New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation.

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.

Establishment and characterization of human uterine poorly differentiated adenocarcinoma cell line (HHUABM).

The cell line designated HHUABM was established from the metastatic region (left Bartholin gland) of human endometrial adenocarcinoma. The cell line grew well, multilayering rapidly without contact inhibition, and 72 serial passages were successively done within 25 months. The cultured cells of HHUABM line were round and spindle in shape, and showed a pavement-like arrangement. The distribution of chromosome number varied narrowly at the diploid range, and the modal chromosome number was 46. The 90% of metaphase cells showed normal karyotype. The HHUABM cells were transplanted easily into the subcutis of BALB/c nude mice and produced poorly differentiated adenocarcinoma resembling the original tumor. The conditioned medium promoted the proliferation of CPAE (endothelial cells). The estradiol-17 beta and progesterone receptors were not detected.

Establishment and characterization of human choriocarcinoma cell line derived from a metastatic focus of a testicular mixed germ cell tumor.

A human testicular choriocarcinoma cell line HKRT-II was established by the single-cell cloning method from a mixed cell culture system derived from a retroperitoneal metastatic germ cell tumor composed of a yolk-sac tumor, a choriocarcinoma, and an immature teratoma. Its primary tumor rose from the testis and was comprised of a seminoma, a yolk-sac tumor, a choriocarcinoma and an immature teratoma. The HKRT-II cells were spindle or polygonal in shape and contained multi-nucleated giant cells showing neoplasticity and pleomorphism. The cells proliferated in a stable manner, and the population doubling time was 42 hours. The chromosome numbers showed a wide distribution of aneuploidy, while the mode was in the hypertetraploid range. Double minute chromosomes and homogeneously staining regions were recognized in about 5% to 10% of the metaphase plates, respectively. Heterotransplantation was not difficult. Subcutaneous transplantation of 1 x 10(7) cells into nude mice formed a tumor composed of only a choriocarcinoma. The most noteworthy characteristics of the cell line were that it produced human chorionic gonadotropin (hCG) in an in vitro culture system and in in vivo grafted cells, and that the N-myc gene was amplified about 10 times.

Establishment and characterization of melanoma cell line derived from malignant melanoma of human uterine cervix.

A cell line designated HOMM (human Okuno Malignant Melanoma) was established from the uterine cervical malignant melanoma of a 65-year-old Japanese woman. The cell line has grown well and serial passages were successively carried out 32 times within 19 months. The monolayer cultured cells revealed anaplastic and pleomorphic features, and grew in multilayers. They had long cell protrusions and many dark brown pigments. Immunocytochemical stain revealed that S-100 protein existed in the cytoplasm. Electron micrographs also revealed that they had a number of pre-melanosomes and melanosomes in the cytoplasm. All cultured cells were triploid, the modal chromosome number was 68 and the marker chromosomes were presented. The cells were transplanted into an immune-suppressed hamster's cheek pouch and produced a malignant melanoma resembled original tumor.

Effects of feeder cells (human cancer cell lines) on the development of mouse embryos by co-culture.

In order to establish the best co-culture system on embryogenesis such as egg fertilization, egg cleavage, blastocyst formation, hatching and implantation etc., several kinds of cell lines as a feeder cell and mouse fertilized eggs (zygotes) were co-cultured in the organ culture dish, and embryotrophic effects of feeder cells were investigated. Best feeder cell on the embryogenesis was SKG-II cell line derived from squamous cell carcinoma of human uterine cervix which was chosen from 10 of the human tumor cell lines. Furthermore, in order to isolate and determinate embryotrophic factors produced by feeder cells, we established a SKG-II SF subline which was grown in serum free medium derived from SKG-II cell line. The SKG-II SF cell line secreted an epidermal growth factor (EGF) into the medium. Also, cleavaged egg produced and secreted interleukin (IL)-1 alpha into the medium.

Tissue reconstruction of gynecologic tumor cells in the rotation culture system.

Tissue reconstruction of various kinds of gynecologic malignant tumor cell lines was studied using the rotation-culture system. The reconstructed cell aggregates were examined histologically using both light and electron microscopy. Our established cell lines used in this study were uterine cervical epidermoid carcinoma, endometrial adenocarcinoma, ovarian malignant tumor and uterine sarcoma. All of the reconstructed aggregates from each cell line were very similar to the original tumor tissue. In the case of a well differentiated type of adenocarcinoma derived from the ovarian cancers or the endometrial cancers, papillary cell aggregates (grape-like structures) and/or hollow cell ball (gland alveolus-like) structures were observed. The individual cells were adjoined with by desmosomes and well developed microvilli protruded from the free surface of the cells. On the other hand large cell non-keratinizing squamous cell carcinoma cells formed spherical-shaped aggregates that showed a stratified structure similar to pearl formation. Sarcoma cells formed solid clusters while desmosomes or desmosome-like junctions were not detected. Rotation culture is an excellent method to reveal diagnosis of the original tumor and tumorigenesis by investigating a reconstructed tissue from peritoneal effusions because the reconstructed tissue is similar to the original tumor.

Histogenesis of hollow cell ball structure of ovarian and endometrial adenocarcinoma cells in vivo and in vitro.

Hollow cell ball structure is often found in the ascites of adenocarcinoma patients. How to form a hollow cell ball structure was studied in vivo and in vitro, using the human cell lines derived from ovarian and endometrial adenocarcinomas. The hollow cell ball structure was formed by horizontal rotation culture of 1 x 10(7) single-suspended cells for 24 hours or by transplanting 1 x 10(6) single-suspended cells into the peritoneal cavity of nude mouse for 24 hours. At one month after transplantation hemi-cyst and hollow cell ball structure were formed in the outermost layer of the grafted tumor on the intraperitoneal serous membrane in the nude mouse. And also great number of floating hollow cell ball structure in the ascites were observed. These results suggest that mechanisms of formation of hollow cell ball structure found in the ascites; one by cell aggregate of single cells, sometimes inner cells of cell aggregate fall into necrosis or secretes mucus inside and make a hollow cell ball structure and another by the removed as the hollow cell ball structure grown from hemi-cyst on the surface of intraperitoneal grafted tumor.

Transmission of hepatitis C virus from mothers to infants. The Vertical Transmission of Hepatitis C Virus Collaborative Study Group.

BACKGROUND: Although there are case reports of vertical transmission of hepatitis C virus (HCV), it remains uncertain to what extent infected mothers transmit this virus to their infants. METHODS: We investigated the transmission of HCV from infected mothers to their babies by analyzing HCV RNA in the blood. Three independent studies were performed. First, 7698 parturient women were tested for anti-HCV antibodies; 53 were positive. Their 54 infants (including one set of twins) were followed prospectively for at least six months and tested for HCV disease were prospectively studied. Third, the families of three HCV-infected infants were examined retrospectively. RESULTS: Of the 53 antibody-positive mothers, 31 were also positive for serum HCV RNA: Three of the 54 babies born to these mothers (5.6 percent) became positive for HCV RNA during the follow-up period. None of the babies of the 22 women who were antibody-positive but HCV RNA-negative became positive for HCV RNA: In the second study, HCV RNA was detected in one of the six infants of infected mothers. In the third study, HCV RNA was detected in the mothers of the three HCV-infected infants. In each of the seven infected infants we studied, the genomic sequence of HCV was almost identical to that from the mother. These seven mothers had significantly higher titers of HCV RNA than did the mothers of infants with no evidence of infection (mean [+/- SD], 10(6.4 +/- 0.5) vs. 10(4.4 +/- 1.5) per milliliter; P < 0.001). CONCLUSIONS: HCV is vertically transmitted from mother to infant, and the risk of transmission is correlated with the titer of HCV RNA in the mother.

Presence of human papillomavirus genome in human tumor cell lines and cultured cells.

A series of 47 human carcinoma cell lines and their cultured cells were examined for human papillomavirus (HPV) genomes with the use of an HPV detection kit (DNA-RNA hybridization, mixed HPV DNA probe of types 6, 11, 16, 18, 31, 33 and 35). Four of 8 cases of mild dysplasia, 3 of 9 cases of severe dysplasia, 3 of 7 cases of carcinoma in situ, 3 of 15 cases of uterine carcinoma and 5 of 6 cases of condyloma acuminatum were shown to contain the HPV DNA genome in primary cultured cells, while HPV was not detected in the third-passage cells except for the three cases of large cell, nonkeratinizing squamous cell carcinoma. HPV was also not detected in such normal tissues as uterine cervical squamous epithelium, uterine cervical columnar epithelium and endometrium. The presence of HPV DNA genomes was detected consistently in the passages of three lines (SKG-II, HKMUS and HKTUS; large cell nonkeratinizing squamous cell carcinomas of the uterine cervix) with the use of the Southern Blot method (DNA-DNA hybridization, mixed HPV probe of types 6, 11, 16 and 18). HPV type 16 DNA was detected in HKTUS, and HPV type 18 DNA was found in SKG-II and HKMUS. The other 44 cell lines, including ovarian carcinoma, endometrial carcinoma, sarcoma, gastric cancer, pancreatic cancer and rectal cancer, were negative for the HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 genomes under stringent hybridization conditions.

N-myc gene amplification and neuron specific enolase production in immature teratomas.

The primary tumour tissues from 13 teratomas were investigated, 3 cases of immature teratoma (1 of pure type and 2 of mixed type), 5 cases of dermoid cyst, and 5 cases of mature solid teratoma, with special reference to N-myc gene amplification, productivity of neuron specific enolase (NSE), and the presence of double minutes (DMs). The possible relationship between these variables and malignancy of the tumours was also examined. N-myc gene amplification and NSE production were recognized in the primary tumour tissues and the first and the third passage cultured cells of all of the 3 cases of immature teratoma containing immature neural tissues. In 2 cases DMs were recognized. In dermoid cysts and mature teratoma, neither N-myc gene amplification nor NSE production were recognized in either the primary tumour tissues or cultured cells. The chromosomes were normal. Malignancy of teratoma is generally decided by the clinical stage and histological grade, but a more securely based decision is necessary where an immature teratoma contains immature neural tissues. The presence of N-myc gene amplification, NSE productivity and the presence of DMs may be valuable.

Carcinoembryonic proteins produced by Wilms' tumor cells in vitro and in vivo.

A Wilms' tumor cell line (HFWT) was established after tissue culture of the Wilms' tumor which had developed in the left kidney of a five-month-old boy. The HFWT line has the following cyto-biological characteristics: 1. The cells have a spindle, round or polygonal shape with neoplastic and pleomorphic features that grew in multilayers without contact inhibition. 2. The cells show a stable proliferation, giving 95 passages within 4 years. 3. The chromosomes show a wide aneuploidy distribution, and the modal number was found in diploid range. The stem cells have a normal karyotyping, 46, XY. 4. Heterotransplantation into nude mice can be easily made, and anaplastic Wilms' tumor resembling the original tumor forms. 5. The principal tumor markers produced by the cells in large amounts in the conditioned culture media were carbohydrate antigen 125 (CA 125) and tissue polypeptide antigen (TPA). 6. A good correlation was found between enlargement of the tumor formed by heterotransplantation into nude mice and the levels of CA 125 and TPA in the serum of the nude mouse. No report on the study of tumor markers widely used in clinical treatment of Wilms' tumor has been available to us, but CA 125 and TPA are considered to be useful in the diagnosis and treatment of Wilms' tumor.

Differences between cell lines of uterine cervical glassy cell carcinoma and large cell nonkeratinizing squamous cell carcinoma.

Cell lines were established from two uterine cervical cancers, a glassy cell carcinoma (GCC) and a large cell nonkeratinizing squamous cell carcinoma (LCSC), and studied by a variety of techniques, including histology, chromosome analysis, heterotransplantation and tumor marker analyses. There were radical differences in the morphology, heterotransplantability, production of tumor markers, etc., between the cultures of these morphologically similar cancers. The LCSC line (HKMUS) consisted of polygonal and round cells containing tonofilaments; these cells discharged tumor antigen-4 (TA-4) into the conditioned media. HKMUS was heterotransplantable into the subcutis of nude mice to form LCSC. On the other hand, the GCC line (HOKUG) consisted of round or spindle-shaped cells. HOKUG was easily transplanted into the subcutis or intraabdominal cavity of nude mice and metastasized easily. It discharged TA-4, carbohydrate antigen 125 (CA125) and neuron-specific enolase (NSE) into the conditioned media. The histologic picture of GCC revealed numerous blood vessels and a rapid proliferation of the cells. GCC, which is considered to be a mixed carcinoma having the characteristics of both squamous carcinoma and adenocarcinoma, seems to be a cancer of unpredictable prognosis as compared to LCSC, possibly due to its rapid proliferation and easy metastasis, leading to peritonitis carcinomatosa.

Effect of tumour angiogenesis factor on proliferation of endothelial cell and tube formation.

The effect of a tumour angiogenesis factor on proliferation of various kinds of cells was examined in vitro. The factor (TAF) a polypeptide of 14000 molecular weight, was extracted and purified from the conditioned medium of ovarian clear cell carcinoma cell line (HUOCA-II). TAF at concentrations of 10 ng/ml and 100 ng/ml promoted proliferation of the endothelial cells and induced tube formation. However, it had no stimulatory effect on the proliferation of fibroblasts, endometrial columnar cells, squamous epithelial cells or cancer cells.

Characterization of Krukenberg tumor cell line, especially the biological relationship between cancer and stromal cells.

The signet-ring cell adenocarcinoma cell line (HSKT-C) and the fibroblast cell strain (HSKT-F) were established from a Krukenberg tumor. The HSKT-C cells are small, roundish or spindle-like in shape and form monolayer sheets of epithelial pavement cells and produce carcinoembryonic proteins such as carbohydrate antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), etc. They show a stable proliferation after successful passages of 45 times within 13 months. The chromosome number varied widely and showed aneuploidy. The HSKT-C cells were transplanted to hamster cheek pouches and produced a tumor (signet-ring cell adenocarcinoma). On the other hand, HSKT-F cells are fibroblast-like. Their chromosome number is 46, and no karyological abnormality was observed. They could not be transplanted in the nude mouse or the hamster and did not produce carcinoembryonic proteins. It should be noted that they produce estrogens (estrone and 17 beta-estradiol). Sarcomatous morphological change of the stromal cells in Krukenberg tumor is considered to be a reactive change against invasion of the signet-ring cell adenocarcinoma into stromal tissues.

[Establishment and characterization of human neuroblastoma cell line (HSNB)].

We cultured an aspiration fluid of the sternal bone marrow of the patient having adrenal neuroblastoma and established a neuroblastoma cell line (HSNB). The HSNB line has the following biological properties. 1. They are small round in shape and proliferate in flotation while forming cell aggregate, and often they attach the bottom of plastic dish and process the nerve-like fibers. A rough-endoplasmic reticulum are poorly developed, however, a lot of free ribosomes are scattered in the cytoplasm. In the peripheral area of the cells, small spherical secretory granules (60-140 nm in diameter) are existed. One characteristic of this cell is existence of microtubules in the cell-projections. 2. They show a stable growth and the doubling time is about 50 hours. 3. Their chromosome number varied widely and the mode is 46. The double minute chromosomes were present in 50% of cells. 4. When they are transplanted in the cheek pouch of hamster, they produced the neuroblastoma. 5. They produce neuron specific enolase. 6. N-myc gene was amplified ca 250 folds.

[Establishment and characterization of a human malignant schwannoma cell line (HKMS)].

The malignant schwannoma cell line (HKMS) was established from the subcutaneous tumor of Axilla region of a 48-year-old Japanese woman. The HKMS line has the following biological properties. 1. The HKMS cells were spindle in shape and showed neoplastic and pleomorphic features. The monolayer sheet of HKMS cells showed the resemble cell-arrangement with that of the original tumor tissue. 2. The cells showed a stable growth and the serial passages were successively carried out 150 times within 3 years. Their population doubling time is about 40 hours. 3. The chromosome number varied widely, and the modal number was stable at the 78-80. The marker chromosomes were present. 4. The cells were transplanted into the subcutis of nude mice and produced the malignant schwannoma.

[Establishment and characterization of a human malignant fibrous histiocytoma line serially transplanted in nude mice].

Serial heterotransplantation of human malignant fibrous histiocytoma (MFH) derived from tibia was attempted in BALB/c nu/nu mice, and HKMFH-nu transplantable tumor line was established. This line had the following biological properties. (1) Eighteen serial passages were carried out in 41 months. (2) Morphological changes of the grafts occurred in nude mice with serial passages: During the first 6 passages, histiological picture was consistent with the common type of MFH similar to that of the original tumor, then after the 7th passage, the myxoid type coexisted with the common type, and finally the myxoid type occupied the entire grafts to form large cysts. (3) The common type grafts grew more rapidly than the myxoid type grafts. (4) Granulocytosis (neutrophilia) was observed in mice bearing the common type tumor, but not in mice bearing the myxoid type tumor.

N-myc amplification and neuron-specific enolase production of a neuroblastoma cell line and germ cell tumor cell lines.

N-myc gene amplification of the gynecological malignant tumor cell lines and a neuroblastoma cell line was studied by the Southern hybridization method along with the production of neuron-specific enolase (NSE) by these cell lines. N-myc amplification and NSE production were observed side by side in three cell lines: neuroblastoma cell line HSNB, endodermal sinus tumor cell line HAEST, and malignant teratoma cell line HUOT. However, N-myc amplification and NSE production disappeared gradually following successive passages of the HAEST and HUOT lines. With respect to the HUOT line, these parameters disappeared along with the cells of nervous origin. N-myc amplification and NSE production were not observed in nine other cell lines.