A tribute to Dr. Gordon Hisashi Sato (December 24, 1927-March 31, 2017).

Gordon H. Sato, an innovator in mammalian tissue culture and integrated cellular physiology, passed away in 2017. In tribute to Dr. Sato, In Vitro Cellular and Developmental Biology-Animal presents a collection of invited remembrances from six colleagues whose associations with Dr. Sato spanned more than 40 years. Dr. Sato was a past president of the Tissue Culture Association (now the Society for In Vitro Biology), editor-in-chief of In Vitro Cellular and Developmental Biology (1987-1991), and the recipient of the lifetime achievement award from the Society for In Vitro Biology (2002). He was elected to the US National Academy of Sciences in 1984.

Serum and glucocorticoid-inducible kinase1 increases plasma membrane wt-CFTR in human airway epithelial cells by inhibiting its endocytic retrieval.

BACKGROUND: Chloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells. METHODS AND RESULTS: We report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR). CONCLUSION: This study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane.

Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium.

Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase, and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.

Biomedical advances from tissue culture.

The demonstration that the "dedifferentiation" of cells commonly observed in the early days of tissue culture was due to selective overgrowth of fibroblasts led to enrichment culture techniques (alternate animal and culture passage) designed to give a selective advantage to functionally differentiated tumor cells. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types. These results gave rise to the hypothesis that cells in culture accurately represent cells in vivo but without the complex in vivo environment. This concept has been strengthened with the development of hormonally defined culture media in combination with functionally differentiated clonal cell lines, which have augmented the potential of tissue culture studies. The use of hormonally defined media in place of serum-supplemented media demonstrates that hormonal responses and dependencies can be discovered in culture. Discoveries of hormonal dependencies of cancer cells has led to therapies targeting intracellular signaling pathways while discoveries of hormonal responses of pluripotent cells are helping to identify the potential application of stem cells. In these and other ways tissue culture technology will continue to contribute to solving problems of human health.

Nedd4-2 does not regulate wt-CFTR in human airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel in airway epithelial cells, plays an important role in maintaining the volume of the airway surface liquid and therefore mucociliary clearance of respiratory pathogens. A recent study has shown that the E3 ubiquitin ligase Neural precursor cells expressed developmentally downregulated (Nedd4-2) ubiquitinates ΔF508-CFTR in pancreatic epithelial cells and that siRNA-mediated silencing of Nedd4-2 increases plasma membrane ΔF508-CFTR. Because the role of Nedd4-2 in regulating wild-type (wt)-CFTR in airway epithelial cells is unknown, studies were conducted to test the hypothesis that Nedd4-2 also ubiquitinates wt-CFTR and regulates its plasma membrane abundance. We found that Nedd4-2 did not affect wt-CFTR Cl(-) currents in Xenopus oocytes. Likewise, overexpression of Nedd4-2 in human airway epithelial cells did not alter the amount of ubiquitinated wt-CFTR. siRNA knockdown of Nedd4-2 in human airway epithelial cells had no effect on ubiquitination or apical plasma membrane abundance of wt-CFTR. Thus Nedd4-2 does not ubiquitinate and thereby regulate wt-CFTR in human airway epithelial cells.

A novel aquaporin 3 in killifish (Fundulus heteroclitus) is not an arsenic channel.

The Atlantic killifish (Fundulus heteroclitus) is a model environmental organism that has an extremely low assimilation rate of environmental arsenic. As a first step in elucidating the mechanism behind this phenomenon, we used quantitative real-time PCR to identify aquaglyceroporins (AQPs), which are arsenite transporters, in the killifish gill. A novel homolog killifish AQP3 (kfAQP3a) was cloned from the killifish gill, and a second homolog was identified as the consensus from a transcriptome database (kfAQP3b). The two were 99% homologous to each other, 98% homologous to a previously identified killifish AQP3 from embryos (kfAQP3ts), and 78% homologous to hAQP3. Expression of kfAQP3a in Xenopus oocytes significantly enhanced water, glycerol, and urea transport. However, kfAQP3a expressed in HEK293T cells did not transport significant amounts of arsenic. All sequence motifs thought to confer the ability of AQP3 to transport solutes were conserved in kfAQP3a, kfAQP3b, and kfAQP3ts; however, the C-terminal amino acids were different in kfAQP3a versus the other two homologs. Replacement of the three C-terminal amino acids of kfAQP3 (GKS) with the three C-terminal amino acids of kfAQP3b and kfAQP3ts (ANC) was sufficient to enable kfAQP3a to robustly transport arsenic. Thus, the C-terminus of kfAQP3b and kfAQP3ts confers arsenic selectivity in kfAQP3. Moreover, kfAQP3a, the only AQP expressed in killifish gill, is the first aquaglyceroporin identified that does not transport arsenic, which may explain, in part, why killifish poorly assimilate arsenic and are highly tolerant to environmental arsenic.

Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation.

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.

Tissue culture: the unlimited potential.

Lack of tissue-specific differentiated functions of cells in tissue culture, once thought to be due to "dedifferentiation", was shown to be due to selective overgrowth of fibroblasts by a series of simple experiments that challenged the prevailing dogma. Following this insight, enrichment culture techniques (alternate animal and culture passage) were designed to give functionally differentiated tumor cells selective advantage over the fibroblasts. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types, providing the final point of destruction of the dogma of "dedifferentiation." Instead, the hypothesis was proposed that cells in culture accurately represent cells in vivo, but without the complex in vivo environment. With the development of hormonally defined media and its combination with functionally differentiated clonal cell lines, this concept has been strengthened and the potential of tissue culture studies has been greatly augmented. Hormonally defined media allow the culture of cell types that cannot be grown in conventional, serum-supplemented media. These approaches demonstrate that hormonal responses and dependencies can be discovered in culture. Following this thinking and the discovery of hormonal dependencies of cancer cells has led to a new rationale for therapy. Tissue culture and cell technology continue to play an important role in solving human health problems.

The role of SGK and CFTR in acute adaptation to seawater in Fundulus heteroclitus.

Killifish are euryhaline teleosts that adapt to increased salinity by up regulating CFTR mediated Cl(-) secretion in the gill and opercular membrane. Although many studies have examined the mechanisms responsible for long term (days) adaptation to increased salinity, little is known about the mechanisms responsible for acute (hours) adaptation. Thus, studies were conducted to test the hypotheses that the acute homeostatic regulation of NaCl balance in killifish involves a translocation of CFTR to the plasma membrane and that this effect is mediated by serum-and glucocorticoid-inducible kinase (SGK1). Cell surface biotinyation and Ussing chamber studies revealed that freshwater to seawater transfer rapidly (1 hour) increased CFTR Cl(-) secretion and the abundance of CFTR in the plasma membrane of opercular membranes. Q-RT-PCR and Western blot studies demonstrated that the increase in plasma membrane CFTR was preceded by an increase in SGK1 mRNA and protein levels. Seawater rapidly (1 hr) increases cortisol and plasma tonicity, potent stimuli of SGK1 expression, yet RU486, a glucocorticoid receptor antagonist, did not block the increase in SGK1 expression. Thus, in killifish SGK1 does not appear to be regulated by the glucocorticoid receptor. Since SGK1 has been shown to increase the plasma membrane abundance of CFTR in Xenopus oocytes, these observations suggest that acute adaptation (hours) to increased salinity in killifish involves translocation of CFTR from an intracellular pool to the plasma membrane, and that this effect may be mediated by SGK1.

Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium.

A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serum-free medium, hESF9, for the culture of HESCs on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), l-ascorbic acid-2-phosphate (0.1 microg/ml), and heparin. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/ml although higher concentrations were deleterious in the presence of heparin.

Integrins regulate mouse embryonic stem cell self-renewal.

Extracellular matrix (ECM) components regulate stem-cell behavior, although the exact effects elicited in embryonic stem (ES) cells are poorly understood. We previously developed a simple, defined, serum-free culture medium that contains leukemia inhibitory factor (LIF) for propagating pluripotent mouse embryonic stem (mES) cells in the absence of feeder cells. In this study, we determined the effects of ECM components as culture substrata on mES cell self-renewal in this culture medium, comparing conventional culture conditions that contain serum and LIF with gelatin as a culture substratum. mES cells remained undifferentiated when cultured on type I and type IV collagen or poly-D-lysine. However, they differentiated when cultured on laminin or fibronectin as indicated by altered morphologies, the activity of alkaline phosphatase decreased, Fgf5 expression increased, and Nanog and stage-specific embryonic antigen 1 expression decreased. Under these conditions, the activity of signal transducer and activator of transcription (STAT)3 and Akt/protein kinase B (PKB), which maintain cell self-renewal, decreased. In contrast, the extracellular signal-regulated kinase (ERK)1/2 activity, which negatively controls cell self-renewal, increased. In the defined conditions, mES cells did not express collagen-binding integrin subunits, but they expressed laminin- and fibronectin-binding integrin subunits. The expression of some collagen-binding integrin subunits was downregulated in an LIF concentration-dependent manner. Blocking the interactions between ECM and integrins inhibited this differentiation. Conversely, the stimulation of ECM-integrin interactions by overexpressing collagen-binding integrin subunits induced differentiation of mES cells cultured on type I collagen. The results of the study indicated that inactivation of the integrin signaling is crucial in promoting mouse embryonic stem cell self-renewal. Disclosure of potential conflicts of interest is found at the end of this article.

Regulation of human cystic fibrosis transmembrane conductance regulator (CFTR) by serum- and glucocorticoid-inducible kinase (SGK1).

BACKGROUND: Serum- and glucocorticoid-inducible kinase-1 (SGK1) increases CFTR Cl currents in Xenopus oocytes by an unknown mechanism. Because SGK increases the plasma membrane expression of other ion channels, the goal of this paper was to test the hypothesis that SGK1 stimulates CFTR Cl currents by increasing the number of CFTR Cl channels in the plasma membrane. METHODS: CFTR Cl currents were measured in Xenopus oocytes by the two-electrode voltage clamp technique, and CFTR in the plasma membrane was determined by laser scanning confocal microscopy. RESULTS: wt-SGK1 stimulated CFTR Cl currents by 42% and increased the amount of CFTR in the plasma membrane by 35%. A kinase-dead SGK mutant (K127N) had a dominant-negative effect on CFTR, reducing CFTR Cl currents by 38%. In addition, deletion of the C-terminal PDZ-interacting motif (SGK1-DeltaSFL) increased CFTR Cl currents by 108%. Thus, SGK1-DeltaSFL was more effective than wt-SGK1 in stimulating CFTR Cl currents. Neither wt-SGK nor the K127N mutant had any effect on Cl currents in oocytes when expressed alone in the absence of CFTR. CONCLUSION: SGK1 stimulates CFTR Cl currents in Xenopus oocytes by increasing the number of channels in the plasma membrane. Moreover, the effect of SGK may be mediated by protein-protein interactions involving the PDZ interacting motif.

Immunohistochemical expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) as an angiogenic factor in head and neck tumorigenesis.

Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) is a secreted protein that releases immobilized fibroblast growth factor-2 (FGF-2) from the extracellular matrix and plays a critical role in FGF bioactivation. In the present study co-localization of FGF-2 and HBp17/FGFBP-1 was observed in oral tissues including normal mucosa, hyperplasia, dysplasia of different degrees and oral squamous cell carcinoma (OSCC). The expression score for HBp17/FGFBP-1, FGF-2 as well as vascular endothelial growth factor A (VEGF-A) became higher with the severity of epithelial dysplasia and was highest in severe dysplasia. The expression of HBp17/FGFBP-1, FGF-2 and VEGF-A showed significant association with microvessel density, but no correlation with TNM stages or OSCC recurrence interval. Our results demonstrated that HBp17/FGFBP-1, like VEGF-A and FGF-2, might also promote the induction of tumor angiogenesis. The strongest expression of angiogenic factors in severe dysplasia suggests a potential point for targeting novel anti-angiogenic therapeutic strategies.

Role of glucocorticoid receptor in acclimation of killifish (Fundulus heteroclitus) to seawater and effects of arsenic.

Killifish are euryhaline teleosts that adapt to rapid changes in the salinity of the seawater. It is generally accepted that acclimation to seawater is mediated by cortisol activation of the glucocorticoid receptor (GR), which stimulates CFTR mRNA expression and CFTR-mediated Cl- secretion by the gill. Because there is no direct evidence in killifish that the GR stimulates CFTR gene expression, quantitative PCR studies were conducted to test the hypothesis that cortisol activation of GR upregulates CFTR mRNA expression and that this response is required for acclimation to seawater. Inhibition of the GR by RU-486 prevented killifish from acclimating to increased salinity and blocked the increase in CFTR mRNA. In contrast, inhibition of the mineralocorticoid receptor by spironolactone had no effect on acclimation to seawater. Thus acclimation to increased salinity in killifish requires signaling via the GR and includes an increase in CFTR gene expression. Because arsenic, a toxic metalloid that naturally occurs in the aquatic environment, has been shown to disrupt GR transcriptional regulation in avian and mammalian systems, studies were also conducted to determine whether arsenic disrupts cortisol-mediated activation of CFTR gene expression in this in vivo fish model and thereby blocks the ability of killifish to acclimate to increased salinity. Arsenic prevented acclimation to seawater and decreased CFTR protein abundance. However, arsenic did not disrupt the GR-induced increase in CFTR mRNA. Thus arsenic blocks acclimation to seawater in killifish by a mechanism that does not disrupt GR-mediated induction of CFTR gene expression.

Inhibition of human tumor xenograft growth in nude mice by a conjugate of monoclonal antibody LA22 to epidermal growth factor receptor with anti-tumor antibiotics mitomycin C.

Anti-EGFR monoclonal antibodies LA22 and Erbitux bind to different epitopes of EGFR. The chemimmunoconjugates of MMC with LA22 or Erbitux were prepared, and in vitro cytotoxicity assays with A549 cells showed that LA22-MMC was much more potent than Erbitux or Erbitux-MMC. Viabilities of A549 cells treated with LA22-MMC, Erbitux or Erbitux-MMC were 35%, 94%, and 81%, respectively. Immunoscintigraphy of xenografts of human A431 and A549 cells in nude mice both showed that (125)I-labeled-LA22-MMC enriched in tumor sites prominently. Most importantly, in vivo assays showed LA22-MMC was significantly more effective than free drug MMC in the treatment of subcutaneous xenografts of human A431 cells in nude mice (83% inhibition for LA22-MMC and 30% for MMC). We concluded that LA22-MMC could be a very potent drug for treatment of solid tumors.

Leukemia inhibitory factor as an anti-apoptotic mitogen for pluripotent mouse embryonic stem cells in a serum-free medium without feeder cells.

We have developed a serum-free medium, designated ESF7, in which leukemia inhibitory factor (LIF) clearly stimulated murine embryonic stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-1 and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription factor Oct-3/4, the stem cell marker SSEA-1, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell differentiation. Addition of BMP4 promoted ES cell differentiation into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell differentiation into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell differentiation, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to growth factors under defined conditions.

Constitutive activating mutation of the FGFR3b in oral squamous cell carcinomas.

A G to T mutation at nucleotide position 2128 in the human FGFR3b coding region resulting in a Cys for Gly substitution (G697C) in the tyrosine kinase domain was observed in 62% (44/71) of oral squamous cell carcinomas (OSCC) examined. Immunostained FGFR3b was found in the cytoplasm of prickle cells in normal epithelia, and FGFR3b was localized in the cytoplasm and nucleus in non-FGFR3b mutant OSCC. Overexpressed FGFR3b protein on plasma membranes was noted in OSCC bearing the FGFR3b mutation. Enhanced tyrosine kinase activity of G697CFGFR3b was confirmed. Our results indicate that G697C is an activating mutation causing constitutive ligand-independent FGFR3b signaling. This mutation may be involved in the progression of OSCC and thus the FGFR3b coding sequence may have diagnostic or prognostic value for OSCC.

Treatment with siRNA and antisense oligonucleotides targeted to HIF-1alpha induced apoptosis in human tongue squamous cell carcinomas.

Overexpression of hypoxia inducible factor-1alpha (HIF-1alpha) in cancers has been correlated to a more aggressive tumor phenotype. We investigated the effect of HIF-1alpha knockout on the in vitro survival and death of human tongue squamous cell carcinomas (SCC-4 and SCC-9). Under normoxic condition, a basal level of HIF-1alpha protein was constitutively expressed in SCC-9 cells, albeit an undetectable level of HIF-1alpha messages. Exposure to hypoxia induced only a transient increase in mRNA transcript but a prolonged elevation of HIF-1alpha protein and its immediate downstream target gene product, VEGF. Under normoxic or hypoxic conditions, treatment of SCC-9 cells with AS-HIF-1alpha ODN suppressed both constitutive and hypoxia-induced HIF-1alpha expression at both mRNA and protein levels. Knockout of HIF-1alpha gene expression via either AS-HIF-1alpha ODN or siRNA (siRNAHIF-1alpha) treatment resulted in inhibition of cell proliferation and induced apoptosis in SCC-4 and SCC-9 cells. We also demonstrated that exposure of SCC-9 cells to hypoxia led to a time-dependent increase in the expression of bcl-2 and IAP-2, but not p53. The attenuated levels of bcl-2 and IAP-2, and the enhanced activity of caspase-3 after treatment with AS-HIF-1alpha ODN may contribute partly to the effects of HIF-1alpha blockade on SCC-9 cell death. Collectively, our data suggest that a constitutive or hypoxia-induced expression of HIF-1alpha in SCC-9 and SCC-4 cells is sufficient to confer target genes expression essential for tumor proliferation and survival. As a result, interfering with HIF-1alpha pathways by antisense or siRNA strategy may provide a therapeutic target for human tongue squamous cell carcinomas.

Pluripotent differentiation in vitro of murine ES-D3 embryonic stem cells.

Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro during a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1, and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro.