Transformation of Hamster Kidney Cells by BK Papovavirus DNA

Supercoiled BK papovavirus DNA was shown to transform hamster kidney cells using the calcium phosphate co-precipitation technique. The transformed cells contained intranuclear T-antigen(s) and rescuable virus and produced progressively growing tumors when inoculated into hamsters. A novel finding was the production in tumor-bearing animals of antinuclear antibody, which reacted against normal, untransformed cells; in addition, tumor serum contained antibody against virus-specific T-antigen(s).     

Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis

We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.     

Complementation Between BK Human Papovavirus and a Simian Virus 40 tsA Mutant

Complementation tests between BK human papovavirus and SV40 temperature-sensitive mutants tsA58 and tsB11 were performed. Under the reported experimental conditions, BKV complemented the "early" mutant tsA58 but failed to complement the "late" mutant tsB11.     

Primary Culture of Human Embryonic Kidney Cells as the Medium for Isolation of ECHO 6 Virus

A large epidemic of aseptic meningitis due to ECHO 6 virus swept over Japan in the summer months of 1965. In our studies on 89 cases of aseptic meningitis in the epidemic, primary culture of human embryonic kidney cells was shown to provide a highly sensitive host system for isolation of ECHO 6 virus from clinical materials. Virus was recovered from cerebrospinal fluid in 72% of 89 cases, from throat swabs in 64% of 36 cases, and from rectal swabs in 59% of 37 cases. Most significant is the finding that the rate of virus isolation from cerebrospinal fluid in this host system was considerably higher as compared with that obtained by other investigators in other host systems such as primary monkey kidney cells or human amniotic cells (primary or EL). This finding should be emphasized particularly because isolation of a virus from cerebrospinal fluid, in contrast to throat secretions or feces, is of much greater importance in establishing the etiologic relationship to the disease in the diagnosis of aseptic meningitis. The neutralization test was shown to be efficient in detecting ECHO 6 virus infection; acute serums should be taken preferably by the 4th day of illness and convalescent serums in the second week. Epidemiologic findings, such as the predominance of male patients and occurrence of the epidemic in summer months, generally coincide with the previous reports. However, our cases were in much younger age groups in contrast with the previous reports; 90% of our 89 patients were 6 years of age or younger, and 15 children or 17% were less than 1 year of age, including 3 less than 6 months of age. The clinical observations on our cases confirmed the previous reports.     

人乳头状瘤病毒诱导人胚食管上皮永生化细胞恶性转化

为了证实ＨＰＶ１８Ｅ６Ｅ７基因诱导的人胚食管上皮永生化细胞（ＳＨＥＥ）６１代（ＳＨＥＥ６１）部份细胞（ＳＨＥＥ６１Ａ）已经恶性转化,以寻找监控细胞早期恶性转化的方法。对培养的ＳＨＥＥ第１０代（ＳＨＥＥ１０）和６１代（ＳＨＥＥ６１）细胞,用光学是为微镜和电子显微镜观察细胞形态及生长形式;用流工细胞仪分析细胞周期;用染色体Ｇ带核型分析和间期核染色体１、７、８号着丝粒探针荧光原位杂交９ＦＩＳＨ）检测细胞     

Replication of Oral BK Virus in Human Salivary Gland Cells

BK polyomavirus (BKPyV) is the most common viral pathogen among allograft patients. Increasing evidence links BKPyV to the human oral compartment and to HIV-associated salivary gland disease (HIVSGD). To date, few studies have analyzed orally derived BKPyV. This study aimed to characterize BKPyV isolated from throat wash (TW) samples from HIVSGD patients. The replication potential of HIVSGD-derived clinical isolates HIVSGD-1 and HIVSGD-2, both containing the noncoding control region (NCCR) architecture OPQPQQS, were assessed and compared to urine-derived virus. The BKPyV isolates displayed significant variation in replication potential. Whole-genome alignment of the two isolates revealed three nucleotide differences that were analyzed for a potential effect on the viral life cycle. Analysis revealed a negligible difference in NCCR promoter activity despite sequence variation and emphasized the importance of functional T antigen (Tag) for efficient replication. HIVSGD-1 encoded full-length Tag, underwent productive infection in both human salivary gland cells and kidney cells, and expressed viral DNA and Tag protein. Additionally, HIVSGD-1 generated DNase-resistant particles and by far surpassed the replication potential of the kidney-derived isolate in HSG cells. HIVSGD-2 encoded a truncated form of Tag and replicated much less efficiently. Quantitation of infectious virus, via the fluorescent forming unit assay, suggested that HIVSGD BKPyV had preferential tropism for salivary gland cells over kidney cells. Similarly, the results suggested that kidney-derived virus had preferential tropism for kidney cells over salivary gland cells. Evidence of HIVSGD-derived BKPyV oral tropism and adept viral replication in human salivary gland cells corroborated the potential link between HIVSGD pathogenesis and BKPyV.     

Temperature-Dependent Transformation of Cells Infected with a Mutant of Bryan Rous Sarcoma Virus

Chick embryo cells infected with a mutant (Ta) of the Bryan high-titer strain of Rous sarcoma virus (RSV-BH) are morphologically transformed at 36 C but appear similar to uninfected cells at 41 C. When cells infected with RSV-BH-Ta are switched from 41 to 36 C, morphological changes characteristic of transformation are observable within 10 min. The transformation is reversible; cells shifted from 36 to 41 C have been observed to lose their transformed morphology within 1 hr. The transformation after a shift in temperature is unaffected by inhibition of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein synthesis, demonstrating that the proteins involved in the morphological change are already present. Transformed cells infected with RSV-BH or RSV-BH-Ta take up hexose and synthesize hyaluronic acid at higher rates than uninfected cells or RSV-BH-Ta-infected cells grown at 41 C. However, inhibition of either protein or RNA synthesis, but not DNA synthesis, prevented the induction of increased hexose uptake and hyaluronic acid synthesis after a shift of RSV-BH-Ta-infected cells from 41 to 36 C. Therefore, these biochemical changes are secondary to a more basic change responsible for morphological transformation.     

乙型肝-T炎病毒和黄曲霉素协同在裸鼠体内诱发人胎肝细胞癌变的研究

为了研究乙型肝炎（乙肝）病毒（HBV）和黄曲霉素（AFB1）在肝癌发生过程中的作用,用HBV感染人胚胎肝细胞移植至裸鼠背部皮下,以后每周注射AFB1,能诱发裸鼠成瘤,实验分为4组：A组为HV＋AFB1组,即用HBV感染的人胚胎肝细胞移植于裸鼠,同时注射AFB1;B组为HBV^ 组,用HBV感染的人胚胎肝细胞移植于裸鼠,不注射AFB1;C组为AFB1^ 组,用不感染HBV的人胚胎肝细胞移植于裸鼠,注射AFB1;D组为对照组,用不感染HBV的人胚胎肝细胞移植于裸鼠,也不注射AFB1。结果：A组成瘤率为27．3％（6／22）,B组0％,C组13．3％（2／15）,D组0％,所有肿瘤病理诊断均为肝细胞癌。用EMA单抗检测,证实为人来源细胞。PCR和DNA狭缝印迹显示：HBV X和HBV S基因阳性,证明HBV基因已在瘤细胞中。细胞首次用人乙肝病毒协同AFB1在裸鼠体内诱发成功人肝细胞癌,证明了ABV协同AFB1,在人肝细胞癌发生过程中的病因作用。同时,为进一步研究肝癌的发生及防治了良好的动物模型。     

Transformation and Productive Infection of Human Osteosarcoma Cells by a Feline Sarcoma Virus

FELINE sarcoma virus (FSV) transforms human embryo cells in vitro¹; it therefore seemed interesting to determine whether this virus could transform human osteosarcoma cells. Defective Moloney sarcoma virus genome can be rescued from non-producer hamster tumour cells by feline leukaemia virus (FeLV)² and because FSV stocks also contain excess FeLV (ref. 1 and unpublished observations of R. V. G.), it was hoped that human osteosarcoma cells transformed by FSV and co-infected with FeLV might yield a human sarcoma virus.     

Induction of Specific Chromosomal Aberrations by Adenovirus Type 12 in Human Embryonic Kidney Cells

Nonrandom chromosomal breaks in chromosomes 1 and 17 were provoked in human embryonic kidney cells 24 hr after infection with adenovirus type 12. These chromosomal changes disappeared in persistently infected cultures. Neutralization of the virus with type-specific antiviral serum prior to infection prevented the occurrence of chromosomal aberrations. No viral deoxyribonucleic acid (DNA) synthesis, as determined by autoradiography, was seen in metaphases containing adenovirus type 12-induced chromosomal aberrations. Ultraviolet irradiation of the virus reduced chromosomal aberrations linearly. This reduction in aberrations was fourfold slower than the inactivation of viral infectivity. At 24 hr after infection of cells with purified (3)H-labeled adenovirus type 12, the isotope was found to be associated with the nuclei. The uptake of isotope was reduced ninefold when the labeled virus was neutralized with type-specific antiviral serum. This difference is considered to account for neutralization of labeled virions. In metaphases infected with labeled viruses, most of the clustered grains were seen only on one arm of the chromatid, even after 72 hr. Isochromatid labeling was found, however, in a small percentage of chromosomes, and increased with time after infection. This increase was threefold between 24 and 72 hr after infection, whereas the mean grain counts decreased twofold during the same period. This has been tentatively interpreted to mean that most of the viral DNA molecules or parts thereof are merely attached to cellular chromatin, but a small fraction of them becomes gradually integrated as time proceeds. Certain chromosomal sites appeared to be preferentially labeled when chromosome 2 was used as a model for evaluation.     

Isolation of a Transformation-Defective Deletion Mutant of Moloney Murine Sarcoma Virus

A transformation-defective (td) deletion mutant of Moloney murine sarcoma virus (td Mo-MSV) and a transforming component termed Mo-MSV 3 were cloned from a stock of clone 3 Mo-MSV. To define the defect of the transforming function, the RNA of td Mo-MSV was compared with those of Mo-MSV 3 and of another transforming variant termed Mo-MSV 124 and with helper Moloney murine leukemia virus (Mo-MuLV). The RNA monomers of td Mo-MSV and Mo-MSV 3 comigrated on polyacrylamide gels and were estimated to be 4.8 kilobases (kb) in length. In agreement with previous analyses, the RNA of Mo-MSV 124 measured 5.5 kb and that of Mo-MuLV measured 8.5 kb. The interrelationships among the viral RNAs were studied by fingerprinting and mapping of RNase T₁-resistant oligonucleotides (T₁-oligonucleotides) and by identification of T₁-oligonucleotides present in hybrids formed by a given viral RNA with cDNA's made from another virus. The nontransforming td Mo-MSV RNA lacked most of the Mo-MSV-specific sequence, i.e., the four 3′-proximal T₁-oligonucleotides of the six T₁-oligonucleotides that are shared by the Mo-MSV-specific sequences of Mo-MSV 3 and Mo-MSV 124. The remaining two Mo-MSV-specific oligonucleotides identified td Mo-MSV as a deletion mutant of MSV rather than a deletion mutant of Mo-MuLV. td Mo-MSV and Mo-MSV 124 exhibited similar deletions of gag, pol, and env sequences which were less extensive than those of Mo-MSV 3. Hence, td Mo-MSV is not simply a deletion mutant of Mo-MSV 3. In addition to their MSV-specific sequences, all three MSV variants, including td Mo-MSV, shared the terminal sequences probably encoding the proviral long terminal repeat, which differed from their counterpart in Mo-MuLV. This may indirectly contribute to the oncogenic potential of MSV. A comparison of td Mo-MSV sequences with either Mo-MSV 124 or Mo-MSV 3 indicated directly, in a fashion similar to the deletion analyses which defined the src gene of avian sarcoma viruses, that Mo-MuLV-unrelated sequences of Mo-MSV are necessary for transformation. A definition of transformation-specific sequences of Mo-MSV by deletion analysis confirmed and extended previous analyses which have identified Mo-MuLV-unrelated sequences in Mo-MSV RNA and other studies which have described transformation of mouse 3T3 fibroblasts upon transfection with DNAs containing the Mo-MSV-specific sequence.     

Transformation of African Green Monkey Kidney Cells by Irradiated Adenovirus 7-Simian Virus 40 Hybrid

The ability of adenovirus 7-simian virus 40 (SV40) hybrid (strain LL "E-46") to replicate decreased exponentially as a function of the amount of gamma-irradiation; the ability to induce SV40 and adenovirus 7 T antigen decreased at a much slower rate. Nevertheless, the virus was still able to transform African green monkey kidney cells at a radiation dosage that had completely destroyed its replication ability. All transformed colonies were positive for SV40 T antigen but were negative for adenovirus 7 T antigen. The adenovirus 7-SV40 hybrid transformed cells were superinfectible with SV40 virus. Two of the three transformed cell populations apparently did not sensitize hamsters against the appearance SV40 primary tumors, thus suggesting a deficiency in the SV40 transplantation antigen.     

Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection.     

Natural Scaffolds for Renal Differentiation of Human Embryonic Stem Cells for Kidney Tissue Engineering

Despite the enthusiasm for bioengineering of functional renal tissues for transplantation, many obstacles remain before the potential of this technology can be realized in a clinical setting. Viable tissue engineering strategies for the kidney require identification of the necessary cell populations, efficient scaffolds, and the 3D culture conditions to develop and support the unique architecture and physiological function of this vital organ. Our studies have previously demonstrated that decellularized sections of rhesus monkey kidneys of all age groups provide a natural extracellular matrix (ECM) with sufficient structural properties with spatial and organizational influences on human embryonic stem cell (hESC) migration and differentiation. To further explore the use of decellularized natural kidney scaffolds for renal tissue engineering, pluripotent hESC were seeded in whole- or on sections of kidney ECM and cell migration and phenotype compared with the established differentiation assays for hESC. Results of qPCR and immunohistochemical analyses demonstrated upregulation of renal lineage markers when hESC were cultured in decellularized scaffolds without cytokine or growth factor stimulation, suggesting a role for the ECM in directing renal lineage differentiation. hESC were also differentiated with growth factors and compared when seeded on renal ECM or a new biologically inert polysaccharide scaffold for further maturation. Renal lineage markers were progressively upregulated over time on both scaffolds and hESC were shown to express signature genes of renal progenitor, proximal tubule, endothelial, and collecting duct populations. These findings suggest that natural scaffolds enhance expression of renal lineage markers particularly when compared to embryoid body culture. The results of these studies show the capabilities of a novel polysaccharide scaffold to aid in defining a protocol for renal progenitor differentiation from hESC, and advance the promise of tissue engineering as a source of functional kidney tissue.     

Efficient Production and Characterization of Recombinant Human NELL1 Protein in Human Embryonic Kidney 293-F Cells

NELL1 is a secretory protein that induces osteogenic differentiation and bone formation by osteoblastic cells. Because of its potent osteoinductive activity, NELL1 may be useful for bone regeneration therapy. However, at present, we have little knowledge regarding NELL1 receptors and NELL1-mediated signaling pathways. We have previously produced NELL1 using an insect’s cell expression system; however, the protein was relatively unstable and was degraded by proteases released from dead cells. In the present study, NELL1 protein was expressed in human embryonic kidney 293-F cells. Stable cell lines expressing NELL1 fused to a C-terminal hexahistidine-tag were obtained by G418 selection of transfected cells. Cells grown in serum-free medium showed high levels of NELL1 protein production (approximately 4 mg/l cell culture) for up to 6 months. NELL1 protein was purified from culture medium using a one-step nickel-chelate affinity chromatography protocol. Purified NELL1 protein immobilized onto culture dishes induced the expression of both early and late osteogenic markers on mouse mesenchymal C3H10T1/2 cells. When NELL1-expressing 293-F cells were grown on gelatin-coated glass cover slips, recombinant NELL1 was deposited in the extracellular matrix after detachment of cells. These results suggest that NELL1 acts as an extracellular matrix component. Recombinant NELL1 formed multimers and was glycosylated. An abundant source of functionally active NELL1 protein will be useful for more advanced studies, such as the development of novel techniques for bone regeneration.     

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 10⁵ hESCs are needed to form a 16 cm³ teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.