Derivation of embryonic stem cells across avian species

Avians, especially chickens, are crucial model organisms in developmental biology, virology, immunology, epigenetics, conservation biology and genomics related to domestication^1,2,3. Moreover, the chicken model organism has enhanced our understanding of human diseases⁴. However, due to the lack of genetic tools in avians, the use of this species in research has been greatly limited.

Embryonic stem (ES) cells derived from mice and rats can be maintained in culture indefinitely while retaining the ability to contribute to chimera formation and transmit through the germline^5,6,7,8. This property has enabled us to generate numerous genetically modified animals, substantially advancing our understanding of biology in various aspects. However, attempts to establish chimeric and germline-competent ES cell lines from species other than rodents have, thus far, proved unsuccessful.

Blastodermal cells isolated from freshly laid chicken eggs (EGK.X stage) can contribute to both somatic and germline lineages when reintroduced into EGK.X-stage chicken embryos, indicating that the chicken blastoderm contains germline-competent pluripotent cells⁹. ES cell-like cell lines have been previously established from chicken blastodermal cells; however, these cells tend to lose their ability to form chimeras and transmit through the germline after prolonged culture^10,11,12,13. This has led to the prevailing hypothesis that germline specification in birds may be predetermined by maternal inheritance and cannot be induced from epiblast cells or ES cells derived from them¹⁴.

It is worth noting that primordial germ cells (PGCs), the progenitors of the gamete lineage, have emerged as a focal point in avian reproductive biology due to their unique role in preserving germline integrity. Unlike their mammalian counterparts, avian PGCs transiently circulate in the bloodstream during early embryogenesis, allowing for their isolation at well-defined developmental stages (Hamburger–Hamilton stages 14–16). Pioneering studies in chickens have led to the development of in vitro culture systems that support long-term propagation of PGCs under conditions both with and without serum and feeders, while retaining germline competency^15,16. These advances have established the utility of avian PGCs for genetic conservation, biodiversity preservation and transgenesis, particularly for applications such as avian bioreactors and genome-edited poultry models. However, substantial limitations remain. Despite decades of effort, reliable PGC propagation is largely restricted to chickens, with only limited success in other avian species¹⁷. In contrast to PGCs, ES cell derivation may be more readily adaptable across diverse avian species. Overcoming species barriers using ES cells may permit applications across diverse avian populations, including endangered species, nonmodel avian species with limited embryo availability and other economically important birds. Furthermore, the capacity of ES cells to differentiate into both germline and somatic lineages broadens their use in basic biological studies and commercial applications such as cultivated meat production. As such, pluripotent or ES cells retain complementary value for germline preservation, offering greater flexibility and scalability, despite differences in differentiation potential.

Recent studies on mouse pluripotent stem cells have revealed the existence of multiple pluripotent states¹⁸. Even pluripotent cells derived from closely related developmental stages can exhibit distinct responses to signaling pathways that govern self-renewal¹⁸. The precise pluripotent state of avian blastodermal cells remains elusive in comparison to mammals. Nonetheless, it is well established that key transcription factors and signaling pathways crucial for early development and lineage specification are often conserved across diverse animal species, including birds and mammals^{19,20,21,22,23}.

Given this evolutionary conservation, we hypothesized that, akin to mammalian pluripotent cells, the pluripotent state of avian blastodermal cells can be captured and sustained in vitro under appropriate culture conditions.

Optimizing chicken ES cell culture conditions

To develop culture conditions for deriving chicken ES cells, we isolated blastodermal cells from freshly laid Rhode Island Red (RIR) chicken eggs at the EGK.X stage. We then screened a panel of small molecules, growth factors and cytokines previously shown to support self-renewal in mouse, rat or human pluripotent stem cells (Supplementary Table 1). Morphologically undifferentiated ES cell colonies emerged when the culture medium was supplemented with either the Wnt/β-catenin signaling inhibitor IWR-1 or the protein kinase C (PKC) inhibitor Gö6983 (Fig. 1a). Similar effects were observed with other Wnt and PKC inhibitors (Extended Data Fig. 1a). Notably, the combination of IWR-1 and Gö6983 produced a synergistic effect in promoting self-renewal of chicken ES cells (Extended Data Fig. 1b). ES cells derived in the presence of IWR-1 and Gö6983 expressed Nanog messenger RNA (mRNA), a key pluripotency marker^24,25 (Extended Data Fig. 1c). However, chicken ES cells began to differentiate on passaging, and could not be maintained beyond two passages in the presence of IWR-1, Gö6983 or both (Extended Data Fig. 1d).

We observed that chicken ES cells exhibited improved growth when a larger amount of egg yolk was carried over during embryo isolation. Moreover, ES cells retained their undifferentiation state for up to 2 weeks of initial plating when the culture medium, which contained residual yolk, was left unchanged and cells were not passaged (Extended Data Fig. 1e). This observation led us to speculate that egg yolk might contain crucial component(s) for promoting chicken ES cell self-renewal. Indeed, by supplementing the culture medium with yolk in combination with IWR-1, and Gö6983 (referred to as Yolk/2i), we were able to continuously passage chicken ES cells while maintaining their undifferentiated morphology (Fig. 1b). Notably, both fertilized and unfertilized egg yolks supported chicken ES cell self-renewal equally well (Fig. 1b and Extended Data Fig. 1f). The pluripotent identity of the expanded chicken ES cells was confirmed by high expression of Nanog mRNA, which was rapidly downregulated upon withdrawal of the self-renewal cocktail (Extended Data Fig. 1g).

Next, we aimed to identify the self-renewal-promoting component(s) within the yolk using the experimental strategy outlined in Fig. 1c. Initially, we fractionated the yolk into sedimented granules and plasma. Our results indicated that the self-renewal-promoting effect was attributed to the component(s) present in the plasma rather than the granules (Extended Data Fig. 2a). We then used molecular-weight cutoff membrane filters to fractionate the plasma into three fractions: <50 kDa, 50–100 kDa and>100 kDa. Only the 50–100-kDa fraction retained the self-renewal-promoting effect (Extended Data Fig. 2a). Further fractionation of the 50–100-kDa fraction using ammonium sulfate precipitation revealed that the protein precipitates recovered at different ammonium sulfate concentrations exhibited distinct effects on maintaining chicken ES cells. Notably, the precipitates obtained at 80% saturation of ammonium sulfate (80p) exerted the strongest effect in preserving the undifferentiated state of chicken ES cells (Extended Data Fig. 2b). SDS–PAGE analysis of the 80p fraction revealed an enriched band with an approximate size of ~75 kDa (Fig. 1d). Subsequent mass-spectrometry analysis confirmed the presence of ovotransferrin in the 80p fraction, which was identified as the protein responsible for the self-renewal-promoting effect (Extended Data Fig. 2c and Supplementary Table 2). Note that minor factors observed in the 80% fraction, such as vitellogenin, serum albumin and ovomucoid, were present at high levels in the 50% and 70% fractions, as indicated by the trend box (Extended Data Fig. 2c) and Supplementary Table 2. Neither of these fractions supported self-renewal (Extended Data Fig. 2b).

Furthermore, we successfully derived and maintained chicken ES cells from multiple breeds in the presence of ovotransferrin, IWR-1 and Gö6983 (hereafter referred to as OT/2i) (Fig. 1e and Extended Data Fig. 2d). These findings indicate that ovotransferrin is the primary self-renewal-promoting component in egg yolk. The function of ovotransferrin in the OT/2i condition could not be substituted by its mammalian ortholog, highlighting a species-specific role for ovotransferrin in supporting chicken ES cell self-renewal and survival^16,26,27 (Extended Data Fig. 2e,f). Chicken ES cells cultured in OT/2i retained alkaline phosphatase activity, although at a lower intensity than that observed in mouse ES cells (Extended Data Fig. 2g). Notably, the withdrawal of either IWR-1 or Gö6983 resulted in a rapid loss of self-renewal capacity in vitro, suggesting that all components of the OT/2i cocktail are essential for the successful establishment and maintenance of chicken ES cells (Extended Data Fig. 2h).

Optimizing culture conditions for ES cells across avian species

We investigated the feasibility of deriving ES cells from various avian species using the OT/2i culture system. Blastodermal cells from fertilized eggs of pheasant, duck and turkey were cultured in OT/2i. While ES cell colonies emerged within 2–3 days, they rapidly differentiated, primarily into beating cardiomyocytes (Extended Data Fig. 3a and Supplementary Video 1). SB431542, a potent and selective inhibitor of activin receptor-like kinases −4, −5 and −7, previously shown to block cardiomyocyte differentiation²⁸effectively prevented this differentiation a nd maintained pheasant, duck and turkey ES cells in an undifferentiated state (Extended Data Fig. 3b–d). However, long-term self-renewal of quail ES cells could not be sustained using OT/2i + SB431542 (hereafter referred to as OT/3i). For quail ES cell maintenance, the addition of chicken leukemia inhibitory factor (LIF) was essential (Extended Data Fig. 3e,f). The self-renewal-promoting effect of chicken LIF on quail ES cells could not be replicated using human LIF, likely due to the limited sequence homology between avian and mammalian LIF, as previously reported²⁹ (Extended Data Fig. 3e).

While the combination of OT/3i and chicken LIF (referred to as OT/3i/chLIF) was sufficient for the derivation and maintenance of ES cells from chicken, quail, goose, duck, turkey, pheasant and peafowl, we found that ostrich ES cells could be derived and maintained in OT/3i, and that the addition of chLIF induced differentiation (Fig. 2a,b and Extended Data Fig. 3g,h). Two ES cell lines, one from chicken and one from quail, established in OT/3i/chLIF, were subjected to karyotype analysis. G-banding revealed that both lines were predominantly diploid (50 out of 50 cells for chicken ES cells and 44 out of 48 cells for quail ES cells), with approximately 10% of the quail cells exhibiting haploid or near-haploid karyotypes (Extended Data Fig. 3i,j).

Next, we compared the efficiency of chicken ES cell derivation and maintenance between OT/3i/chLIF and OT/2i. Both conditions successfully facilitated single-embryo derivation, resulting in eight stable ES cell lines from nine embryos in either condition and supported long-term maintenance of chicken ES cells. Notably, chicken ES cells propagated more robustly in OT/3i/chLIF than in OT/2i, suggesting a synergistic effect of SB431542 and chicken LIF with IWR-1 and Gö6983 in promoting chicken ES cell self-renewal (Fig. 2c). It is worth noting that some established chicken ES cell lines occasionally lost their characteristic colony morphology, defined by tightly packed, uniform cells, and became prone to differentiation. This phenotype typically occurred after extended culture (>20 passages) and when cells became overly confluent before passaging (Extended Data Fig. 3k). Similar to observations in stem cell cultures from other species, high cell density may create a local microenvironment that promotes sustained MAPK (MAP-kinase) signaling via combined autocrine secretion, paracrine support and extracellular matrix stabilization^30,31. To address this, we tested whether inhibition of MAPK signaling, which is not directly targeted in the OT/3i/chLIF condition, could help stabilize clonal morphology during long-term culture. We found that the addition of CP-673451, a selective inhibitor of platelet-derived growth factor receptor, effectively prevented spontaneous differentiation and stabilized long-term in vitro maintenance of chicken ES cells (Extended Data Fig. 3k). Furthermore, CP-673451 improved the clonogenic efficiency of chicken ES cells (Extended Data Fig. 3l).

In the chicken embryo, a subset of epiblast cells retains pluripotency during primitive streak formation (HH2 stage)³². To determine whether these cells could be maintained under chicken ES cell culture conditions, we isolated and cultured cells from HH2-5 embryos in OT/2i, OT/3i or OT/3i/chLIF. However, all cells underwent differentiation (Extended Data Fig. 3m), suggesting that the EGK.X stage represents a restricted developmental window for successful derivation of chicken ES cells using the described conditions.

Chicken ES cells derived in OT/3i/chLIF are pluripotent

Chicken ES cells exhibit heterogeneous expression of pluripotency-associated surface markers, including stage-specific embryonic antigen-1 (SSEA-1) and epithelial membrane antigen-1, across individual colonies (Extended Data Fig. 4a). This pattern is consistent with previously reported characteristics of pluripotent stem cells in some nonmammalian systems³³. To further assess the pluripotency of chicken ES cells, we induced their differentiation through embryoid body (EB) formation, a process during which ES cells spontaneously differentiate into derivatives of all three germ layers³⁴. Quantitative PCR with reverse transcription (RT–qPCR) analysis revealed significant downregulation of the core pluripotency genes Nanog, Pou5f3 and Sall4 on EB formation^{24,25,35,36,37,38}along with upregulation of lineage-specific markers for ectoderm, mesoderm and endoderm (Extended Data Fig. 4b). Immunostaining further confirmed the differentiation of chicken ES cells into all three germ layers, as evidenced by the presence of TUJ1-positive (ectoderm), MHC-positive (mesoderm) and GATA4-positive (endoderm) cells (Fig. 3a). In addition, Oil Red O staining verified the differentiation of chicken ES cells into adipocytes (mesoderm) (Extended Data Fig. 4c). Together, these results demonstrate that chicken ES cells are pluripotent and possess the capacity to differentiate into all three germ layers in vitro.

To further characterize the cellular identity, we conducted RNA sequencing (RNA-seq) analysis on OT/3i/chLIF chicken ES cells. Unbiased principal component analysis of bulk RNA-seq data from chicken ES cells, as compared to cells at EGK.VIII, EGK.X and HH4 stages, revealed a close relationship between chicken ES cells and blastodermal cells at the EGK.X stage, from which the ES cells were derived (Fig. 3b, based on PC1). However, notable differences were observed between ES cells and EGK.X cells, resulting in their separation on PC2. As an extra test, we identified signature genes from EGK.I to HH4 stages and performed heatmap analysis, which demonstrated that ES cells and EGK.X cells clustered together and were distinct from cells at other developmental stages (Extended Data Fig. 4d).

Subsequently, we conducted a comprehensive transcriptomic analysis of chicken ES cells, their in ovo counterpart EGK.X cells and HH4 cells at the gastrulation stage. At the whole-transcriptome level, chicken ES cells exhibited a higher linear correlation with EGK.X cells (R = 0.8), compared to HH4 cells (R = 0.73) (Extended Data Fig. 4e). Gene set enrichment analysis further confirmed that the expression of EGK.X signature genes, rather than HH4 signature genes, was significantly upregulated in chicken ES cells (Extended Data Fig. 4f).

By comparing gene expression in chicken ES cells with cells from EGK.X and HH4 embryos, we categorized all genes into three groups: A, B and C (Fig. 3c and Extended Data Fig. 4g). Group A contained 1,386 genes, with 358 upregulated and 1,028 downregulated in both chicken ES cells and EGK.X stage embryos relative to HH4 stage embryos. Within this group, core pluripotency genes, such as Nanog, Pou5f3 and Klf4 were highly expressed in chicken ES cells and EGK.X embryos (Fig. 3d). Gene ontology term enrichment analysis of the downregulated genes in group A revealed numerous differentiation and development-related gene ontology terms, suggesting that the OT/3i/chLIF condition effectively suppressed ES cell differentiation (Fig. 3e,f).

Group B contained 1,046 genes that showed no statistical difference between EGK.X and HH4 stage embryos but were significantly upregulated (384 genes) or downregulated (662 genes) in chicken ES cells. Gene ontology term analysis revealed that the upregulated genes were primarily involved in metabolic processes, suggesting that the differential expression between chicken ES cells and cells from EGK.X and HH4 stage embryos could be partially attributed to the environmental differences between in vitro and in ovo conditions (Extended Data Fig. 4g,h). Group C included the remaining 21,944 genes. Of these, 20,913 showed no statistically significant differences among chicken ES cells, EGK.X and HH4 embryos. The other 1,031 genes were differentially expressed between EGK.X and HH4 stage embryos but showed no significant differences between chicken ES cells and HH4 embryos. However, a gene ontology term analysis of these 1,031 genes did not reveal any statistically significant gene clusters (P_adjust < 0.05).

In our bulk RNA-seq experiment, EGK.X embryos were collected in their entirety, comprising not only epiblast cells but also hypoblast cells and cells from the extra-embryonic region (area opaca). To further investigate the identity of chicken ES cells, we analyzed the expression of marker genes specif ic to the hypoblast and area opaca regions in both EGK.X embryos and cultured chicken ES cells^39,40. This comparative analysis revealed that chicken ES cells exhibit low expression of markers associated with those two lineages, suggesting that the cultured ES cells closely resemble epiblast cells at the EGK.X stage (Fig. 3g).

In mice, at least three pluripotent states (naive, formative and primed) have been proposed¹⁸. To assess the pluripotent state of chicken ES cells, we conducted transcriptome correlation analysis using overlapping gene orthologs between two species and observed that chicken ES cells exhibited a closer resemblance to mouse formative pluripotent stem cells than to mouse naive or primed pluripotent stem cells (Fig. 3h).

In summary, our findings demonstrate that the OT/3i/chLIF condition effectively captures and maintains chicken ES cells in a pluripotent state resembling that of in ovo chicken EGK.X-stage epiblast cells.

Chicken ES cells are amenable to precise genome editing

To assess the genome-editing capability of chicken ES cells, we used a GFP-to-blue fluorescent protein (BFP) conversion assay to quantify CRISPR–Cas9-mediated genome editing via nonhomologous end joining and homology-directed repair^41,42. A GFP-positive chicken ES cell line was derived from a transgenic chicken ubiquitously expressing GFP (Roslin Green)⁴³. These cells were electroporated with Cas9 protein, a GFP single-guide RNA, and a single-stranded oligodeoxynucleotide (ssODN) donor to induce GFP-to-BFP knock-in conversion (Extended Data Fig. 5a). Flow cytometry analysis revealed a GFP-to-BFP conversion efficiency (BFP⁺/GFP⁻) of 9.3%, with an extra 3.7% of cells displaying single-allele conversion (BFP⁺/GFP⁺) (Extended Data Fig. 5b). Clonal populations were subsequently established via fluorescence-activated cell sorting and low-density plating, followed by expansion (Extended Data Fig. 5c). Genotypic analysis confirmed precise genome editing consistent with the observed fluorescence profiles (Extended Data Fig. 5d). These findings demonstrate that chicken ES cells are amenable to efficient and precise genome editing, supporting their use as a robust platform for both basic research and biotechnological applications.

Avian ES cells express core pluripotency factors

To characterize the pluripotency identity of avian ES cells beyond chicken, we examined ES cell lines derived from quail, goose and ostrich. Similar to chicken ES cells, these ES cell lines retained alkaline phosphatase activity, although the level and pattern of expression varied among colonies and species (Extended Data Figs. 2g,h, 3f,h and 6a). Quail ES cells demonstrated the capacity for in vitro differentiation into all three germ layers, as evidenced by the expression of TUJ1 (ectoderm), MHC (mesoderm) and GATA4 (endoderm) (Extended Data Fig. 6b). To further explore transcriptional identity, we performed bulk RNA-seq on ES cell lines derived from quail, goose and ostrich, and compared them to chicken ES cells. All four avian ES cell lines expressed high levels of key pluripotency-associated transcription factors, including Nanog or Nanog-like, Lin28a/b and Pou5f3while other Pou family members were not detected (Extended Data Fig. 6c). We also examined the expression of SOX and KLF family genes. Unlike mammalian ES cells, avian ES cells did not express Sox2; instead, Sox3 was consistently expressed in ES cells derived from all four avian species. In addition, Sox4 and Sox11 were coexpressed in quail and goose ES cells. Regarding KLF family genes, avian ES cells predominantly expressed Klf3, Klf5 and KLF6in contrast to the high Klf4 expression observed in mammalian ES cells. Notably, the expression levels of KLF3 / 5/6 varied among the different avian ES cell lines.

Avian ES cells show species-specific responses to LIF signaling

While chicken, quail and goose ES cells exhibited improved self-renewal in the presence of exogenous LIF, ostrich ES cells responded differently: LIF exposure induced differentiation, potentially toward trophectoderm lineages (Extended Data Fig. 3e–h). Transcriptomic profiling revealed that ostrich ES cells uniquely expressed elevated levels of LIF receptor (LIFR) and GP130 (IL6ST) compared to other avian ES cell lines (Extended Data Fig. 6d). This observation aligns with previous mechanistic studies in mouse ES cells, where STAT3 signaling functions as a dose-sensitive ‘rheostat’ rather than a binary switch in regulating cell fate⁴⁴. Together, these findings suggest that while ES cells derived from diverse avian species share core pluripotent features with chicken ES cells, they also display species-specific regulatory response, particularly in their sensitivity to the LIF/STAT3 signaling pathway.

Chicken ES cells derived in OT/3i/chLIF are chimera-competent

The ability to contribute to chimera formation is a defining characteristic of authentic ES cells. To investigate whether OT/3i/chLIF chicken ES cells possess this capacity, we implanted them onto ex ovo incubated chicken embryos (Fig. 4a). GFP-positive chicken ES cells efficiently integrated into the developing chicken EGK.X-stage embryos, leading to the formation of chimeric embryos (Fig. 4b,c). In contrast, chicken ES cells implanted into HH4 stage embryos failed to integrate (Fig. 4d), highlighting the importance of matching developmental stages between donor cells and recipient embryos^45,46.

While the ex ovo culture system provided a straightforward means to assess the chimera formation potential of chicken ES cells, the in ovo system was necessary for long-term monitoring of donor cell development. We used the surrogate open-shell system to observe chimeric embryo development until hatching⁴⁷ (Fig. 4e and Extended Data Fig. 7a). GFP-positive chicken ES cells were injected into the subgerminal cavity of EGK.X-stage chicken embryos. Embryonic development and the distribution of GFP-positive cells were monitored through a window sealed with plastic film (Extended Data Fig. 7b). Chicken ES cells contributed to multiple lineages within the chicken embryos in ovo (Extended Data Fig. 7c and Supplementary Video 2). However, the overall level of in ovo chimerism was relatively low compared to ex ovo chimeras.

Previous studies demonstrated that sublethal irradiation of recipient chicken embryos can enhance the contribution of injected blastodermal cells⁴⁸. Applying this method, irradiation of recipient embryos at 500–550 cGy (rad) before chicken ES cell injection increased the extent of chimerism without substantially compromising embryo viability (Fig. 4f,g and Extended Data Fig. 7d). Long-term cultured chicken ES cells, which included 60 passages on feeders and 15 passages under feeder-free conditions, retained their capacity for chimera formation (Extended Data Fig. 7e). Immunostaining for lineage markers in chimeric embryos demonstrated donor cell contribution to neural tube formation, indicated by SOX2 expression, and to the mesoderm, marked by Brachyury expression (Extended Data Fig. 7f). Notably, chicken ES cells gave rise to both somatic and extra-embryonic cells in the chimeric embryos (Fig. 4g and Supplementary Videos 3 and 4), a feature distinct from mouse ES cells.

To assess the sustainability of functional engraftment over time, we analyzed chimeric embryos at later developmental stages (embryonic day 8–21 and posthatching). Visible coat color chimeras were successfully obtained, consistent with the chimera rate determined by GFP coverage at E4 (Fig. 4h,i). Furthermore, we detected donor cell contribution to organs from multiple germ layers (Fig. 4j). A summary of the avian ES cell–embryo chimeras is provided in Supplementary Table 3, and the ratios of chimerism in chicken ES cell–chicken embryo chimeras generated via nonirradiation and irradiation methods are provided in Extended Data Fig. 7d. Collectively, the ex ovo and in ovo experiments demonstrated the efficient integration of chicken ES cells into embryonic development, contributing to both somatic and extra-embryonic cell lineages.

Chicken ES cells contribute to feather patterning in chimeras

To further monitor the development of engrafted chicken ES cells over the long-term, embryos with feather color chimerism were hatched and raised for observation (Fig. 4i and Extended Data Fig. 8a). In adult feathers, the feather follicle undergoes cyclic renewal, and the pigment patterns depend on the follicle melanocyte stem cells⁴⁹. The White Leghorn chicken strain has a mutation that affects melanocytes’ ability to process melanosome properly, resulting in an apigmented phenotype⁵⁰. We investigated whether chicken ES cells derived from RIR could contribute to the functional melanocyte lineage and exhibit pigmentations in the chimera. Contour feather follicles were collected from adult chimeric chickens and compared to feathers from adult White Leghorn and RIR chickens (Extended Data Fig. 8b–d). Par affin sections were stained with hematoxylin and eosin, revealing normal feather follicle development in chimeric chickens (Extended Data Fig. 8e–g). Bright views of feather follicles displayed pigmented chimeric feathers compared to apigmented White Leghorn feathers (Extended Data Fig. 8h–j). Staining for microphthalmia-associated transcription factor (MITF), a marker for melanocyte progenitor cells⁵¹showed MITF expression in RIR and chimeric chicken feathers, while White Leghorn chicken feathers were MITF-negative (Extended Data Fig. 8k–m). In addition, chimeric chicken feathers were GFP-positive (Extended Data Fig. 8n). All together, these findings indicate that chicken ES cells derived from RIR contributed to the generation of melanocyte progenitor cells inside the recipient White Leghorn feather follicles, resulting in feather pigmentation in chimeric chickens.

Avian ES cells contribute to intra- and interspecies chimeras

To evaluate the developmental potential of ES cells derived from other avian species, we generated quail and goose ES cells that constitutively expressed GFP (Fig. 5a,b). Given that quail-chick chimeras can be created by injecting quail blastodermal cells into chicken embryos⁵²we hypothesized that this interspecies chimera system could be used to assess the chimera competency of avian ES cells. GFP-positive quail and goose ES cells were implanted onto ex ovo incubated EGK.X-stage chicken embryos, resulting in the successful integration of donor cells into the recipient chicken embryos (Fig. 5a,b).

To further confirm the chimera formation capacity of avian ES cells, we injected GFP-positive quail and goose ES cells into chicken embryos in ovo. Both quail and goose ES cells contributed to chimera formation within developing chicken embryos (Fig. 5c,d). Notably, donor ES cells from quail and goose participated in the somitogenesis process in recipient chicken embryos. The stable integration of quail ES cells within chicken embryos was evidenced by coat color chimerism, with black feathers from donor quail cells appearing on an apigmented White Leghorn chicken embryo (Fig. 5e). In addition, GFP-positive donor cells from quail and goose were found in the blood circulation, indicating their contribution to the blood lineage in the recipient chicken embryos (Supplementary Videos 5 and 6).

Finally, substantial integration was observed when quail ES cells were injected into the subgerminal cavity of quail embryos in ovo, with donor cells contributing to organs derived from multiple germ layers (Fig. 5f,g and Supplementary Video 7). These findings demonstrate that quail and goose ES cells can efficiently contribute to chimera formation.

Avian ES cells can differentiate into germ cells

Next, we evaluated the germ cell differentiation potential of chicken ES cells. We generated EBs from chicken ES cells and examined the expression of germ cell markers, including Dazl, Cvh, Cxcr4, Sycp3 and Stra8 (ref. ⁵³). The relative expression levels of these markers significantly increased in day-4 EBs compared to undifferentiated chicken ES cells, but subsequently decreased in day-8 EBs, likely due to the expansion of somatic cells within the EBs (Fig. 6a). This peak in germ cell marker expression identified day 4 as an optimal time point to further investigate germ cell formation in EBs.

To further investigate germ cell differentiation, we performed immunofluorescence staining for the PGC marker DAZL in both undifferentiated chicken ES cells and differentiating EBs. DAZL expression was not detected in chicken ES cells, but a subset of cells (1–3%) in day-3–4 EBs stained positive for DAZL, including those derived from goose ES cells (Fig. 6b, Extended Data Fig. 9a and Supplementary Video 8). Immunostaining of cultured goose ES cells showed a consistent, albeit very low percentage (0.01–0.14%, n = 7) of DAZL-positive cells without the need of EB formation. To determine whether these DAZL-positive cells originated from cultured ES cells or residual PGCs present in the initial culture, goose ES cells were seeded at low density to allow clonal expansion. Individual colonies were picked and expanded to establish clonal lines (n = 4). DAZL-positive cells were still detected in all clonal lines, confirming that cultured goose ES cells possess the capacity to generate DAZL-positive cells in vitro (Extended Data Fig. 9b). To further characterize ES cell-derived PGC-like cells, we designed a probe targeting Cvh for in situ hybridization chain reaction and validated its specificity in chicken HH8 embryo (Extended Data Fig. 9c). Using this probe, we also found Cvh-positive cells inside the chicken EBs (Extended Data Fig. 9d).

To examine germline contribution in ovo, we injected male GFP-positive chicken ES cells into EGK.X-stage chicken embryos pretreated with 500–550-cGy irradiation. Sublethal irradiation has been shown to increase the germline transmission rate of donor PGCs in chickens by reducing the number of endogenous PGCs⁵⁴. All subsequent analysis of donor ES cell-derived germ cells were performed exclusively using male-to-male donor–recipient pairings. GFP-positive donor cells were present in the gonads of chicken embryos injected with chicken ES cells, particularly in high-grade chimeras (Fig. 6c).

We analyzed the presence of ES cell-derived germ cells in chimeric embryos at different developmental stages. In E9 chimeric embryos, DAZL-double-positive and GFP-double-positive cells were present in gonads as small clusters consisting of 1–3 cells (Fig. 6d). In E15 and later chimeric embryos, larger clusters of GFP-positive cells that were also positive for DAZL or SSEA-1, another germ cell marker⁵⁵were found in the gonads (Fig. 6d–f and Extended Data Fig. 9e). In E21 embryos, some PGCs were enclosed within the seminiferous tubules as gonads continued to develop. GFP-positive PGCs, identified by staining with P63, a germ cell marker⁵⁶could be detected within the tubules alongside endogenous PGCs. Furthermore, some seminiferous tubules contained GFP cells that differentiated into Sertoli cells, as indicated by staining with SOX9 (Fig. 6g).

Multiple chicken ES cell lines were confirmed to contribute to the germline lineage within chicken embryos (Supplementary Table 4). Notably, chicken ES cell lines that failed to differentiate into DAZL-positive cells in vitro were able to give rise to germ cells after transplantation into chicken embryos, suggesting a more favorable germline-inductive environment in ovo compared to the spontaneous differentiation environment in vitro. Qualitative analysis indicated that high-grade chimeras exhibited a substantial proportion of donor-derived PGCs (Fig. 6d,e and Supplementary Table 4). In contrast, donor-derived PGCs were present at low frequencies in mid-grade chimeras and were not observed in low-grade chimeras (Supplementary Table 4). In conclusion, by tracking the germline development of avian ES cells, we demonstrated their competence to adopt a germ cell fate both in vitro and in ovo.

This study demonstrates that avian ES cells can be efficiently derived and maintained long-term without losing their pluripotency, including the ability to differentiate into germ cells. The establishment of avian ES cells, coupled with advancements in gene-editing technology⁵⁷creates a robust platform for generating genetically modified avian species. These results hold promise for applications in developmental biology, the bioreactor industry, poultry and related fields. Moreover, the development of avian ES cell self-renewal conditions opens avenues for conserving endangered species and reviving extinct avian animals in combination with cellular reprogramming technology^33,58.

In contrast to mammals, the mode of PGC formation in birds has long been debated^14,59. Our study challenges the prevailing notion of maternal specification in chickens, revealing that in vitro expanded chicken ES cells can give rise to PGCs both in vitro and in ovo. Detailed analysis across developmental stages confirmed chicken ES cell-derived PGC identity, revealing a trajectory that mirrors endogenous PGCs. While avian ES cell lines exhibit variable in vitro differentiation capacity into the germline lineage, efficient adoption of a germline fate on in ovo transplantation suggests a promising avenue for generating transgenic avian species.

A limitation of this study was the absence of definitive germline transmission, despite the demonstration of donor ES cells differentiating in ovo to PGCs. This limitation was due to the failure of high-grade chimeras to hatch. Notably, 32 high-grade chimeras developed normally until 3 days prehatching, but none successfully hatched. Strain-specific incompatibility likely contributed to this outcome, where RIR ES cells were injected into White Leghorn recipient embryos. Previous work using blastodermal donor cells demonstrated reduced hatch rates and somatic chimerism using this RIR-White Leghorn combination compared to other donor–recipient pairings (for example, 7.4% hatch rate for RIR versus 33.8% for Barred Plymouth Rock and 56.1% for Green-legged Partridge-like in White Leghorn recipients)⁶⁰. Our results align with these observations, suggesting that donor–recipient incompatibilities may hinder late embryonic viability in high-grade chimeras. Meanwhile, six low-grade to mid-grade male chimeras were successfully hatched but produced zero donor-derived offspring among 255 fertilized eggs. The absence of donor-derived offspring from these chimeras was consistent with our characterization of chimeric gonads, which indicated an absence of donor-derived PGCs in low-grade chimeras. While these data were not included in the main results due to iteratively evolving methodologies, they are reported here to inform replication efforts. For future experiments, it would be advisable to use genetically tagged donor ES cells injected into recipient embryos of the same strain or more optimally matched strains. This approach is expected to improve the production and viability of high-grade chimeras, a critical step for rigorous assessment of germline transmission. Furthermore, elucidating molecular mechanisms governing ES cell-to-PGC differentiation will be crucial for advancing both our understanding and the practical application of avian germline engineering.

The capture of ES cells across avian species underscores the conserved roles of Wnt and PKC signaling in governing the pluripotency of avian blastodermal cells. However, applying the chicken ES cell self-renewal condition (OT/2i) to other avian species proved ineffective due to subtle differences in cell fate control. Through meticulous adjustments, we identified the self-renewal-promoting effects of SB431542 and chicken LIF, which were initially excluded during the development of the chicken ES cell culture condition. Unlike ES cells from other avian species, ostrich ES cells rapidly underwent differentiation (potentially toward the trophectoderm lineage) in response to chicken LIF. Cross-species RNA-seq analysis revealed elevated expression of both LIF receptor and GP130 (IL6ST) in ostrich ES cells compared to chicken, quail and goose ES cells (Extended Data Fig. 6d). In mouse ES cells, it has been shown that supra-threshold STAT3 activation can shift its role from promoting self-renewal to inducing trophectoderm differentiation via Tfap2c/Cdx2 induction⁴⁴. We propose that ostrich ES cells intrinsically operate near or above this critical STAT3 activation threshold due to increased expression of LIF receptor components. Elevated levels of LIF receptor/Gp130 may amplify LIF-induced STAT3 signaling beyond the optimal range for pluripotency maintenance, thereby driving differentiation, resembling the phenotype observed in hyper-stimulated mouse ES cells. This dose-dependent STAT3 paradigm, initially characterized in murine systems, may underlie the divergent LIF responses observed across avian species. Together, our findings emphasize the importance of tailoring culture conditions to each species and highlight the utility of supportive compounds, such as SB431542, in fine-tuning the balance between self-renewal and differentiation^61,62.

While our study primarily focused on chicken ES cells, further comprehensive investigations are warranted to assess the developmental potential of ES cells from other avian species through chimera formation and germline transmission assays. The lessons learned from developing avian ES cell culture conditions offer valuable insights for deriving pluripotent cell lines from diverse species. Notably, the conservation of key signaling pathways regulating pluripotency across species emphasizes the importance of understanding primary cell signaling responses for successful stem cell line derivation^61,62.

Regarding the state of avian pluripotency, we propose that the chicken epiblast at the EGK.X stage and the ES cells derived from it closely parallel the formative state of mouse pluripotency, based on molecular, functional and developmental characteristics. Similar to mouse formative pluripotent cells, the EGK.X epiblast and chicken ES cells exhibit reduced expression of canonical naive pluripotency markers (for example, Tfcp2l1, NR0B1, Fbxo15) compared to earlier pre-oviposition embryos (EGK.III/VI/VIII). Notably, chicken ES cells show high expression of the de novo DNA methyltransferase Dnmt3bwhile lacking expression of primitive streak markers (for example, Tbxt, Foxa2, Gsc), distinguishing them from mouse primed epiblast stem cells (Extended Data Fig. 10a). Whole-genome transcriptional correlation analysis further supports this classification, revealing a greater similarity between chicken ES cells and mouse formative pluripotent cells than to either the naive or primed states (Fig. 3h).

Functionally, a defining characteristic of formative pluripotency is the ability to undergo direct multi-lineage differentiation without previous ‘capacitation’, a requirement for naive cells^63,64. Consistent with this, chicken ES cells exhibit robust in vitro differentiation capacity, including rapid responsiveness to differentiation cues (Extended Data Fig. 10b) and specification toward the germline lineage. Furthermore, chimera assays confirm their contribution to both somatic and germline lineages, reinforcing their functional similarity to mouse formative cells.

Developmentally, the formative state in mice spans the postimplantation epiblast (E5.0–E6.5), bridging the naive (pre-implantation, ~E3.5) and primed (~E7.5) phases. The chicken embryo at oviposition (EGK.X) mirrors this developmental window, with the epiblast and hypoblast having undergone morphological segregation and epithelialization underway⁶⁵. Apicobasal polarity, a hallmark of advancing epiblast maturation, emerges shortly thereafter (EGK.XII–XIII)⁴⁰.

The signaling requirements further align chicken ES cells with the formative state. In mice, naive pluripotency is maintained by suppressing fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) signaling, whereas primed pluripotency requires its activation. Like mouse formative cells, chicken ES cells display minimal reliance on ERK pathway modulation, as both inhibition and hyperactivation compromise survival or induce differentiation. Zebra finch blastoderm cells form Nanog/PouV-high aggregates on ERK inhibition, possibly reflecting their earlier developmental stage at oviposition (EGK.VI/VIII), which may correspond more closely to the mouse naive state³⁹. In addition, canonical Wnt inhibition, critical for stabilizing mouse formative pluripotency, is also essential for chicken ES cell derivation, further supporting a conserved functional requirement⁶⁴. Despite these converging lines of evidence, the precise classification of avian pluripotency warrants further exploration. Systematic derivation of avian pluripotent stem cells across developmental stages will be critical for elucidating the continuum of pluripotency in birds and refining its correspondence with mammalian states.

In summary, the derivation of germline-competent chicken ES cells addresses a longstanding chicken model system gap, unlocking future research opportunities. Extending this achievement to other avian species will broaden the scope of biological modeling systems and offer new opportunities in the poultry and biotechnology industries.

Avians and avian fertile eggs

All animal procedures were carried out in accordance with protocols approved by the University of Southern California Institutional Animal Care and Use Committee. Specific-pathogen-free fertile chicken eggs (SPAFAS, White Leghorn) were purchased from Charles River. Fertile avian eggs used for ES cell derivation were purchased from the following sources: chicken breeds includes RIR (AA Laboratory Eggs), White Leghorn, Black Plymouth Rock and Frizzle Polish (all from Meyer Hatchery), pheasant and goose eggs (Sunstate Ranch), ostrich eggs (OstrichLand), Japanese quail and turkey eggs (AA Laboratory Eggs), duck eggs (Metzer Farms) and peafowl eggs (Miyazaki City Phoenix Zoo).

Avian ES cell derivation and maintenance

All avian embryos were staged according to the Eyal-Giladi and Kochav staging system and the Hamburger and Hamilton staging system^66,67. Freshly laid avian fertile eggs at EGK.X-XII stages were kept at 16 °C before harvesting embryos. To harvest embryos, a filter paper-based method was applied⁶⁸. Briefly, a filter paper with a hole at the center was placed on top of the embryo. The outer edge of the filter paper was cut together with the vitelline membrane. The embryo was then released from the yolk together with the filter paper and washed in PBS solution. Next, the embryo was transferred into a 15-ml tube and dissociated by pipetting in N2B27 medium⁶⁹. Dissociated cells were cultured in tissue culture plates preseeded with mitotically inactivated mouse embryonic fibroblasts. For feeder-free culturing, culture plates were precoated with laminin511-E8 (iMatrix-511, Nippi, Inc.) at 0.35 µg per cm² for 1 h at 37 °C. The basal medium used for the routine avian ES cell derivation and maintenance was serum-free N2B27 medium supplemented with 10 µg ml⁻¹ ovotransferrin (Sigma, cat. no. C7786), 2.5 µM IWR-1, 2 µM Gö6983, 1 µM SB431542 (all from Selleckchem) and 10 ng ml⁻¹ chicken LIF (Kingfisher). Optionally, 0.5 µM CP-673451 (Selleckchem) can be added to the culture medium to stabilize established chicken ES cells over long-term passaging. For ostrich ES cell culture, chicken LIF was removed from the avian ES cell culture cocktail. All avian ES cells were cultured at 38 °C in 5% C O₂. For passaging, avian ES cells were incubated in 0.05% Trypsin-EDTA (Gibco) for 1 min at room temperature and neutralized with serum-containing medium (DMEM + 10% fetal bovine serum). Cells were then collected by centrifuge and resuspended in fresh avian ES cell media. Avian ES cells were cryopreserved using CELLBANKER 2 freezing media (Amsbio).

Generation of GFP-labeled ES cell lines

Avian ES cells carrying a constitutively expressed GFP transgene were generated by lentivirus infection. Lenti-X 293T cells (Takara) were transfected with the pSIN-EF2-GFP-Puro plasmid⁷⁰ along with pSPAX2 and pVSVG using polyethylenimine (PEI 25,000, Polysciences). Lentivirus particles were subsequently collected and concentrated using the Lenti-X concentrator (Takara) as per the manufacturer’s instructions. Following lentiviral infection, avian ES cells were treated with 0.5 µg ml⁻¹ puromycin (Invitrogen) for 24 h. This selection was repeated for 3–4 times until no obvious cell death occurred on treatment, and a homogenous GFP expression was confirmed under a fluorescence microscope. The sex of each GFP-labeled chicken ES cell line was determined via PCR using Z and W sex chromosome specific primers^71,72. Primer sequences are provided in Supplementary Table 5.

RNAscope in situ hybridization

The detection of Nanog mRNA expression in chicken blastodermal cells was carried out using the RNAscope 2.5 HD-BROWN kit (Advance Cell Diagnostics) following the manufacturer’s instructions. A specific RNAscope probe targeting chicken Nanog mRNA (Gg-Nanog) was designed by Advance Cell Diagnostics. Chicken blastodermal cells were isolated from fertile eggs and cultured under various conditions in four-well chamber slides (Nunc Lab-Tek II Chamber Slide, Thermo Fisher Scientific) preseeded with mitotically inactivated mouse embryonic fibroblasts. After a 3-day culture period, cells were fixed in 4% paraformaldehyde and PBS for 30 min, followed by dehydration and rehydration with EtOH. Subsequently, cells were treated with hydrogen peroxide for 10 min at room temperature and washed with distilled water. Cells were then treated with protease plus for 30 min at 40 °C and again washed with distilled water. Next, cells were hybridized with the chicken Nanog probe, GG-PPIB as a positive control or DapB as a negative control for 2 h at 40 °C. A six-step amplification process were then performed to amplify the signal of the hybridization probe. To detect the signal, cells were stained with 3,3′-diaminobenzidine and counter-stained with 50% hematoxylin staining solution. Last, slides were dehydrated, mounted and examined under a microscope.

In situ hybridization

Hybridization chain reaction fluorescence in situ hybridization was performed using labeling kits from Molecular Technologies, following the manufacturer’s protocol⁷³. The probe set for Cvh was designed with a B1 initiator and detected using amplifier hairpins conjugated to Alexa647.

RT–qPCR

Chicken embryos were harvested using the filter paper-based method as described above. Total RNA from chicken embryos and in vitro expanded cells was extracted using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. Complementary DNA was synthesized from 1 µg of total RNA using the iScript Reverse Transcription Supermix (BIO-RAD) according to the manufacturer’s protocol. RT–qPCR was performed using the iTaq Universal SYBR Green Supermix (BIO-RAD) on an Applied Biosystems QuantStudio 7 Flex Real-Time PCR machine (Applied Biosystems) with melt curve analysis. Cq detection limit is 40 cycles. Gene expression levels were normalized to Gapdh expression. The primers used for RT–qPCR were custom-designed using primer-blast (NCBI) with the criteria of spanning exon–exon junctions, a melting temperature of 60 °C and a PCR product size between 70 bp and 200 bp. Primer sequences are listed in Supplementary Table 5.

Egg yolk fractionation

Here, 20 ml of yolk was collected from 2 unfertilized chicken eggs and further diluted with 80 ml of 0.15 M NaCl (yielding 20% yolk). This 20% yolk was then centrifuged at 30,000g for 2 h at 4 °C. Yolk plasma was collected (designated as 20% plasma) and granules were resuspended in 50 ml of 0.75 M NaCl (as 20% granules). To further fractionate plasma, eight layers of paper towels presoaked with ddH₂O were used for initial filtration, and the flow-through was sequentially passed through 0.8-µm, 0.45-µm and 0.2-µm filters (Pall). The final flow-through, referred to as the 0.2-µm filtered fraction, was then applied to Amicon Ultra-15 Centrifugal Filters (Millipore) with a 100-kDa and 50-kDa molecular-weight cutoff at 10,000g for 1 h at 4 °C. This yielded fractions designated as>100 kDa, 50–100 kDa and <50 kDa. All fractions were considered to be at 20% concentration and were added to cell culture medium as a final concentration of 3% for testing.

An ammonium sulfate precipitation was performed to separate proteins in the 50–100 kDa fraction. Saturated ammonium sulfate solution was slowly added into the fraction to achieve a 30% salt concentration, and the mixture was stirred at 4 °C for 1 h followed by centrifugation at 5,000g for 30 min. The pellet was resuspended in 0.15 M NaCl and marked as 30p. The remaining supernatant was loaded again with saturated ammonium sulfate solution to achieve a 40% salt concentration, followed by mixture and concentration. The same procedure was repeated to collect samples marked as 40p, 50p, 60p, 70p and 80p. All these fractions were centrifuged twice with an Amicon Ultra-0.5 Centrifugal Filter Unit at a 3-kDa molecular-weight cutoff to remove any residual ammonium sulfate before cellular assay.

The ammonium sulfate fractions were separated by SDS–PAGE and stained with Coomassie blue. Gel fractions were cut and sent to Proteomics Core Facility at the University of Southern California for liquid chromatography with tandem mass-spectrometry analysis.

Alkaline phosphatase staining

To perform alkaline phosphatase staining, we used Vector Blue Substrate Kit (SK-5300, Vector Laboratories) and followed the manufacturer’s instruction. Briefly, ES cells were fixed using 4% PFA for 1 min at room temperature and washed once with PBS. Working solution was prepared by adding 16 μl of Reagent 1, 16 μl of Reagent 2 and 9 μl of Reagent 3 to 1 ml of 100 mM Tris-HCl buffer (pH 8.2–8.5) with 0.1% Tween-20. Cells were incubated with working solution for 40 min in dark at room temperature. After staining, cells were washed once with PBS and proceeded to imaging analysis.

Growth curve analysis

To analyze the growth kinetics of stem cells cultured in different conditions, 43,000 of chicken ES cells were seeded in 24-well plates in duplicate. Cell counts were performed at every passage using trypan blue dye. Cell counts were plotted against passage numbers using GraphPad Prism software to generate growth curves.

Chromosome analysis

Metaphase spreads were prepared after colcemid and ethidium bromide treatment. G-banding and karyotype analysis was performed at the Molecular Cytogenetics Laboratory, Texas A&M University.

GFP-to-BFP knock-in conversion assay

The GFP⁺ chicken ES cell line was established from a single transgenic chicken egg ubiquitously expressing GFP (Roslin Green)⁴³. For GFP-to-BFP knock-in conversion, GFP⁺ chicken ES cells were electroporated using the Neon Transfection System (Thermo Fisher) together with Cas9 ribonucleoprotein and ssODN. Briefly, per each reaction, TrueCut HiFi Cas9 Protein (1,500 ng, Thermo Fisher), TrueGuide Synthetic guide RNA (gRNA) (10 pmol, Thermo Fisher) and ssODN (15 pmol, ultramer DNA oligo from IDT) were incubated in Resuspension buffer R at room temperature for 20 min. Cells were detached, counted and washed with PBS, and then added to the Cas9 ribonucleoprotein and ssODN mixture in Resuspension buffer R to reach to a final volume of 12 μl with 150,000 cells. Cell mixture was electroporated using a 10-μl Neon tip with a program of 1,200 V, 20 ms and 2 pulses (or 1,200 V, 30 ms and 1 pulse). Afterward, cells were directly transferred to the culture plate with prewarmed complete culture medium (OT/3i/chLIF/CP67) plus 10 μM Y-27632 (ROCK inhibitor, Selleckchem). Culture medium was completely changed the next day to remove Y-27632 and cells were maintained on feeder layers. Engineered cells were then sorted according to fluorescence expression pattern and 1,000 sorted cells were seeded in a 12-well plate to allow clonal growth. Individual clones were picked and expanded for genotype analysis. Genomic DNA was extracted using DNeasy Blood & Tissue kit (Qiagen). The sequences of the GFP gRNA, ssODN donor and genotyping PCR primers were obtained from a previous study⁴² and are listed in Supplementary Table 5.

RNA-seq sample preparation and analysis

Chicken EGK.X-stage embryos were harvested using the filter paper-based method⁶⁸. Total RNA from chicken ES cells and EGK.X embryos was extracted using the RNeasy Mini Kit (Qiagen). RNA samples were processed for library preparation following the BGISEQ-500 library construction protocol, and library quality was assessed using a Bioanalyzer. Three biological replicates were collected for each cell type, and a minimum of 20 million paired-end reads was generated per replicate. Quail, goose and ostrich ES cell samples were prepared and sequenced using the same protocol.

The sequencing reads were aligned to the Gg6 chicken reference genome (Ensembl GRCg6), quail reference genome (Ensembl Coturnix_japonica.Coturnix_japonica_2.0.113), goose reference genome (Ensembl Anser_cygnoides.GooseV1.0.113) or Ostrich reference genome (Ensembl Struthio_camelus_australis.ASM69896v1.113) using STAR⁷⁴. RSE⁷⁵ was used for read count quantification. Differential expression analysis was conducted with DESeq2 (ref. ⁷⁶), considering genes with a log₂ fold change greater than 1, an adjusted P value less than 0.05 and baseMean more than 200 as significant. Principal component analysis and unsupervised hierarchical clustering were performed using counts transformed by DESeq2’s regularized logarithm (Rlog), with plots generated with ggplot2 (ref. ⁷⁷) and ComplexHeatmap^78,79. Gene ontology analysis was carried out using the clusterProfiler package^80,81 and the results were visualized using the enrichplot package⁸². Gene set enrichment analysis was conducted with fgsea package⁸³ranking genes based on log₂FC × −log₁₀ (adjusted P value). Signature gene sets for each cell type were determined by selecting genes with a log₂ fold change greater than or equal to one and an adjusted P less than 0.1 through pairwise comparison across cell type. The x axis labeled ‘rank’ represents the ranked order of all genes expressed in chicken ES cells, sorted based on their gene expression fold changes (with adjusted P value) compared to the HH4 gene expression profile. Correlation analysis was performed using Pearson correlation, using log₂-transformed average fragments per kilobase of transcript per million mapped reads (FPKM) values across replicates of each cell type.

In vitro differentiation of ES cells

Differentiation of avian ES cells into soma tic and germline lineages was induced by the formation of EBs. Avian ES cells were dissociated using TrypLE, resuspended in N2B27 basal medium and plated into tissue culture plates for 1 h to remove feeder cells. Subsequently, loosely attached or floating avian ES cells were collected and plated into Aggrewell-400 24-well plates (StemCell Technologies) at a density of 70,000–150,000 cells per well to allow EB formation in N2B27 basal medium. EBs were harvested from Aggrewell after 4 days, followed by immunostaining and RT–qPCR analysis to assess the expression of lineage markers, including markers for somatic and germ cells. For longer differentiation, EBs were transferred to nonadherent wells for an additional 4-day incubation before being harvested.

For differentiation into neural lineage, EBs were formed overnight using Aggrewell-400 and then transferred to nonadherent wells for additional 3-day culture in N2B27 medium supplemented with 2.5 µM IWR-1, 2 µM Gö6983 and 2 µM SB431542. The medium was then replaced with N2B27 supplemented with 5% chicken serum for another 7 days. Subsequently, EBs were plated onto Matrigel-coated four-well plates and cultured for two more days in the same medium before immunostaining.

For differentiation into cardiomyocytes, EBs were formed overnight using Aggrewell-400 and then transferred to nonadherent wells for additional 3-day culture in N2B27 medium supplemented with 2.5 µM IWR-1 and 2 µM Gö6983. EBs were plated onto gelatin-coated wells and cultured for another 10 days in monolayer before immunostaining.

For differentiation into adipocytes, EBs were formed overnight using Aggrewell-400 in N2B27 medium alone and then transferred to nonadherent wells for additional 2-day culture in N2B27 medium plus 5% fetal bovine serum. Subsequently, EBs were plated onto feeder-seeded wells and cultured for another 10 days before staining.

Oil Red O staining

Cells were fixed with 10% formalin for 30 min and washed 3 times with PBS. Subsequently, cells were incubated with 60% isopropanol for 5 min, followed by staining with Oil Red O working solution (Sigma-Aldrich) for 20 min on a rotator. After staining, the Oil Red O working solution was removed, and cells were washed three times with PBS. Next, cells were treated with hematoxylin for 1 min and rinsed with PBS 5 times before imaging.

Immunostaining

Chimeric embryos or gonadal tissues were fixed in 4% paraformaldehyde at 4 °C overnight and transferred to 30% sucrose until sinking to the bottom of the vial. Subsequently, embryos or tissues were embedded in optimal cutting temperature compound and cryosectioned for immunofluorescence. For staining of attached cells or EBs, cells were fixed in 4% paraformaldehyde for 20 min at room temperature. After fixation, all samples were blocked with PBS containing 5% normal donkey serum and 0.2% Triton X-100 at 4 °C overnight. Samples were then incubated with primary antibodies overnight at 4 °C. After 3 washes in PBS with 0.1% Tween-20, secondary antibodies were applied overnight at 4 °C. Samples were washed 3 times in PBS with 0.1% Triton X-100 before mounting for imaging. Nuclei were stained with Hoechst 33342 (Invitrogen, 1:50,000). Primary antibodies used include: Myosin (MF-20, DSHB, 1:50), TuJ1 (MAB1195, R&D Systems, 1:100), GATA4 (sc-25310, Santa Cruz, 1:100), Brachyury/TBXT (AF2085, R&D systems, 1:300), SOX2 (ab5603, Sigma, 1:500), DAZL (ab215718, Abcam, 1:750), P63 (ab124762, Abcam, 1:500), GFP (A11120, Invitrogen, 1:500), SOX9 (AF3075, R&D Systems, 1:40), SSEA-1 (MC480, DSHB, 1:10), epithelial membrane antigen-1 (DSHB, 1:10). Secondary antibodies used: Goat Anti-Mouse IgM NorthernLights NL557 (NL019, R&D Systems, 1:200), Donkey Anti-Rabbit IgG (H+L)-Cy5-AffiniPure conjugated (711-175-152, Jackson ImmunoResearch, 1:200), Donkey Anti-Goat IgG (H+L)-Cy5-AffiniPure conjugated (705-175-147, Jackson ImmunoResearch, 1:200), Donkey Anti-Rabbit IgG (H+L) DyLight 549 conjugated (611-742-127, Rockland, 1:5,000), Donkey Anti-Mouse IgG (H+L), Alexa Fluor 488 Conjugated (A-21202, Invitrogen, 1:1,000) and Donkey Anti-Mouse IgG (H+L) DyLight 549 conjugated (610-742-124, Rockland, 1:5,000).

Ex ovo chimera formation assay

Albumin agar plates (0.3% w/v agarose, 0.3% w/v glucose with 20 U ml⁻¹ penicillin and 20 µg ml⁻¹ streptomycin) were freshly prepared and used for avian embryo culture. Avian embryos were explanted and cultured with a filter paper carrier on an albumin agar plate according to the EC culture system⁶⁸. Briefly, a freshly laid egg was poured into a petri dish with the thick albumin being removed to expose the embryo directly to the paper ring. The paper was cut around the perimeter of the embryo, and the embryo was transferred into a dish with PBS to remove any residual yolk. Afterward, the embryo was placed ventral-side up onto the albumin agar plate.

For the chimera formation assay, GFP-labeled avian ES cells were first dissociated using TrypLE for 8 min at room temperature. After neutralization, these cells were again dissociated using 0.025% Trypsin for 3 min at 38 °C to ensure dissociation into single cells. Then, 20,000 GFP-labeled cells were equally distributed on top of the recipient embryo using a glass microcapillary. The embryo was incubated at 38 °C for 2–4 days, and donor cell contribution was monitored by live imaging.

In ovo chimera formation assay

The surrogate eggshell system was employed to culture chimeric chick embryos in ovo⁸⁴. Optionally, fertile eggs can be irradiated at 500–550 cGy before injection. A surrogate chicken egg, 4 g to 5 g heavier than the recipient fertile egg, was used, and the entire contents of the recipient fertile egg were transferred into this larger surrogate shell. Donor ES cells were dissociated into single cells and prepared as described in the ex ovo chimera formation assay. 7,500 cells were injected into the subgerminal cavity using a glass microcapillary, with a volume of 0.75 µl. After injection, the egg was filled with albumen, covered with cling film and secured using PVC plastic rings wrapped with rubber bands. The reconstituted egg was then placed window-side up on a constantly tilted shaker (about 1 rpm) overnight before being transferred upside down into an incubation cabinet set at 37.5 °C with a rocking angle of 90° at 1-h intervals. After 3–4 days of incubation, the chimerism of the injected embryo was determined by live imaging based on GFP signal. To further incubate the chimeric embryo, a jumbo chicken egg weighing 20–25 g heavier than the recipient egg was used as a secondary surrogate shell. The entire content of the chimeric embryo was carefully transferred into this jumbo surrogate shell and sealed with cling film. The chimeric embryo in this jumbo shell was then incubated in an incubator at 37.5 °C until hatching.

The window method was used for quail ES cell transplantation into quail embryos and in ovo culture thereafter⁸⁵. Irradiation at 500 cGy was applied to the recipient eggs if necessary. Wild-type Japanese quail eggs were sanitized with 70% ethanol and placed with the narrow end up for 2 h at room temperature. Subsequently, eggs were placed laterally for 2 h before injection to allow the embryo to float to the lateral apex of the eggs. A 4 mm by 4 mm window was drilled at the lateral apex of the eggshell using a handheld rotary tool (Dremel), and the shell membrane was removed using forceps. Donor ES cells were injected into the subgerminal cavity following the same procedure described in the surrogate method above. After injection, the window was sealed with plastic tape, and eggs were placed with the blunt end up in an incubator cabinet set at 37.5 °C with a rocking angle of 90° at 1-h intervals.

Feather histology and immunohistochemistry

Feather follicle samples were collected from adult roosters and fixed in 4% paraformaldehyde in PBS overnight. Subsequently, the samples were serially dehydrated with alcohol and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin or analyzed by immunohistochemistry staining⁴⁹. The following antibodies were used: MITF (ab12039, Abcam, 1:800) and GFP (A11120, Invitrogen, 1:500). For immunohistochemistry staining, color development was achieved using the AEC peroxidase substrate kit (Vector Laboratories).

Statistics

Statistical significance between groups was assessed using Student’s t-test (two-sided) or one-way analysis of variance, as appropriate. For mass-spectrometry data, statistical significance was determined using one-way analysis of variance followed by Dunnett’s multiple-comparison test. Data are presented as mean ± s.d. from a minimum of three independent experiments unless otherwise noted in the figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Author notes

1. These authors contributed equally: Zheng Guo, Xinyi Tong.

Authors and Affiliations

1. Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

   Xi Chen, Zheng Guo, Xinyi Tong, Xizi Wang, Xugeng Liu, Christina Wu, Lin Cao, Yixin Huang, Zhaohan Zeng, Fan Feng, Nima Adhami, Sirjan Mor & Qi-Long Ying

2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

   Xi Chen, Carol Readhead & Carlos Lois

3. International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan

   Hiroki Nagai & Guojun Sheng

4. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

   Ping Wu, Jiayi Lu & Cheng-Ming Chuong

5. Department of Radiology and Developmental Neuroscience Program, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA, USA

   David Huss, Martin Tran & Rusty Lansford

Contributions

X.C. conceived the project and performed the majority of experiments with help from Z.G. and X.T. X.W. analyzed sequencing data, performed gonad cryosection and helped organizing figures. X.L. performed chicken ES cell in vitro differentiation and RT–qPCR assays with help from Z.Z., N.A. and S.M. H.N. performed chicken and peafowl ES cell derivation and chimera formation assay under supervision by G.S. D.H., M.T. and R.L. provided guidance for the ex ovo chimera formation assay. P.W. performed feather histology experiments and helped cryosection analysis together with J.L. under supervision by C.-M.C. C.R. performed in situ hybridization chain reaction experiments. C.W., L.C., Y.H. and F.F. helped with in ovo chimera formation assay. C.L. interpreted the results and provided research support. Q.-L.Y. conceived the project, provided research support, analyzed and interpreted the results. X.C. and Q.-L.Y. cowrote the paper with input from all coauthors.

Corresponding authors

Correspondence to Xi Chen or Qi-Long Ying.

Competing interests

Q.-L.Y., X.C., Z.G., X.T. and X.L. are inventors on a patent (WO2023158627A3) entitled ‘Methods for derivation and propagation of avian pluripotent stem cells and applications thereof’ arising from this work. The other authors declare no competing interests.

Peer review information

Nature Biotechnology thanks Björn Oback and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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