Characteristic of tumors developed after transplantation of transgenic GFP-positive C57BL/6 mice bone-marrow mesenchymal stem cells to mdx mice muscle

The purpose of the study was the morphological and histochemical characteristics of differentiation of tumors developed after transplantation of GFP-positive mesenchymal bone-marrow stem cells (MSC) of transgenic mice C57BL/6 into M. quadriceps femoris of mdx mice. The tumors occurred only after transplantation of MSCs of 43-45th passages and did not arise after transplantation of MSCs of the 15th passage. No tumors developed also after transplantation of MSCs of 43-45th passages into muscle of C57BL/6 mice. The average weight of tumors appeared in 4 mdx mice studied was 1.3 +/- 0.5 g. All four tumors were classified as mesenchymomas because they originated from mesenchymal stem cells. Most of the periphery of the tumors was classified as fibrosarcomas with mitotic index 0.9 +/- 0.1%. The central parts of tumors had areas with epithelial like morphology of cells. Such cells showed positive reactivity for alcyan blue staining at pH 2.5, which indicated chondrocyte nature of the cells. No mitosis was observed in epithelial like cells. In the tumors, there were also areas with bone trabeculae containing megacaryocytes and foci of myeloid and erythrocyte hematopoiesis. There were also areas with neuronal and glial cells, and accumulations of adipocytes. One of the tumors was classified as a round cells sarcoma. The observed types of tumor cell differentiation in vivo were in accordance with described in literature types of MSCs differentiation after induction in vitro with special inductors. The spectrum of in vivo differentiation of transgenic GFP-positive MSCs after transplantation to mdx mice was broader than the spectrum of in vivo differentiation of transfected or transformed in vitro adult MSCs after transplantation to immunodeficient mice and mdx mice.     

Differentiation of human testicular embryonal carcinoma and teratocarcinoma grown in nude mice and soft-agar cultures

The differentiation pattern of two human germ cell tumors, grown in nude mice and in vitro is described. Tumor A was an embryonal carcinoma (EC) of borderline histology with characteristics of yolk sac tumor and of seminoma; tumor B was a teratocarcinoma with yolk sac elements and syncytiotrophoblastic giant cells. The morphology of an EC as well as cytogenetic characteristics were maintained during 20 passages in nude mice from tumor A and over 11 passages from tumor B. Tumor A did not grow in vitro. Cell suspensions prepared from xenografted tumor B grew into cystic embryoid bodies in semi-solid tissue culture medium. These embryoid bodies showed cuboidal and flattened cells with microvilli, junctional complexes, peripheral microfilaments, and annulated lamellae, reminiscent of the 'inner cell mass' of a blastula and of endoderm, respectively. When such colonies were transplanted into nude mice, however, only tumors with the morphology found in the transplants appeared.     

Human renal cell carcinoma: Establishment and characterization of a new cell line (OS-RC-2)

Summary A cell line, designated as OS-RC-2, has been established from a renal cell carcinoma in a 52-yr-old Japanese male patient and maintained for 23 mo. through 60 in vitro passages. The OS-RC-2 formed monolayers of polygonal epithelial cells and lacked contact inhibition. Doubling time of cells was about 60 h at the 30th passage. Electron microscopic findings indicated numerous long microvilli on the cell surface and many glycogen granules in the cytoplasm of this cell line, which are characteristic structures of renal cell carcinoma. Chromosomal analysis revealed that a small portion of this cell line had a hypodiploid modal number of 40 and a large portion had a hypotetraploid modal number of 75. The characteristics of the karyotype were one detected marker chromosome and the translocation between the Chromosomes 2 and 13. Cell line OS-RC-2 was serially transplantable into nude mice, and histopathological findings of heterotransplanted tumor showed a close similarity to those of the original tumor. Histocompatibility antigens of OS-RC-2 were HLA-A9, Bw52, which were identical to those of the peripheral blood lymphocytes of the patient.     

Establishment of duck cell line derived from experimental tumor induced by 20-methylcholanthrene

Summary Experimental tumors developed in white Pekin ducks after intramuscular implantation of 20-methylcholanthrene. Cells derived from the primary tumor were adapted successfully to grow in vitro and have growth characteristics similar to that of established cell lines of mammalian origin. The cell density rises rapidly and the doubling time is approximately 19 hr. The duck cells have been cultured succesfully for at least 80 passages in vitro. The continuously cultured cells have the characteristic chromosome pattern of duck, and the DNA of the duck cell line hybridized with duck liver DNA. We believe we have established a continuous cell line of avian origin. Electron-microscopic examinations of the tumor cells and RNA-directed DNA polymerase of the cell-free supernate show no evidence of endogenous virus production. This study was supported by Public Health Service Research Grants CA 16479 and CA 20012 from the National Cancer Institute and RR 00890 from the National Institutes of Health.     

Cytogenetic changes in an explanted mouse rhabdomyosarcoma during prolonged cultivation

Quantitative and qualitative chromosome rearrangements, dynamics of distribution of double-minute chromosomes (DMs), and morphological characteristics of tumor rhabdomyoblasts MH-82 during explantation and following in vitro cultivation are analysed. Cells of the 13th and 27th passages of cultivation were characterized by the epithelial type of growth, although their form and size varied. Chromosome analysis of tumor rhabdomyoblasts was carried out on passages 4, 14, 20, 25 and 30 of in vitro cultivation. The modal class with 53-55 chromosomes was established within 20 passages. Heterogeneity of cell population in concern to the chromosome number and content of hypotetraploid cells (72-78) diminished during cultivation. Chromosome rearrangements (marker chromosomes) in hyperdiploid and hypotetraploid cell subpopulations differed. The number of cells with DMs and the number of DMs per cell decreased till the full disappearance by the 30th passage. It is concluded that the establishment of the MH-82 cell line was completed up to the 30th passage of cultivation.     

Establishment and characterization of patient-derived xenograft and its cell line of primary leiomyosarcoma of bone

Primary leiomyosarcoma (LMS) of bone is a rare and aggressive mesenchymal malignancy that differentiates toward smooth muscle. Complete resection is the only curable treatment, and novel therapeutic approaches for primary LMS of bone have long been desired. Patient-derived xenografts (PDXs) and cell lines are invaluable tools for preclinical studies. Here, we established PDXs from a patient with primary LMS of bone and a cell line from an established PDX. Bone primary LMS tissue was subcutaneously implanted into highly immune-deficient mice. After two passages, a piece of the tumor was subjected to tissue culturing, and a morphological evaluation and proteomic analysis were performed on the PDX and the established cell line. Moreover, the responses of the established cell line to anti-cancer drugs were examined. Microscopic observations revealed that the PDX tumors retained their original histology. The cell line was established from the third-generation PDX and named NCC-LMS1-X3-C1. The cells were maintained for over 18 mo and 40 passages. The cells exhibited a spindle shape and aggressive growth. Mass spectrometric protein identification revealed that the original tumor tissue, PDX tumor tissue, and NCC-LMS1-X3-C1 cells had similar but distinct protein expression profiles. We previously established the cell line, NCC-LMS1-C1, from the tumor tissue of same patient. We found that the response to drug treatments was different between NCC-LMS1-X3-C1 and NCC-LMS1-C1, suggesting the heterogeneous traits of tumor cells in the identical tumor tissue. This set of PDXs and stable cell line will be a useful resource for bone LMS research.     

Establishment and Partial Characterization of a Continuous Human Malignant Glioma Cell Line: NG97

1. A human glioma cell line, NG97, was established from tissue obtained from a patient diagnosed with a grade III astrocytoma.2. The NG97 cell line has been subcultured for more than 100 passages in standard culture media without feeder layer or collagen coatings.3. NG97 cells grow in vitro as two subpopulations with distinct morphological appearance: stellate cells with pleomorphic nuclei, and small round cells with few processes. The cells have a doubling time of about 72 h and a plating efficiency of 1%. The injection of NG97 cells into congenitally athymic mice induced the formation of solid tumor masses that could be retransplanted every 4 weeks. The cells obtained from tumor mass when cultivated in vitro had a morphology comparable to those of the initial culture.4. This cell line may prove useful for cellular and molecular studies as well as in studies of gliomas treatment.     

Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain-type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES-NP) for the large-scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES-NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES-NP cell number was increased by > 2 × 10⁴-folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre-synaptic DA neuronal functionality. Furthermore, the hES-NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson's disease rat model, the multi-passaged hES-NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine-induced rotation score of Parkinson's disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES-NP cell expansion is exploitable for a large-scale generation of experimental and transplantable DA neurons of human-origin.     

Two new C3H mouse ascites tumor cell lines capable of proliferation in vivo and in suspension culture: Morphological,karyological, kinetic,and immunological properties

Summary The establishment of mouse tumor cell lines capable of proliferating in vivo and in suspension culture was undertaken. The MM-46 tumor line, initiated from primary mammary carcinoma arising in a C3H/He mouse, was maintained for over 100 generations in the peritoneal cavities of syngenic mice. At the 50th generation of the tumor suspension, cultures were initiated. The established cell lines, designated TMT-1 and TMT-2, were characterized in vitro and in vivo. The morphological finding indicated that TMT-1 and TMT-2 cells from mice closely resembled the MM-46 tumor cells. The oncogenic potential of the cultured cells was comparable to that of the original ascites tumor. The population doubling time of TMT-1 and TMT-2 cell lines was about 12 hr in mice, whereas the population doubling time of both cell lines lengthened to 20 hr in suspension culture. The increase of doubling time in culture was due to the prolongation of the G₁ period. The cell lines, TMT-1 and TMT-2, whether from culture or mice, possessed colony forming ability in soft agar medium. The colony forming ability of the cells decreased gradually through in vivo passages but it recovered upon recultivation of the cells from mouse to culture. Chromosome analysis and cytotoxicity test by anti-MM antiserum indicated that TMT-1 and TMT-2 cell lines closely resembled and had been derived from MM-46 tumor line. Therefore, it is possible to assay cell survival in vitro after in vivo experiments on these cells.     

Establishment and characterization of a human ureteral cancer cell line producing carbohydrate antigen 19-9 and carcinoembryonic antigen

We established a new cell line (FU-UrC-1) derived from a human primary ureteral carcinoma xenografted in a nude mouse. This cell line exhibited epithelial characteristics and formed clusters in monolayer cultures. The cells were subcultured in vitro for more than 20 passages and had a doubling time of 53 hours. The modal number of chromosomes was 66. The cell line, which was xenografted again to nude mice, produced tumors essentially identical to the original tumor. Furthermore, the cultured cells expressed carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) that were secreted in the culture media. This cell line appears to provide a useful system for studying ureteral carcinoma in vivo and in vitro.     

Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer-derived mammary tumor cells in suspension

Summary The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY-beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incoerporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in susfpension resulted in 10- to 400-fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Incrases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 μM hydrocorticone further increased MMTV yield two-to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure. This work was supported by the T. J. Martell Foundation for Cancer and Leukemia Research and by USPHS grant 5P-30CA23102. F. M. is a trainee on MSTP grant GM07280 from the National Institute of Health. This work was submitted in partial fullfillment of the requirements for the Ph. D. degree (F. M.).     

Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro

Summary The lens of the eye is one of the rare organs in which tumors do not occur spontaneously. It therefore appeared to us that lens cells would not present the background of spontaneous transformation toward malignancy found with many other cell cultures. We have cultured C3H/HeA mouse lens explant (MLE) cells for 70 wk an analyzed changes in malignancy-related phenotypes in function of the number of passages. In vitro, we studied morphology, colony forming efficiency on tissue culture plastic substrate (CFEtc) and in soft agar, population doubling time, saturation density, and invasiveness into precultured chick heart fragments. In vivo, tumorigenicity, invasion, and metastasis were analyzed after injection of cell suspensions subcutaneously and intraperitoneally, after implantation of cells aggregated to collagen sponges under the renal capsule and after implantation of cell aggregates subcutaneously into the tail and into the pinna. The CFEtc, population doubling time, and saturation density increased as the number of passages of culture in vitro increased, but colony formation in soft agar was never observed. MLE cells till passage 16 were not invasive in vitro, but hereafter consistently were found to be invasive. After about 17 passages, corresponding to 25 wk of culture, MLE cells acquired the capacity to form tumors in syngeneic mice. These tumors were invasive but metastases were not observed, We concluded that MLE cells acquired in an apparently spontaneous way a number of malignancy-related phenotypes, without, however, reaching the stage of metastasis. L. M. is a recipient of a fellowship from the IWONL, Belgium. This work was supported by the Belgisch Werk Tegen Kanker and the Internationale Stichting Jacques Brel.     

Establishment of a New Human Glioblastoma Multiforme Cell Line (WJ1) and Its Partial Characterization

(1) A new human glioblastoma multiforme (GBM) cell line, WJ1, was established from the tissue derived from a 29-year-old patient diagnosed with a grade IV GBM. (2) The WJ1 cell line has been subcultured for more than 80 passages in standard culture media without feeder layer or collagen coatings. (3) GBM cells grow in vitro with distinct morphological appearance. Ultrastructural examination revealed large irregular nuclei and pseudo-inclusion bodies in nuclei. The cytoplasm contained numerous immature organelles and a few glia filaments. Growth kinetic studies demonstrated an approximate population doubling time of 60 h and a colony forming efficiency of 4.04%. The karyotype of the cells was hyperdiploid, with a large subpopulation of polyploid cells. Drug sensitivities of DDP, VP-16, tanshinone IIA of this cell line were assayed. They showed a dose- and time-dependent growth inhibition effect on the cells. (4) Orthotopic transplantation of GBM cells into athymic nude mice induced the formation of solid tumor masses about 6 weeks. The cells obtained from mouse tumor masses when cultivated in vitro had the same morphology and ultrastructure as those of the initial cultures. (5) This cell line may provide a useful model in vitro and in vivo in the cellular and molecular studies as well as in testing novel therapies for human glioblastoma multiforme.     

大鼠脑神经干细胞系(RNSC-FMU 1)的建立和鉴定

采用无血清培养液分离和培养新生SD 大鼠脑的神经干细胞,以机械分散的方法传代,成功地建立了大鼠脑神经干细胞系(RSNC-FMU 1)。该细胞系可在体外长期传代,至今已在体外连续生长超过21个月(>100代),保持了神经干细胞的特性和正常的核型,经诱导可分化成为神经元、星形胶质细胞和少突胶质细胞,具有较旺盛的自新能力,倍增时间约为20h,并可冷冻保存,裸鼠体内移植证实其不具有成瘤性。该细胞系为神经干细胞研究提供了一个良好的工具。     

MiR-371-5p Facilitates Pancreatic Cancer Cell Proliferation and Decreases Patient Survival

Background

microRNAs (miRNAs) play a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. To date, scientists have obtained a substantial amount of knowledge with regard to miRNAs in pancreatic cancer. However, the expression and function of miR-371-5p in pancreatic cancer has not been clearly elucidated. The aim of this study was to investigate the roles of miR-371-5p in pancreatic cancer and its association with the survival of patients with pancreatic cancer.

Methods

The expression of miR-371-5p was examined in pancreatic duct adenocarcinoma (PDAC) and their adjacent normal pancreatic tissues (ANPT) or in pancreatic cancer cell lines by qRT-PCR. The association of miR-371-5p expression with overall survival was determined. The proliferation and apoptosis of SW-1990 and Panc-1 cells, transfected with miR-371-5p mimics or inhibitor, were assessed using MTT assay and flow cytometry, respectively. The tumorigenicity was evaluated via mice xenograft experiments. miR-371-5p promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP). Protein expression was analyzed by Western blot.

Results

The expression level of miR-371-5p was dramatically upregulated in clinical PDAC tissues compared with ANPT. Patients with high miR-371-5p expression had a significantly shorter survival than those with low miR-371-5p expression. The in vitro and in vivo assays showed that overexpression of miR-371-5p resulted in cell proliferation and increased tumor growth, which was associated with inhibitor of growth 1 (ING1) downregulation. Interestingly, we also found that ING1, in turn, inhibited expression of miR-371-5p in the promoter region.

Conclusions

our study demonstrates a novel ING1-miR-371-5p regulatory feedback loop, which may have a critical role in PDAC. Thus miR-371-5p can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.     

大鼠脑神经干细胞系（RNSC—FMU 1）的建立和鉴定

A neural stem cell line (RNSC-FMU 1) from rat brain have been established successfully by isolating and culturing neural stem cells from newborn SD rat brain in vitro with free-serum medium and passaging with mechanical division. The cell line cultured can continuously generate in vitro for long-term and it is 21 months (>100 passages) so far. These cells keep the feature of neural stem cell and normal karyotype. These neural stem cells can be induced to differentiate into neurons, astrocytes and oligodendrocytes. The cells have an extensive self-renewal capacity; its doubling time of proliferation is about 20 h. The cells are also cryopreservable. Tumor formation is not observed in nude mice that explanted with the cells. This cell line is a good tool for research of neural stem cell.     

Long-term in vitro cell culture of sinclair swine melanoma

Summary The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in culture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell culture of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18. This work was supported by a grant from the NIH, NCI (2 P01 CA 08023-11A1).