Sensitivities of a Rapid Test Versus an ELISA Kit for Detecting Anti-SARS-CoV-2 IgM/IgG in Sera from an Egyptian Cohort.

This study aimed to compare diagnostic sensitivities of a rapid test (Rt) and an ELISA kit for detecting anti-SARS-CoV-2 IgM/IgG in virus-RT-PCR-positive (VPP) and virus-RT-PCR-unchecked (VPU) subjects in an Egyptian cohort during the first wave of SARS-CoV-2 infection. The results revealed higher sensitivity of the Rt for detecting IgM/IgG in the VPP subjects. Both the Rt and ELISA showed identical sensitivities for IgM detection in the VPU subjects. The ELISA was more sensitive for detecting IgG in the VPU subjects. Generally, within both the VPP and the VPU groups, Rt was more sensitive for detecting IgM/IgG among the symptomatic (S) compared to asymptomatic (AS) subjects than ELISA. Within the VPP group, the Rt was more sensitive for detecting both IgM/IgG among the AS subjects than ELISA. In the VPU group, the Rt was more sensitive for detecting IgM among the S subjects than ELISA. The ELISA was more sensitive for detecting IgM/IgG among AS subjects than the Rt. From these results we concluded that, despite the limitation of sample size, this study indicates suitability of the used Rt for detecting anti-SARS-CoV-2 IgM/IgG among S subjects and sheds light on possibility of relying on the used ELISA for IgG detection among AS human subjects.

Postmortem identification of spermatozoa on human skin based on fluorescent monoclonal antibody method.

Identification of semen residues has relevant consequences, especially for what concerns the ascertainment of possible sexual assault. Forensic scientists usually focus on the vaginal swab for semen detection despite the importance of semen deposition on the skin. Postmortem identification of spermatozoa on putrefied human skin is still under investigation. Sperm Hy-Liter™ is an antibody technique, used to identify human spermatozoa heads in forensic stains. This approach has the potential to eliminate spermatozoa visualization problems in a traditional method. Therefore, the present study aimed to compare between the traditional method (light microscope and staining via hematoxylin/eosin) and a fluorescence-based method (by using fluorescent microscope and staining via Sperm Hy-Liter™) for postmortem identification of spermatozoa on human skin at different time intervals. A piece of human skin was divided into three strips; the first was a negative control while semen was spread on the second and third skin strips. The first (control) and second groups were stained by hematoxylin/eosin for light microscopic examinations. The third group was stained by Sperm Hy-Liter™ then examined under fluorescent microscope. The results revealed that the spermatozoa identifiability was up to 110 days based on Sperm Hy-Liter™ and fluorescent microscope, while it was up to 12 days via using hematoxylin/eosin and light microscope. Further studies are recommended in order to verify not only the accuracy of the used method on skin of dead victims but also to evaluate persistence of spermatozoa on different body sites and fabrics.

Insulinoma-Associated Protein 1 Is a Crucial Regulator of Neuroendocrine Differentiation in Lung Cancer.

Insulinoma-associated protein 1 (INSM1) is expressed exclusively in embryonic developing neuroendocrine (NE) tissues. INSM1 gene expression is specific for small-cell lung cancer (SCLC), along with achaete-scute homolog-like 1 (ASCL1) and several NE molecules, such as chromogranin A, synaptophysin, and neural cell adhesion molecule 1. However, the underlying biological role of INSM1 in lung cancer remains largely unknown. We first showed that surgically resected SCLC samples specifically expressed INSM1. Forced expression of the INSM1 gene in adenocarcinoma cell lines (H358 and H1975) induced the expression of ASCL1, brain-2 (BRN2), chromogranin A, synaptophysin, and neural cell adhesion molecule 1; in contrast, knockdown of the INSM1 gene by siRNA in SCLC (H69 and H889) decreased their expression. However, forced/knockdown expression of ASCL1 and BRN2 did not affect INSM1 expression. A chromatin immunoprecipitation study revealed that INSM1 bound to the promoter region of the ASCL1 gene. A xenotransplantation assay using tet-on INSM1 gene-transfected adenocarcinoma cell lines demonstrated that INSM1 induced NE differentiation and growth inhibition. Furthermore, we found that INSM1 was not expressed in non-small-cell lung cancer and some SCLC cell lines expressing Notch1-Hes1. By forced/knockdown expression of Notch1 or Hes1 genes, we revealed that Notch1-Hes1 signaling suppressed INSM1, as well as ASCL1 and BRN2. INSM1, expressed exclusively in SCLC, is a crucial regulator of NE differentiation in SCLCs, and is regulated by the Notch1-Hes1 signaling pathway.

Molecular characterization of double-stranded RNA virus in Trichomonas vaginalis Egyptian isolates and its association with pathogenicity.

Trichomoniasis is a common human sexually transmitted infection caused by Trichomonas vaginalis. The parasite can be infected with double-stranded RNA viruses (TVV). This viral infection may have important implications on trichomonal virulence and disease pathogenesis. This study aimed to determine the prevalence of T. vaginalis virus among isolates obtained from infected (symptomatic and asymptomatic) women in Ismailia City, Egypt, and to correlate the virus-infected isolates with the clinical manifestations of patients. In addition, the pathogenicity of TVV infected isolates on mice was also evaluated. T. vaginalis isolates were obtained from symptomatic and asymptomatic female patients followed by axenic cultivation in Diamond's TYM medium. The presence of T. vaginalis virus was determined from total extraction of nucleic acids (DNA-RNA) followed by reverse transcriptase-PCR. Representative samples were inoculated intraperitoneally in female albino/BALB mice to assess the pathogenicity of different isolates. A total of 110 women were examined; 40 (36.3 %) samples were positive for T. vaginalis infection. Of these 40 isolates, 8 (20 %) were infected by TVV. Five isolates contained TVV-2 virus species, and the remaining three isolates were infected withTVV-4 variant. A significant association was found between the presence of TVV and particular clinical manifestations of trichomoniasis. Experimental mice infection showed varying degrees of pathogenicity. This is the first report on T. vaginalis infection by TVV in Egypt. The strong association detected between TVV and particular clinical features of trichomoniasis and also the degree of pathogenicity in experimentally infected mice may indicate a possible clinical significance of TVV infection of T. vaginalis isolates.

Proinflammatory Cytokine Profiles in Both Mild Symptomatic and Asymptomatic SARS-CoV-2-Infected Egyptian Individuals and a Proposed Relationship to Post-COVID-19 Sequela.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection is associated with proinflammatory cytokine release as mediators of host antiviral response to the infection. Cytokine persistent elevation leads to post-Coronavirus disease-2019 (COVID-19) post-COVID-19 sequela (PCS) reported in about 60% of patients affecting individual's normal life after recovery. This study evaluates relationship of cytokines and chemokines pattern during and postinfection to PCS events. Serum samples collected from 82 individuals with symptomatic, asymptomatic, or no SARS-CoV-2 infection were classified as recently or formerly infected groups according to levels of anti-2019nCoV Immunoglobulin G/Immunoglobulin M. Levels of interleukin (IL)-1α, IL-1ß, IL-6, IL-8, interferon alpha (IFN-α), tumor necrosis factor alpha (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein-1 were assessed via ELISA for each individual. All asymptomatic groups showed nonsignificant differences in cytokines' levels than control group. Significant elevation of IFN-α, TNF-α, and GM-CSF levels were observed in recent symptomatic, while IFN-α and TNF-α levels were significant in former symptomatic groups. We observed an association between fever with IL-1α and IFN-α levels, fatigue with TNF-α and GM-CSF, dyspnea with IFN-α, TNF-α, and GM-CSF, and chest-wheezing with GM-CSF. Individuals were surveyed 12 months postsampling for PCS events. Among 35 responders to survey, 8 (22.8%) reported PCS events, 6 of which were females. Upon studying PCS events, IL-8, IFN-α, TNF-α, and GM-CSF levels showed significant elevation in active infection, that was not seen in a resolved state of infection. Cytokines patterns suggest that either a persistent elevation in levels or damage caused during infection contributes to PCS. Although with the limited sample size, our study emphasizes the importance to conduct medical approaches targeting the associated cytokines to improve the PCS symptoms.

Vitamin D and Swimming Exercise Prevent Obesity in Rats under a High-Fat Diet via Targeting FATP4 and TLR4 in the Liver and Adipose Tissue.

The prevalence of obesity has risen in the last decades, and it has caused massive health burdens on people's health, especially metabolic and cardiovascular issues. The risk of vitamin D insufficiency is increased by obesity, because adipose tissue alters both the requirements for and bioavailability of vitamin D. Exercise training is acknowledged as having a significant and long-term influence on body weight control; the favorable impact of exercise on obesity and obesity-related co-morbidities has been demonstrated via various mechanisms. The current work illustrated the effects of vitamin D supplementation and exercise on obesity induced by a high-fat diet (HFD) and hepatic steatosis in rats and explored how fatty acid transport protein-4 (FATP4) and Toll-like receptor-4 antibodies (TLR4) might be contributing factors to obesity and related hepatic steatosis. Thirty male albino rats were divided into five groups: group 1 was fed a normal-fat diet, group 2 was fed an HFD, group 3 was fed an HFD and given vitamin D supplementation, group 4 was fed an HFD and kept on exercise, and group 5 was fed an HFD, given vitamin D, and kept on exercise. The serum lipid profile adipokines, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were analyzed, and the pathological changes in adipose and liver tissues were examined. In addition, the messenger-ribonucleic acid (mRNA) expression of FATP4 and immunohistochemical expression of TLR4 in adipose and liver tissues were evaluated. Vitamin D supplementation and exercise improved HFD-induced weight gain and attenuated hepatic steatosis, along with improving the serum lipid profile, degree of inflammation, and serum adipokine levels. The expression of FATP4 and TLR4 in both adipose tissue and the liver was downregulated; it was noteworthy that the group that received vitamin D and was kept on exercise showed also improvement in the histopathological picture of this group. According to the findings of this research, the protective effect of vitamin D and exercise against obesity and HFD-induced hepatic steatosis is associated with the downregulation of FATP4 and TLR4, as well as a reduction in inflammation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulins using chemiluminescence immunoassay and its correlation with neutralizing antibodies.

BACKGROUND: Neutralizing antibodies (NAbs) against SARS-CoV-2 infection have a pivotal role in protective immune response; however, their measurement requires specialized facilities. We evaluated the degree of correlation between NAbs and anti-SARS-CoV-2 IgG/total Ig antibodies detected by chemiluminescent immunoassay in asymptomatic and previously symptomatic SARS-CoV-2 patients. METHODS: A total of 1241 participants (previously symptomatic patients and asymptomatic individuals), who were screened for SARS-CoV-2 infection by RT-PCR or serology, were enrolled in our study. Sera were analyzed for the presence of anti-spike-1(S1)-SARS-CoV-2 IgG/total Ig antibodies, using Ortho Clinical Diagnostics, USA. A signal/cut-off value (S/CO) ≥ 1 was considered reactive. NAbs were measured in 103 random samples from groups using microneutralization assay, with titer ≥ 1:10 being considered positive. RESULTS: Asymptomatic (n = 229) and 261 previously symptomatic individuals with positive serology and negative RT-PCR were finally included. Significant higher anti-S1-IgG titers were seen in asymptomatic individuals (P < 0.0001). Conversely, anti-S1-total Ig titers were significantly higher in previously symptomatic (P < 0.0001). NAbs were detected in both groups, however, higher titers were seen in previously symptomatic patients. There is a correlation between NAbs and both IgG/total anti-S1-SARS-CoV-2 antibodies (r = 0.47, P < 0.0001 and r = 0.49, P < 0.0001, respectively). IgG and total Ig could predict a neutralization titer of ≥ 1:160 at S/CO >4.44 and >65 with AUC 0.69 and 0.67, respectively. CONCLUSION: Asymptomatic SARS-CoV-2 infection can produce comparable antibodies response to previously symptomatic individuals, however higher neutralization activity was seen in the previously symptomatic. Anti-S1-SARS-CoV-2 IgG/total Ig antibodies showed a correlation with neutralization activity and can be used to estimate the presence of protective immunity.

Safety and immunogenicity evaluation of inactivated whole-virus-SARS-COV-2 as emerging vaccine development in Egypt.

BACKGROUND: Current worldwide pandemic coronavirus disease 2019 (COVID-19) with high numbers of mortality rates and huge economic problems require an urgent demand for safe and effective vaccine development. Inactivated SARS-CoV2 vaccine with alum. Hydroxide can play an important role in reducing the impacts of the COVID-19 pandemic. In this study, vaccine efficacy was evaluated through the detection of the neutralizing antibodies that protect mice from challenge with SARS-CoV 2 3 weeks after the second dose. We conclude that the vaccine described here has safety and desirable properties, and our data support further development and plans for clinical trials. METHODS: Characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069; and MW250352 at GenBank were isolated from Egyptian patients SARS-CoV-2-positive. Development of inactivated vaccine was carried out in a BSL-3 facilities and the immunogenicity was determined in mice at two doses (55 and 100 µg per dose). RESULTS: The distinct cytopathic effect induced by SARS-COV-2 propagation on Vero cell monolayers and the viral particles were identified as Coronaviridae by transmission electron microscopy and RT-PCR on infected cells cultures. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless of the dose concentration, with excellent safety profiles and no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by the wild virus challenge of the vaccinated mice and viral replication detection in lung tissues. CONCLUSIONS: Vaccinated mice recorded complete protection from challenge infection via inhibition of SARS-COV-2 replication in the lung tissues of mice following virus challenge, regardless of the level of serum neutralizing antibodies. This finding will support future trials for the evaluation of an applicable SARS-CoV-2 vaccine candidate.

SARS-CoV-2 infection among asymptomatic healthcare workers of the emergency department in a tertiary care facility.

BACKGROUND: Healthcare workers (HCWs) represent a high-risk category during the coronavirus disease 2019 (COVID-19) pandemic crisis, with frontline HCWs at emergency departments (EDs) may be at an even higher risk. Determining the spread of infection among HCWs may have implications for infection control policies in hospitals. This study aimed to detect severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection among asymptomatic HCWs of the ED of a large tertiary center in Cairo, Egypt. METHODS: The study was conducted from June 1st to June 14th, 2020. All the recommended national and international indications on infection control measures were followed. Two hundred and three HCWs were included in the study and tested by nasopharyngeal swab (NPS) and rapid serological test (RST). Descriptive statistical analyses were used to summarize the data. RESULTS: Of the 203 HCWs, 29 (14.3 %) tested positive by real-time reverse transcription polymerase chain reaction (RT-PCR). Thirty-seven (18.2 %) HCWs tested positive with RST: 20 with both IgM and IgG; 14 with IgM only, and 3 with IgG only. Age, gender, and/or occupation were not risk factors for SARS-CoV-2 infection. CONCLUSIONS: Point prevalence of COVID-19 in asymptomatic HCWs in ED of tertiary care facility is 14.3 % by RT-PCR. This illustrates the importance of screening all HCWs regardless of symptoms, and the need for strict measures in securing HCWs to reduce transmission from healthcare facilities to the community during the current pandemic.

Prevalence of SARS-CoV-2 infection and dynamics of antibodies response among previously undiagnosed healthcare workers in a university hospital: A prospective cohort study.

BACKGROUND: Healthcare workers (HCWs) are a presumed high-risk population for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Identifying factors associated with seroprevalence can help establish better practices in healthcare settings. In this study, we evaluate prevalence of SARS-CoV-2 infection among previously undiagnosed HCWs and describe profiling of antibody responses against SARS-CoV-2, including neutralizing antibodies (NAbs). METHODS: We analyzed a cohort of 386 HCWs in a university hospital in Egypt and 725 volunteers not affiliated to any healthcare facility (non-healthcare workers - NHCWs). Participants provided a nasopharyngeal swab and serum samples for SARS-CoV-2 nucleic acid and SARS-CoV-2-specific antibodies, respectively. HCWs who tested positive by either test were sequentially monitored. RESULTS: At baseline, point prevalence of viral carriage was 11.4% in HCWs (n = 44/386) and 11.9% in NHCWs (86/725). The cumulative prevalence of SARS-CoV-2 infection among HCWs considering all studies was 25.6%, which was statistically lower than in NHCWs (41.0%). Prevalence was greatest among janitorial staff (45.9%) and the most affected departments were gastroenterology (31.1%), and emergency medicine (30.0%). Prior anosmia, fever or headache were associated with higher odds of positivity for SARS-CoV-2 infection. Regarding serial antibody measurements, RT-PCR-positive HCWs displayed IgG detection rates of 29.5%, 70% and 60% at visit 1, visit 2 and visit 3, respectively with slow decline of median IgG antibody titers, whereas, corresponding detection rates for total Ig antibodies were 50%, 90.3%, and 88.9%, respectively with increasing median titers. NAbs measured at each time point were positively correlated with total Ig levels, whereas IgG levels were positively correlated with NAbs at visit 1 and visit 3. CONCLUSION: Our results demonstrate lower cumulative prevalence of SARS-CoV-2 infection in HCWs than general population and suggest that asymptomatic HCWs exhibit considerable IgG and total Ig antibodies response as well as NAbs for up to 120 days, with positive correlation in between.

SARS-CoV-2 infection among healthcare workers of a gastroenterological service in a tertiary care facility.

BACKGROUND AND STUDY AIMS: Frontlines healthcare workers (HCWs) during the coronavirus disease 2019 (COVID-19) pandemic are at increased risk of infection by SARS-CoV-2, but there are limited data on the prevalence of COVID-19 among HCWs in Egypt. This study aimed to assess SARS-CoV-2 infection among HCWs providing gastroenterological services. SUBJECTS AND METHODS: Seventy-four HCWs at the gastroenterological service of Al-Manial University Hospital, the main hospital of the largest tertiary university hospitals complex in Egypt (Kasr Al-Ainy Faculty of Medicine, Cairo University) were tested using real-time reverse transcription-polymerase chain reaction (RT-PCR) on nasopharyngeal samples, and rapid serological IgM/IgG tests (RST). A questionnaire was used to collect demographic, occupational and clinical data. RESULTS: Of the 74 HCWs, 10 tested positive by RT-PCR (13.5%). In 9/74 (12.2%) HCWs, antibodies could be detected by RST: three with both IgM and IgG lines; six with IgM line only and none with IgG line only. Frequency of positive tests was more among subjects with minor symptoms compared to completely asymptomatic HCWs (50% vs 16.1%, respectively). Neither age, gender or occupation was a risk factor for SARS-CoV-2 infection. CONCLUSIONS: Point prevalence of COVID-19 in gastroenterology HCWs is 13.5% by RT-PCR. Continued measures are warranted to assure HCWs safety and reduce transmission from healthcare settings to the community during COVID-19 pandemic. Presence of positive test results among asymptomatic HCWs illustrates the importance of screening all HCWs irrespective of symptoms.

Study of the pathogenic potential of Dientamoeba fragilis in experimentally infected mice.

Dientamoebafragilis (D. fragilis) is a protozoan parasite whose pathogenic potential is still disputable. The aim of this study was to illustrate the pathogenicity of D. fragilis infection and to determine the infective dose for experimental mice infection. Three groups of mice (8/each) were orally inoculated with in vitro cultured D. fragilis. The infected groups (G1- G3) received 103, 105 and 4 × 106D. fragilis/0.5 ml culture, respectively. A control group (G4) only received parasite-free culture. Two weeks post-inoculation all mice were euthanized for histopathological examination. All mice of G3 (100%) and three mice of G2 (37.5%) were infected, and the results were confirmed by PCR and different staining methods. On the other hand, all mice from group G1 showed a completely negative result. Histopathological examination of the colon and caecum of the highly infected group G3 showed active colitis, with infiltration of mixed inflammatory cells such as eosinophils, neutrophils and lymphocytes within the lamina propria of the intestinal wall. The parasite was not invading the colonic mucosa. This study revealed that infection with D. fragilis is dose-dependent. Moreover, a dose of 105D. fragilis/mouse or higher is necessary to infect mice through the oral route. In addition, this route of infection, although non-invasive, can induce severe inflammatory changes to the colonic and caecal mucosa in experimentally infected mice.

Comparative analysis of endoderm formation efficiency between mouse ES cells and iPS cells.

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.

Hepatic differentiation of mouse iPS cells in vitro.

Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.

Bone repair by transplantation of hTERT-immortalized human mesenchymal stem cells in mice.

BACKGROUND: Human mesenchymal stem cells (hMSCs) are multipotent stem cells found in the adult bone marrow that have the capacity to differentiate into various mesenchymal cell types. The hMSCs may provide a potential therapy to restore damaged tissues or organs of mesenchymal origin; however, a drawback is their limited life span in vitro. METHODS: We immortalized normal hMSCs with retrovirally transmitted human telomerase reverse transcriptase cDNA. One of the immortalized clones (YKNK-12) was established, and the biological characteristics were investigated in vitro and in vivo. RESULTS: YKNK-12 cells were capable of differentiating adipocytes, osetoblasts, and chondrocytes. Osteogenically differentiated YKNK-12 cells produced significant levels of growth factors BMP4, BMP6, FGF6, FGF7, transforming growth factor-beta1, and transforming growth factor-beta3.. Microcomputer tomography T and soft X-ray assays showed an excellent calvarial bone healing in mice after transplantation of osteogenically differentiated YKNK-12 cells. These cells expressed human-specific osteocalcin and increased the gene expression of runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osterix in the bone regenerating area. YKNK-12 cell transplant corrected the bone defect without inducing any adverse effects. CONCLUSIONS: We conclude that hMSCs immortalized by transduction with human telomerase reverse transcriptase may provide an unlimited source of cells for therapeutic use in bone regeneration.