMEF feeders in bFGF-medium supplemented with 10\u0026micro;M Y-27632, or in trophectoderm stem cell (TSC) medium, and refreshed daily without Y-27632. TSC medium contained DMEM/F12 GlutaMAX, 0.2% characterized FBS (HyClone, SH30071.03), 0.3% BSA, 0.1mM β-mercaptoethanol, 0.5% Penicillin-Streptomycin, 1% ITS-X, 0.5mM VPA, 50ng/ml EGF, 2\u0026micro;M CHIR99021, 1.5\u0026micro;g/ml L-ascorbic Acid, 0.5\u0026micro;M A83-01, 1\u0026micro;M SB431542, 5\u0026micro;M Y-27632. ESCLCs and TSCLCs were passaged with ReLeSR\u0026trade; or TrypLE\u0026trade; respectively every 4\u0026ndash;6 days and split 1:10\u0026ndash;20 into their respective media, which was refreshed daily or every other day. 4\u0026ndash;8 ESCLC and TSCLC lines were generated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eattachment assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe extended \u003cem\u003ein vitro\u003c/em\u003e attachment assay was adapted from previously published protocols \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Blastoids were collected and deposited onto 0.1% gelatin- or Matrigel\u0026reg;-coated optical-grade tissue culture plates, and cultured in \u003cem\u003ein vitro\u003c/em\u003e culture medium 1 (IVC1); IVC1 contained Advanced DMEM/F12, 1% ITS-X, 2mM L-glutamine, 0.5% Penicillin-Streptomycin, 20% characterized FBS, 25\u0026micro;M \u003cem\u003eN-\u003c/em\u003eacetyl-L-cysteine, 8nM β-oestradiol, and 200ng/ml Progesterone. 48h later, media was refreshed to \u003cem\u003ein vitro\u003c/em\u003e culture medium 2 (IVC2); IVC2 was identical to IVC1 with the exception of FBS, which was replaced by 30% knockout serum replacement (Gibco, 10828028). Blastoids were cultured in this manner for 5 days.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003ehCG-analogue assay\u003c/h2\u003e\u003cp\u003eTo assess the presence and concentration of chorionic gonadotrophin (CG) produced by syncytiotrophoblast-like cells, IVC1 and IVC2 were collected from the \u003cem\u003ein vitro\u003c/em\u003e attachment assay at Days 2 and 5 respectively, and subjected to commercially available human pregnancy test trips (Guardian, 622125) or a human CG (hCG) beta DuoSet solid phase sandwich enzyme-linked immunosorbent assay (ELISA) (R\u0026amp;D Systems, DY9034-05), following manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using Monarch\u0026reg; Total RNA Miniprep Kit (New England BioLabs Inc., T2010) and reverse transcribed into cDNA with iScript\u0026trade; Reverse Transcription Supermix (Bio-Rad, 1,708,840). qPCR was performed using KAPA SYBR\u0026reg; FAST qPCR Master Mix (2X) Universal (Kapa Biosystems, KK4618) with 3.3ng cDNA with a primer concentration of 115nM. Primers are listed in Supplementary Table\u0026nbsp;1; these were designed to be specific only to predicted orangutan sequences. Samples were run in either duplicates or triplicates and \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eACTB\u003c/em\u003e were used as internal controls. CFX Maestro Software was used for qPCR analyses.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eAttached cells were washed in PBS, floating organoids were washed in 0.04% BSA in PBS. Fixing was performed with 4% PFA for 10-12min. Blocking was performed at room temperature for 2h; blocking buffer contained 2% BSA, 0.3% triton X-100, in PBS. Incubation with primary antibodies was performed at 4\u0026deg; Celsius overnight, before washing with 0.1% tween-20 in wash buffer. Incubation with secondary antibodies and DAPI was performed in the dark at room temperature for 1h. Images were acquired on a fluorescence microscope (Nikon ECLIPSE Ts2, 4X, 10X, 20X) or on an inverted confocal microscope (ZEISS LSM 700, 10X, 20X). ImageJ or ZEISS ZEN Microscopy Software (blue edition) (RRID:SCR_013672) were used for fluorescence or confocal microscopy images respectively, to overlay images and to improve visualisation of the scale bars when necessary. Antibodies are listed in Supplementary Table\u0026nbsp;2.For blastoids, each immunostaining condition was performed in at least 10 blastoids. Images are only presented if\u0026thinsp;\u0026gt;\u0026thinsp;50% of the blastoids examined demonstrated spatial localization of embryonic and extraembryonic lineages that resembled those of blastocysts.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eSingle cell RNA-sequencing library generation\u003c/h2\u003e\u003cp\u003eFor single cell RNA-sequencing (scRNA-seq), 210 F\u003csup\u003e+\u003c/sup\u003eblastoids and 200 bi-blastoids were selected on the basis of morphological assessment. The selection criteria for blastoids were the presence of a blastocoel, the presence of an ICM-like structure, and similarity in size to human blastocysts. The selected blastoids were dissociated to obtain single-cell suspensions as described before, with the addition of 0.05X DNAse I (STEMCELL Technologies, #07900). Cell numbers were quantified, and a minimum cell viability of 70% was ensured before proceeding for library generation. For each type of blastoid, approximately 10,000 cells were loaded into a Chromium Next GEM Chip G (10X Genomics, 2000177) for isolation, encapsulation, and construction of gel beads in-emulsions with the Chromium controller (10X Genomics, 1000204) according to the manufacturer\u0026rsquo;s instructions; GEM was checked for homogenous emulsion and no clog formation, indicating successful GEM generation. Libraries were generated according to the manufacturer's instructions using the Chromium Next GEM Single Cell 3\u0026rsquo; GEM and Library \u0026amp; Gel Bead Kit v3.1 (10X Genomics, PN-1000121). Libraries were pooled and sequencing was performed by Mayo Clinic, on the NovaSeq 6000 System (Illumina) at a read depth of approximately 700M pair-end reads of 150bp per sample/library.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eSingle cell RNA-sequencing analyses\u003c/h2\u003e\u003cp\u003eWith 10X Genomics data obtained, alignment and UMI counting were executed using the Cell Ranger (RRID:SCR_017344) program under default parameters and the ponAbe3 genome. The feature-barcode count matrix generated by Cellranger was utilized for downstream analyses with Seurat in R. During quality control, cells expressing fewer than 500 genes or having over 10% of mitochondrial gene content were considered abnormal and excluded. Genes expressed in fewer than three cells were excluded. Uniform manifold approximation and projection (UMAP) visualization was performed with 20 dimensions following dimensionality reduction using principal component analysis (PCA). Differentially expressed genes (DEGs) in each cluster or cell type were identified using the FindAllMarkers function. DEGs between samples were identified using the FindMarkers function with Wilcoxon\u0026rsquo;s rank sum tests, requiring a minimum upregulation of 0.1 log-fold and an adjusted p-value threshold of 0.01. Average expression values for each cell type in each sample were computed. These steps were performed in collaboration with a bioinformatician.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eIntegrated single cell RNA-sequencing analyses\u003c/h2\u003e\u003cp\u003eFor the integration of publicly available datasets from human blastocysts \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e and blastoids \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, Seurat\u0026rsquo;s FindIntegrationAnchors and IntegrateData functions were employed. UMAP was used for dimensionality reduction. Transcriptome correlation was calculated using integrated gene expression values, and average expression values for each cell type in each sample were computed. Based on these average expression values, PCA and hierarchical clustering were conducted across samples. These steps were performed in collaboration with a bioinformatician.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eBulk RNA-sequencing analyses\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using Monarch\u0026reg; Total RNA Miniprep Kit. Qualitative control was performed to ensure that total RNA samples were \u0026ge;\u0026thinsp;400ng in \u0026ge;\u0026thinsp;20\u0026micro;l for a concentration of \u0026ge;\u0026thinsp;20ng/\u0026micro;l, with an RNA Integrity Number of \u0026ge;\u0026thinsp;6.8 (Agilent 2100\u0026trade; Bioanalyzer) and a purity (OD260/280 and OD260/230) of \u0026ge;\u0026thinsp;2.0 (NanoDrop\u0026trade; ND-1000 Spectrophotometer, Thermo Scientific, RRID:SCR_016517).\u003c/p\u003e\u003cp\u003eFor bulk RNA-seq, sequencing was performed by Novogene, at 40M PE150 reads per sample. Reads were mapped to the ponAbe3 genome using STAR with default parameters. The GTF annotation file for genes was downloaded from the UCSC genome browser. Cufflinks (v2.2.1) was used for gene assembly and quantification to generate fragments per kilobase per million mapped reads (FPKM) tables based on mapped BAM files and the GTF annotation file. Transcript counts were computed using Htseq (v.0.12.4). DESeq2 was employed for differential gene analysis in samples with replicates, with a p-value cutoff of 0.05 and a fold change cutoff of 1.5. For samples without replicates, differential gene analysis was performed using EdgeR, with a p-value cutoff of 0.05. These steps were performed in collaboration with a bioinformatician.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePrincipal component analysis\u003c/h3\u003e\n\u003cp\u003ePrincipal component analysis (PCA) was conducted on RNA-seq counts with at least five mapped reads across at least 10% of samples. Batch effects were corrected using the ComBat function from the sva Bioconductor package (v.3.34.0). PCA analysis was performed using the FactoMineR R package. A cluster dendrogram was plotted following hierarchical clustering with the hclust function.\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eStatistics and reproducibility\u003c/h2\u003e\u003cp\u003eThe experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessments. Data and statistical analyses presented, if not otherwise stated, were plotted and performed using GraphPad Prism. For qPCR, relative normalized gene expression data between two genes were compared using a two sample t-test with a two-tailed p threshold of 0.05, with a Levene\u0026rsquo;s test for equality of variances with a p threshold of 0.05; relative normalized gene expression data between more than two genes were compared using a one-way ANOVA with a p threshold of 0.05, followed by a post-hoc Tukey\u0026rsquo;s multiple comparisons test with a p threshold of 0.05. For efficiency, morphology, and ELISA data, a Shapiro-Wilk test for normality was employed with a p threshold of 0.05; normally distributed data was compared using a one-way ANOVA with a p threshold of 0.05, followed by a post-hoc Tukey\u0026rsquo;s multiple comparisons test with a p threshold of 0.05; and non-normally distributed data compared using either a Mann-Whitney test or a Kruskal-Wallis test with a p threshold of 0.05, followed by a post-hoc Dunn\u0026rsquo;s multiple comparisons test with a p threshold of 0.05; means were compared with reference means using a one-sample two-tailed t-test with a p threshold of 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHORSHIP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNLT:\u003c/strong\u003e conceptualization, methodology, software, validation, formal analysis, investigation, data curation, writing (original draft), visualization. \u003cstrong\u003eQB:\u003c/strong\u003e methodology, validation, formal analysis, investigation. \u003cstrong\u003eUMK, YZ:\u003c/strong\u003e methodology, software, formal analysis, investigation, visualization. \u003cstrong\u003eKWW:\u003c/strong\u003e methodology, investigation. \u003cstrong\u003eCYYL, WKLT, HY:\u003c/strong\u003e investigation, project administration. \u003cstrong\u003eKP:\u003c/strong\u003e investigation. \u003cstrong\u003eDHHC, CH:\u003c/strong\u003e resources. \u003cstrong\u003eSX:\u003c/strong\u003e writing (review \u0026amp; editing). \u003cstrong\u003eMC, JYL, SCN:\u0026nbsp;\u003c/strong\u003econceptualization, supervision. \u003cstrong\u003eOP:\u003c/strong\u003e data curation, writing (original draft), visualization, supervision.\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhokha MK, Liu KJ, Wallingford JB (2020) Challenges and opportunities at the interface of birth defects, human genetics and developmental biology. 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Nat Methods 9:671\u0026ndash;675. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.2089\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.2089\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8154543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8154543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding early primate embryogenesis remains limited by ethical and technical constraints. To address this, we established a stem cell-based embryo model from the North Bornean orangutan (\u003cem\u003ePongo pygmaeus pygmaeus\u003c/em\u003e). Using integration-free induced pluripotent stem cells (iPSCs), we generated blastoids that self-organized into structures resembling human blastocysts in morphology, lineage composition, and transcriptomic identity. Initial aggregation of primed iPSCs produced blastoids with organized lineage segregation. We enhanced this approach by combining isogenic iPSCs maintained under different culture conditions at optimized ratios, generating \"bi-blastoids\" with high formation efficiency, balanced lineage specification, and dimensions matching human blastocysts. Single-cell RNA sequencing revealed epiblast-, trophectoderm-, and hypoblast-like populations with transcriptomic profiles closely resembling human E6-7 blastocysts. Comparative analysis demonstrated that orangutan blastoids exhibited stronger transcriptional fidelity to human embryos than existing macaque models, establishing their unique value for developmental studies. Functional validation confirmed that orangutan blastoids could generate embryonic stem cell-like and trophectoderm stem cell-like lines, recapitulate trophoblast differentiation and secrete chorionic gonadotropin. However, primitive endoderm/hypoblast-like cells failed to form a distinct compartment, mirroring limitations observed across other primate models. This orangutan blastoid system provides a less ethically restrictive platform for studying human-relevant embryogenesis while maintaining high developmental fidelity. Beyond developmental insights into primate development, this model may inform future reproductive interventions for critically endangered great apes as conservation technologies advance.\u003c/p\u003e","manuscriptTitle":"Bridging the gap in embryogenesis research: North Bornean orangutan-derived blastoids as models for human development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 13:05:41","doi":"10.21203/rs.3.rs-8154543/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a58169cd-89a7-4902-9ed8-215b20dc5a14","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59111213,"name":"Biological sciences/Developmental biology/Embryogenesis/Cell lineage"},{"id":59111214,"name":"Biological sciences/Developmental biology/Pluripotency"},{"id":59111215,"name":"Biological sciences/Developmental biology/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells"},{"id":59111216,"name":"Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells"},{"id":59111217,"name":"Biological sciences/Stem cells/Reprogramming"}],"tags":[],"updatedAt":"2026-02-04T14:47:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 13:05:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8154543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8154543","identity":"rs-8154543","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}