Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion.

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.

SFTSV Gn-Head mRNA vaccine confers efficient protection against lethal viral challenge.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus, causing thrombocytopenia and hemorrhagic fever, with a fatality rate ranging from 12% to 30%. SFTSV possesses Gn and Gc glycoproteins, which are responsible for host cell receptor attachment and membrane fusion, respectively, to infect host cells. We have previously reported a protein subunit vaccine candidate (sGn-H-FT) of the SFTSV soluble Gn head region (sGn-H) fused with self-assembling ferritin (FT) nanoparticles, displaying strong protective immunogenicity. In this study, we present messenger RNA (mRNA) vaccine candidates encoding sGn-H or sGn-H-FT, both of which exhibit potent in vivo immunogenicity and protection capacity. Mice immunized with either sGn-H or sGn-H-FT mRNA lipid nanoparticle (LNP) vaccine produced strong total antibodies and neutralizing antibodies (NAbs) against sGn-H. Importantly, NAb titers remained high for an extended period. Finally, mice immunized with sGn-H or sGn-H-FT mRNA LNP vaccine were fully protected from a lethal dose of SFTSV challenge, showing no fatality. These findings underscore the promise of sGn-H and sGn-H-FT as vaccine antigen candidates capable of providing protective immunity against SFTSV infection.

Viral codon optimization on SARS-CoV-2 Spike boosts immunity in the development of COVID-19 mRNA vaccines.

Life-long persistent herpesviruses carry "trans-inducers" to overcome the unusual codon usage of their glycoproteins for efficient expression. Strikingly, this "trans-inducibility" can be achieved by simply changing the codon-usage of acute virus glycoproteins to that of persistent herpesvirus glycoproteins with herpesviral trans-inducer. Here, we apply the "persistent viral codon-usage-trans-inducer" principle to SARS-CoV-2 Spike mRNA vaccine platform, in which the codon-usage of Spike is changed to that of Herpes Simplex Virus-1 (HSV-1) glycoprotein B (gB) with its "trans-inducer" ICP27. The HSVgB-ICP27-codon-optimized Spike mRNA vaccine induced markedly high antigen expression and stability, total IgG, neutralizing antibody, and T cell response, ultimately enhancing protection against lethal SARS-CoV-2 challenge. Moreover, the HSVgB- codon-optimized Delta (B.1.617.2) strain Spike mRNA vaccine provided significant enhancements in antigen expression and long-term protection against SARS-CoV-2 challenge. Thus, we report a novel persistent viral codon-usage-trans-inducer mRNA vaccine platform for enhanced antigen expression and long-term protection against lethal viral infection.

Comparing Free-Flap Reconstruction After Robot-Assisted Neck Dissection via a Retroauricular Approach and a Traditional Transcervical Approach: Single-Surgeon Experiences of 90 Consecutive Cases.

BACKGROUND: Predominant traditional approaches for most patients who have advanced-stage oral cancer with transcervical incision lines left irreversible scars. To address this, surgeons have continuously refined minimally invasive surgery (MIS) techniques, including robot-assisted neck surgeries. This article introduces and discusses the feasibility, versatility, and availability of free-flap reconstruction via the retroauricular approach (RA), considered difficult to date. METHODS: This study retrospectively analyzed 90 consecutive patients who had free-flap reconstruction performed by a single surgeon (D.K.) in the Department of Oral and Maxillofacial Surgery, Yonsei University, from March 2021 to April 2022. The type of defects and flaps, hospitalization days, total operation time, and type of vessels and anastomoses were compared statistically. RESULTS: The type of vessels used did not differ between the RA and the transcervical approach (TA) groups, nor in duration of hospital stays. Likewise, the total reconstruction time did not differ significantly between the TA group (240 min) and the RA group (245 min) (p = 0.756). However, the total operation time was about 1 h less in the TA group, a statistically significant difference (TA group [593 ± 152 min] vs. RA group [655 ± 117 min]; p = 0.044). All flaps were successful in the RA group, whereas one flap in the TA group led to a total loss (TA group [98.3%] vs. RA group [100.0%]; p = 1.000). CONCLUSIONS: Even for patients with advanced oral cancer who require massive tumor ablation, it is feasible to obtain an aesthetic and functional surgical outcome by performing free-flap reconstruction via the retroauricular approach.

Bcl-2 is a prognostic marker and its silencing inhibits recurrence in ameloblastomas.

OBJECTIVES: Ameloblastomas are the most common odontogenic epithelial tumors with high recurrence rate. The aim of this study was to identify apoptosis-related genes with recurrence of ameloblastomas and to evaluate its feasibility as a prognostic marker and as a target molecule preventing from recurrence. MATERIALS AND METHODS: Public microarray data were analyzed. To evaluate their expression in ameloblastoma patients, immunohistochemical staining was performed in 89 human ameloblastoma tissues. Quantitative PCR was performed by use of ameloblastoma cell line (AM-1). Fluorescence activated cell sorting analysis and western blotting were conducted following transfection with siRNA. Further, AM-1 cells were implanted in the renal subcapsular layer of immunodeficient mice. RESULTS: Microarray data analysis revealed that osteoprotegerin (OPG) and B-cell lymphoma 2 (Bcl-2) were the two most upregulated genes in ameloblastoma. Only Bcl-2 expression was significantly (p = 0.020) associated with recurrence in conservative treatment group (n = 17) among 89 patients. Silencing of Bcl-2 increased apoptosis in AM-1 cells in vitro and inhibited tumor nodule formation of AM-1 cells in vivo. CONCLUSION: These results suggest that Bcl-2 expression is a useful biomarker to predict recurrence of ameloblastomas, and as a therapeutic target molecule to prevent recurrence of ameloblastoma.

Role of microcracks in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVES: The aim of this study was to investigate the potential role of microcrack accumulation in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (ONJ) through an animal model. MATERIALS AND METHODS: Twenty-four ovariectomized rats were randomly divided into a bisphosphonate group (n = 19) and control group (n = 5) and weekly injected with zoledronic acid and normal saline, respectively. After 6 weeks, surgical intervention was performed, and the injections were continued for eight additional weeks. Then, the animals were sacrificed, and ONJ lesions were inspected for the presence of microcracks using scanning electron microscopy. Measurements included bone dimension, number of cracks, crack length, and normalized indices; crack density (Cr.Dn) and crack surface density (Cr.S.Dn) were used for group comparison. RESULTS: Both number of cracks and crack length in the bisphosphonate group were greater than those in the control group (P < 0.05). Of the 19 rats injected with bisphosphonates, 13 rats (68.4 %) were classified into the ONJ group. Cr.Dn and Cr.S.Dn were significantly greater in the ONJ group than in the non-ONJ group, indicating accumulation of unrepaired microcracks (P < 0.05). Seventy-two percent of microcracks in the ONJ group conformed to the defined length that was considered significant according to a previous literature (30-80 µm); whereas 12 % of microcracks in the non-ONJ group were considered significant (P < 0.05). CONCLUSION: Accumulation of unrepaired microcracks was significantly associated with the development of bisphosphonate-related ONJ. Further research is required to determine the role of microcracks in the pathogenesis of bisphosphonate-related ONJ. CLINICAL RELEVANCE: Long-term bisphosphonates use may deteriorate the biomechanical and physiological bone integrity, contributing to the pathogenesis of bisphosphonate-related ONJ.

Apoptotic effect of pheophorbide a-mediated photodynamic therapy on DMBA/TPA-induced mouse papillomas.

Pheophorbide a (Pa) is a chlorine-based photosensitizer, and Pa-mediated photodynamic therapy (PDT) reportedly exhibits antitumor activity against various malignancies. The aim of our study was to investigate the therapeutic effect of Pa-mediated PDT on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorobol-13-acetate (TPA)-induced mouse papillomas. Thirty mice received a topical application of DMBA/TPA on their backs to induce mouse papillomas. One week after two sessions of Pa-mediated PDT, immunohistochemical stains and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed to evaluate the apoptotic effects thereof on the papillomas. Among 63 mouse papillomas treated with Pa-mediated PDT, 17.5% of the lesions were completely removed 1 week after the first treatment, while 31.7% disappeared 1 week after the second treatment. Statistical analyses revealed significant differences in therapeutic outcomes for the Pa-mediated PDT group in comparison to a solvent-PDT group and a Pa group. Additionally, a marked downregulation of proliferating cell nuclear antigen expression, as well as upregulation of cleaved caspase 3 and cleaved poly(ADP-ribose) polymerase expression, was noted in the Pa-PDT group, compared to the solvent-PDT group and Pa group. TUNEL assay revealed higher apoptotic cell counts in the Pa-PDT group, although the difference was not statistically significant. Our data demonstrated that Pa-mediated PDT is effective in treating DMBA/TPA-induced mouse papillomas.

Combined genomic expressions as a diagnostic factor for oral squamous cell carcinoma.

Trends in genetics are transforming in order to identify differential coexpressions of correlated gene expression rather than the significant individual gene. Moreover, it is known that a combined biomarker pattern improves the discrimination of a specific cancer. The identification of the combined biomarker is also necessary for the early detection of invasive oral squamous cell carcinoma (OSCC). To identify the combined biomarker that could improve the discrimination of OSCC, we explored an appropriate number of genes in a combined gene set in order to attain the highest level of accuracy. After detecting a significant gene set, including the pre-defined number of genes, a combined expression was identified using the weights of genes in a gene set. We used the Principal Component Analysis (PCA) for the weight calculation. In this process, we used three public microarray datasets. One dataset was used for identifying the combined biomarker, and the other two datasets were used for validation. The discrimination accuracy was measured by the out-of-bag (OOB) error. There was no relation between the significance and the discrimination accuracy in each individual gene. The identified gene set included both significant and insignificant genes. One of the most significant gene sets in the classification of normal and OSCC included MMP1, SOCS3 and ACOX1. Furthermore, in the case of oral dysplasia and OSCC discrimination, two combined biomarkers were identified. The combined expression revealed good performance in the validation datasets. The combined genomic expression achieved better performance in the discrimination of different conditions than a single significant gene. Therefore, it could be expected that accurate diagnosis for cancer could be possible with a combined biomarker.

Current Progress of Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) Vaccine Development.

SFTSV is an emerging tick-borne virus causing hemorrhagic fever with a case fatality rate (CFR) that can reach up to 27%. With endemic infection in East Asia and the recent spread of the vector tick to more than 20 states in the United States, the SFTSV outbreak is a globally growing public health concern. However, there is currently no targeted antiviral therapy or licensed vaccine against SFTSV. Considering the age-dependent SFTS pathogenesis and disease outcome, a sophisticated vaccine development approach is required to safeguard the elderly population from lethal SFTSV infection. Given the recent emergence of SFTSV, the establishment of animal models to study immunogenicity and protection from SFTS symptoms has only occurred recently. The latest research efforts have applied diverse vaccine development approaches-including live-attenuated vaccine, DNA vaccine, whole inactivated virus vaccine, viral vector vaccine, protein subunit vaccine, and mRNA vaccine-in the quest to develop a safe and effective vaccine against SFTSV. This review aims to outline the current progress in SFTSV vaccine development and suggest future directions to enhance the safety and efficacy of these vaccines, ensuring their suitability for clinical application.

Estimating the adjuvant chemotherapy effect in elderly stage II and III colon cancer patients in an observational study.

PURPOSE: Adjuvant chemotherapy has been known as a standard treatment for patients with resected colon cancer. However, in elderly colon cancer patients, the characteristics of patients are heterogeneous with regard to life expectancy and comorbidities. Thus, with regard to the effectiveness of adjuvant chemotherapy for colon cancer, it is difficult to extrapolate data of clinical trials from the younger into the older general population. METHODS: Data for 382 elderly colon cancer patients were analyzed: 217 in Stage II and 165 in Stage III. The efficacy of adjuvant chemotherapy was evaluated in elderly colon cancer patients after a match by the propensity score method. RESULTS: For matched patients with Stage II colon cancer, there was no significant efficacy of adjuvant chemotherapy in the risk of death during all follow-up periods (P-value, 0.06-0.37). Though there was a tendency that the adjuvant chemotherapy reduces the death rate during the follow-up periods, it was not statistically significant. In the case of Stage III, the adjuvant chemotherapy was significantly effective in matched patients for 5-year (hazard ratio [HR], 0.69; 95% confidence interval [CI], 0.30-0.90) and overall survival (HR, 0.56; 95% CI, 0.34-0.94). CONCLUSIONS: Adjuvant chemotherapy for elderly patients with Stage II colon cancer is not effective, whereas elderly patients with Stage III with adjuvant chemotherapy appear to have a better survival rate in the general population.

Robot-assisted radial forearm free flap harvesting: a propensity score-matched case-control study.

Although some surgeons prefer anterolateral thigh and latissimus dorsi flap for soft tissue reconstruction in the head and neck area because it minimizes donor site complications, the radial forearm flap remains the workhorse for soft tissue reconstruction due to its reliability. To reduce donor site morbidity, the authors developed a novel technique for radial forearm flap harvesting using a robotic device. 42 radial forearm free flap reconstruction cases were studied, consisting of 31 conventional and 11 robot-assisted cases. 1:1 propensity score matching was done according to age, sex, previous and postoperative radiation therapy history and method used for vein anastomosis. There was no significant difference in flap outcome, which was 100% vitality in the robot-assisted group and 90.9% vitality in the conventional group. The robot-assisted group showed significantly longer mean harvesting time than did the conventional group, being 107.2 min and 67.0 min, respectively. Robot-assisted radial forearm flap harvesting can reduce donor site complications by minimizing incision. When more surgical experience is gained under appropriate case selection, we expect our robot-assisted method will yield a harvesting time similar to that of the conventional method and thus become more reliable and feasible.

Perforators Detected in Computed Tomography Angiography for Anterolateral Thigh Free Flap: Am I the Only One Who Feels Inaccurate?

BACKGROUND: The number, location, and pattern of perforators in anterolateral thigh(ALT) flap vary and predicting them preoperatively will aid in reconstructing complex head and neck defects. This article suggests guidelines for utilizing CTA imagery to predict perforators of ALT-free flaps. METHODS: We retrospectively analyzed 53 Korean patients who underwent reconstruction with ALT flap in our department from March 2021 to July 2022. The location, course, origin, and pedicle lengths predicted in CTA and confirmed in the operation field were recorded and compared. RESULTS: Among the 85 intraoperatively-found perforators, 79 were also identified in CTA. Six perforators unidentified in CTA were newly found intraoperatively. The positive predictive value of CTA for the perforator was 100%, with a sensitivity of 79/85 = 92.9%. Of the 79 perforators depicted by the CTA for the flap, CTA and intraoperative findings for the course were consistent in 52 cases, a 9.6 mm median discrepancy being noted between the actual location and CTA. CONCLUSIONS: The overall pattern or location of perforation was not significantly different between the two, although some differences were observed. It is suggested that the addition of Doppler imaging, in conjunction with CTA, can aid in perforator detection and help minimize such discrepancies.

Self-assembling Gn head ferritin nanoparticle vaccine provides full protection from lethal challenge of Dabie Bandavirus in aged ferrets.

Dabie Bandavirus (DBV), previously known as Severe Fever with Thrombocytopenia Syndrome (SFTS) Virus, induces a characteristic thrombocytopenia with a mortality rate ranging from 12% to as high as 30%. The sero-prevalence of DBV in healthy people is not significantly different among age groups, but clinically diagnosed SFTS patients are older than ~50 years, suggesting that age is the critical risk factor for SFTS morbidity and mortality. Accordingly, our immune-competent ferret model demonstrates an age (>4 years old)-dependent DBV infection and pathogenesis that fully recapitulates human clinical manifestation. To protect the aged population from DBV-induced SFTS, vaccine should carry robust immunogenicity with high safety profile. Previous studies have shown that glycoproteins Gn/Gc are the most effective antigens for inducing both neutralizing antibody (NAb)- and T cell-mediated immunity and, thereby, protection. Here, we report the development of a protein subunit vaccine with 24-mer self-assembling ferritin (FT) nanoparticle to present DBV Gn head region (GnH) for enhanced immunogenicity. Anion exchange chromatography and size exclusion chromatography readily purified the GnH-FT nanoparticles to homogeneity with structural integrity. Mice immunized with GnH-FT nanoparticles induced robust NAb response and T-cell immunity against DBV Gn. Furthermore, aged ferrets immunized with GnH-FT nanoparticles were fully protected from DBV challenge without SFTS symptoms such as body weight loss, thrombocytopenia, leukopenia, and fatality. This study demonstrates that DBV GnH-FT nanoparticles provide an efficient vaccine efficacy in mouse and aged ferret models and should be an outstanding vaccine candidate targeted for the aged population against fatal DBV infection.

Self-assembling Gn head ferritin nanoparticle vaccine provides full protection from lethal challenge of Dabie bandavirus in aged ferrets.

IMPORTANCE: Dabie bandavirus (DBV) is an emerging tick-borne virus that causes severe fever with thrombocytopenia syndrome (SFTS) in infected patients. Human SFTS symptoms progress from fever, fatigue, and muscle pain to the depletion of white blood cells and platelets with fatality rates up to 30%. The recent spread of its vector tick to over 20 states in the United States increases the potential for outbreaks of the SFTS beyond the East Asia. Thus, the development of vaccine to control this rapidly emerging virus is a high priority. In this study, we applied self-assembling ferritin (FT) nanoparticle to enhance the immunogenicity of DBV Gn head domain (GnH) as a vaccine target. Mice immunized with the GnH-FT nanoparticle vaccine induced potent antibody responses and cellular immunity. Immunized aged ferrets were fully protected from the lethal challenge of DBV. Our study describes the GnH-FT nanoparticle vaccine candidate that provides protective immunity against the emerging DBV infection.

Which risk factors are associated with neurosensory deficits of inferior alveolar nerve after mandibular third molar extraction?

PURPOSE: Mandibular third molar extraction is a commonly performed procedure and is recognized as a relatively frequent cause of inferior alveolar nerve (IAN) injury. The aim of the present study was to investigate the specific risk factors for neurosensory deficits, including age, gender, impaction depth, angulation of the third molar, and various radiographic superimposition signs. MATERIALS AND METHODS: In a case-control study of patients who had undergone mandibular third molar extraction, a case group was developed of patients showing neurosensory deficits of the IAN, and a control group was formed of randomly selected patients without any neurosensory symptoms. Bivariate analyses were performed to assess the relationship between each variable and IAN injury. A multivariate logistic regression model was used to compute the odds ratios, P values, and predictive values of the radiographic superimposition signs. RESULTS: Of 12,842 total patients, the study group included 104 cases and 135 controls. The results indicated that older age and deeper impaction status were significant risk factors (P < .05). Darkening of the roots, deflection of the roots, narrowing of the roots, dark and bifid apexes of the roots, and narrowing of the canal were also significant risk factors. The positive predictive values ranged from 0.7% to 6.9% and the negative predictive values from 99% to 100%, with adjustment for the definitive prevalence of IAN injury (0.81%, 104/12,842 patients). However, the relatively low positive predictive value renders questionable the predictability of superimposition signs on orthopantomography. In the absence of specific radiographic signs, the risk of neurosensory deficit of the IAN could be negligible. The sensory symptoms disappeared after 6 months in 92.3% of the patients and 98.1% showed recovery after 1 year. CONCLUSIONS: The results of the present study have demonstrated a significant association between several risk factors and neurosensory deficits of the IAN after third molar extraction. With a case group of 104 patients, the number of subjects was significantly greater than that in previous studies, increasing the reliability of these results.

Risk factor analysis of dental implants in patients with irradiated head and neck cancer.

BACKGROUND: We investigated dental implant outcomes in patients who had previously received radiotherapy (RT) for head and neck malignancies. METHODS: We reviewed 90 dental implants in 27 patients who received RT for head and neck cancer and received dental implants afterwards. The cumulative implant survival rate (CISR) was calculated. In addition, the implant quality was assessed using "Health Scale for Dental Implants." RESULTS: The CISR at 3 years was 79.6%. The mean radiation dose at the implant site (Dmean ) was identified as an independent prognostic factor for implant survival. No implant failed if Dmean was less than 38 Gy. Regarding implant quality, dental implants in grafted bone and Dmean were independent risk factors. CONCLUSIONS: Dmean was identified as an independent prognostic factor for implant survival and quality. Dental implants can be safely considered when Dmean is lower than 38 Gy.

Risk Factors of Microscopically Tumor-Free Surgical Margins for Recurrence and Survival of Oral Squamous Cell Carcinoma Patients.

Objectives: The concept of adequate surgical margins remains controversial in oral squamous cell carcinoma (OSCC) surgery. This study aimed to identify surgical margin-related indicators that might impact recurrence and survival of OSCC patients. Materials and Methods: Histopathological examination was performed using hematoxylin-eosin-stained surgical margin tissue sections in 235 OSCC patients. Axin2 and Snail expression at the surgical margin was detected by immunohistochemistry. The impact of the Axin2-Snail cascade on tumorigenesis of the immortalized human oral keratinocyte (IHOK) line was investigated in vivo. Results: The width and dysplasia of surgical margins were not significantly associated with the outcome of OSCC patients. In a multivariate analysis using variable clinicopathologic factors and with Axin2 and Snail expression as cofactors, higher age (hazard ratio [HR]:1.050; P=0.047), Axin2 (HR:6.883; P=0.014), and Snail abundance (HR:5.663; P=0.009) had independent impacts on worsened overall survival. Similarly, lesion site in retromolar trigone (HR:4.077; P=0.010), upper (HR:4.332; P=0.005) and lower gingiva (HR:3.545; P=0.012), presence of extranodal extension (HR:9.967; P<0.001), perineural invasion (HR:3.627; P=0.024), and Snail abundance (HR:3.587; P<0.001) had independent impacts on worsened recurrence-free survival. Furthermore, Axin2 knockdown induced decreased Snail expression and attenuated tumorigenesis in the IHOK line. Conclusion: Histopathological examination of surgical margins may not be reliable to predict OSCC patient outcome. Molecular analysis may provide a more accurate risk assessment of surgical margins in OSCC. In particular, Axin2 and Snail are potential predictive biomarkers for the risk assessment of surgical margins in OSCC.

Hepatic differentiation from human embryonic stem cells using stromal cells.

OBJECTIVE: The derivation of hepatocytes from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report the generation of hepatocyte-like cells derived from hES cells. METHODS: Hepatic endoderm cells were generated by adding activin A for 5 d- to 1-d-old embryoid bodies formed from hES cells. The hepatic endoderm cells were cocultured with mitomycin treated 3T3-J2 feeder cells. RESULTS: After co-culture with mitomycin treated 3T3-J2 feeder cells, these hepatic endodermal cells yielded hepatocyte-like cell colonies, which possessed the proliferation potential to be cultured for an extended period of more than 30 d. With extensive expansion, they co-expressed the hepatic marker AFP and albumin, indicating that they were hepatocyte-like cells. CONCLUSIONS: We report the generation of proliferative hepatocyte-like cells from hES cells. These hES cell derived hepatic cells can effectively be used as in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.