T-cell lymphoma lines derived from rat thymomas induced by Moloney murine leukemia virus: phenotypic diversity and its implications.

The phenotype of 27 Moloney murine leukemia virus-induced rat thymic lymphomas and 36 cell lines derived from these tumors was determined by using 18 monoclonal antibodies directed against hematopoietic cell surface determinants. The cell lines and the primary tumors from which they were derived were clonally related as determined by the pattern of provirus integration and the pattern of rearrangement of the T-cell receptor beta and delta and Igh loci. The differentiation phenotype of the primary tumors and the cell lines derived from them were related. The differences observed between the primary tumors and the cell lines could be explained either by the selection of subpopulations of tumor cells during establishment in culture or by the phenotypic instability of the tumor cells. One cell line (LE3Sp) underwent the transition from a CD4+ CD8+ to a CD4+ CD8- phenotype following exposure to interleukin-2 in culture. Both the primary tumors and the cell lines derived from them express a wide range of phenotypes which correspond to multiple stages in T-cell development. This observation suggests that the pleiomorphism of retrovirus-induced lymphomas, which had been suggested previously from the analysis of mouse tumors, is an intrinsic property of the process of oncogenesis and is not due to the transformation of different types of cells by spontaneously arising leukemogenic variants of the inoculated virus. The wide spectrum of phenotypes expressed by these tumors suggests that Moloney murine leukemia virus may infect and transform T cells at various stages of development. Alternatively, the target cells may be immature T-cell precursors which, following transformation, continue to differentiate. A host of early findings, suggesting that the repertoire of target cells is restricted to poorly differentiated hematopoietic progenitors, and the ability of the LE3Sp cell line to differentiate in culture indicate that the latter possibility may be more likely. The data in this report address the extent and mechanism of the phenotypic variability of retrovirus-induced rodent T-cell lymphomas. In addition, they demonstrate the potential usefulness of the T-cell lymphoma lines we have established in studies of oncogenesis and T-cell differentiation.     

Estrogen sensitive cell lines: Establishment and characterization of new cell lines from estrogen-induced rat pituitary tumors

Rat pituitary tumors were induced by the monthly injection of high doses of estradiol valerate. Of 12 animals which received the estrogen, 10 developed tumors. From these 10 tumors, 4 cell lines were successfully established and they have been maintained in culture for over 18 months. Several clones have been isolated from these established cell lines. Three of the 4 cell lines produce tumors in females considerably faster than in males or castrated females. Tumor-bearing animals have significantly increased amounts of rat growth hormone and prolactin in their serum. The 4 established cell lines, as well as clones derived from them, and tumors obtained in situ by injection of these cells growing in culture or successive transplants have shown the presence of an estrogen-binding protein of high affinity and low capacity. This estrogen-binding protein is similar to that described in other target organs (uterus, mammary glands, etc). These cell lines will be used to study the mechanism of action of estrogen in target cells as well as the synthesis and secretion of pituitary hormones.     

Increased tumor-specific CTL activity in human tumor-infiltrating lymphocytes stimulated with autologous tumor lines

Two long-term tumor-infiltrating lymphocyte (TIL) lines and their autologous tumor lines have been established from solid tumors derived from different patients with metastatic melanoma. In 4-hr 51Cr release assays, each TIL culture lysed only the autologous cryopreserved fresh or established melanoma line, but failed to lyse other melanoma tumors or K562 cells. Repeated stimulation of TIL with the autologous melanoma lines resulted in significant increases in anti-tumor CTL activity with no apparent loss in specificity. Stimulated cells have retained cytotoxic activity for up to 5 months in culture. Tumor cell CTL activity for both long-term TIL lines is inhibited by several mAbs, including those against CD3, CD8, and class I MHC molecules, indicating that the effector cells are class I-restricted CD8+, CTL. Furthermore, recognition of Ag on one of the established melanoma lines by TIL is restricted by HLA A-2. The availability of autologous tumor lines may prove clinically useful for the selective stimulation and expansion of cells with anti-tumor activity within a heterogeneous TIL population.     

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. ¹, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.     

Isolation of cloned Syrian hamster insulinoma cell lines with limited capacity for insulin production.

Cell lines derived from a Syrian hamster insulinoma have been cloned in agar and maintained continuously in culture in vitro for 1 1/2 years. The cell lines have average doubling times of 48 h, they have modal chromosome numbers between 38 and 39, and they retain the ability to form tumors when injected into Syrian hamsters. The cells do not produce immunoreactive insulin when grown in vitro (less than 0.5 ng/mg protein), but do produce immunoreactive insulin when grown in vivo as tumors (mean value from six determinations = 7.1 +/- 1.5 ng/mg protein).     

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.     

Analysis of matrix metalloproteinase activity during differentiation of mesenchymal stem cells isolated from different tissues of one donor

Mesenchymal stem cells established from bone marrow (FetMSC) and limb bud (M-FetMSC) of early human embryo, as well as spheroids derived these cells, were induced to undergo osteogenic and adipogenic differentiation. Differentiated cells exhibited the activity of metalloproteinase (MMP)-9, -2, and -1. Its activity was different in osteogenic and adipogenic cells, as well as in monolayer cultures (2D) and cell spheroids (3D). The direct correlation between the level of adipogenic differentiation and gelatinases MMP-9 and MMP-2 activities in both cell lines in 2D and 3D culture was shown. M-FetMSC cells in 2D culture 12 days in culture during showed low potential for adipogenesis and reduced activity of MMP-2 and MMP-9. The low level of adipogenic differentiation in 2D M-FetMSC culture was accompanied with increased MMP-1 activity and enhanced differentiation (3D culture) resulted in a significant increase of both MMP activities. MMP-1 activity varied oppositely. MMP-1 activity declined in 3D cultures with a higher level of adipogenic differentiation. The level of osteogenic differentiation was similar in both cell lines during 2D and 3D cultivation. MMP-1 and -9 activities in both cell lines were not associated with osteogenic differentiation. MMP-2 and MMP-2 activity in these cells remained unchanged. The results suggest MMP implication in FetMSC and М-FetMSC differentiation. The difference in MMP activities during the cell differentiation may be caused by variations in the microenvironment or ECM properties in 2D and 3D cultures.     

Establishment of four mouse mastocytoma cell lines

Four mastocytoma cell lines were isolated from four different mouse mastocytoma tumors. The tumors had been induced in mice treated with tetramethylpentadecane (pristane) and infected with Abelson murine leukemia virus. The cell lines have been carried in culture for over a year and can induce tumors when injected into the mouse strain in which the tumor originated. The cells contain histamine, have high affinity IgE receptors and release histamine by IgE, immune complex or ionophore A23187-induced reactions. This histamine release reaction requires Ca2+, is optimal at 37 degrees C, and is blocked by a number of metabolic inhibitors. There is no requirement for phosphatidylserine. Cloned sublines have been obtained which will be useful for Fc epsilon R, Fc gamma R; and histamine release studies.     

Source, alterations, characteristics and use of a new dog cell line (Cf2Th).

A cell line derived from normal fetal canine thymus (Cf2Th) has been in culture since 1967. During cultivation the cells have changed morphologically from a fibroblast-like to flat, fusiform appearance and karyologically from diploid (2n=78) with 76 telocentric autosomes to hypodiploid with newly formed atelocentric chromosomes. The cells retain canine characteristic enzyme activity (G6PD and LDH) as well as cell membrane fluorescence and are free of mycoplasma. High passage cells produce tumors in ATST mice. No endogenous viruses have been detected in these cells. No original publication exists, to date, on the origin of this line, but seed stocks thereof have been distributed to many laboratories and the cells have served as experimental substrates in a number of published works on oncology albeit under different designations. The present information is offered in order to establish the provenance of this valuable cell line and to list characteristics which may serve to monitor for its purity and to distinguish it from other existing cell lines of dog origin also in common use.     

The development relationship between osteocytes and osteoclasts: a study using the quail-chick nuclear marker in endochondral ossification.

Interspecific grafts of limb buds and femurs on the chorioallantoic membrane of 5-day-old hosts and into the somatopleure of 3-day-old hosts were carried out between quail and chick embryos. Due to their different nuclear features, the cells of the two species can be identified in the chimeric bones resulting from the endochondral ossification which occurs in the explanted tissues. By following the cell lineage in the bone and marrow we were able to show that the hemopoietic and the osteogenic (comprising osteoblasts, osteocytes, and chondrocytes) cell lines have different embryological origins. The osteogenic line is derived from the limb bud mesenchyme, while the hemopoietic cells are brought into the bone marrow via the circulation. In the fixed cells of the marrow two categories have to be distinguished: the reticular cells originating from the bone rudiment and the endothelial cells which invade the cartilage and are of hematogenous origin. The osteoclasts belong to the hemopoietic cell line and are not derived from any cell type of the osteogenic line.     

Retention of Differentiated Function in Clonal Animal Cell Lines, Particularly Hormone-Secreting Cultures

Clonal hormone-secreting cell lines have been established fromanimal tumors. These lines include adrenal steroid-secretingcells, growth hormone-secreting cells, steroid-secreting Leydigcells, and ACTH-secreting cells that retain their differentiatedfunction for prolonged periods in continuous culture. In addition,fibroblast cell lines that secrete a material which stimulatessteroidogenesis by adrenal cells and Leydig cells are described.A systematic approach to obtain functional cultures and thegeneral problem of retention of differentiated function in culturedcells are discussed.     

Derivation of 30 human embryonic stem cell lines—improving the quality

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process, we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal, but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked, and they are available for researchers.     

Cultivation of human plasmacytoma cells

Current knowledge of primary culturing and establishment of continuous cell lines from human plasmacytomas are reviewed. The cells derived from chronic monoclonal tumors are capable of proliferating in culture no more than for 3 weeks. The enrichment of the media with nutrient supplements and growth factors does not improve the cell survival. Most of the described stable cell lines were derived from malignancies of the late stages of tumor progression. Cell lines producing complete molecules of immunoglobulins were established with the aid of sophisticated culturing methods. The microenvironment and growth factors in culture system favoured the survival of transformed plasma cells which retained a high level of differentiation.     

Isolation and Characterization of Multipotential Mesenchymal Cells from the Mouse Synovium

The human synovium contains mesenchymal stem cells (MSCs), which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs) have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.     

Characteristics of two cell lines derived from a histologically undifferentiated bronchial carcinoma expressing neuroendocrine markers

Summary This study investigates the characteristics of two human cell lines—1PT and 1PT VARIANT A—both derived from the same histologically undifferentiated, neuroendocrine positive, non-small cell lung carcinoma (NSCLC) and capable of growth in unsupplemented serum-free minimum essential medium. In stationary culture, the cells of both lines grew both attached to a plastic substratum and in suspension; the 1PT VARIANT A line formed three-dimensional clusters of loosely adherent cells. The cell lines differed in their DNA content, the 1PT having 1.44 times and the 1PT VARIANT A having 2.39 times the normal human diploid DNA content. Chromosome counts supported this observation, the ploidy of the 1PT and VARIANT A lines being 1.11 and 1.64, respectively. On transmission electron microscopy the cells of both lines had dense core granules and immature desmosomes, whereas only the 1PT VARIANT A line had mucin granules. Both lines formed, in nude mice, tumors that, like the original tumor from which they were derived, were histologically undifferentiated and showed local invasion. The original tumor and both lines had demonstrable neuroendocrine markers. Cytokeratins were apparent in the tumor but not the cell lines, and neurofilaments were present in the cell lines only. Staining for epithelial membrane antigen, neural cell adhesion molecule, and desmoplakin differentiated between the two lines. These lines provide a useful model for the investigation of the biology of the neuroendocrine positive subgroup of NSCLC, which is clinically important because of the possible responsiveness of these tumors to chemotherapy.     

Selective lethal effect of thymidine on human and mouse tumor cells

Human cell lines derived from a melanoma and a colon carcinoma, and cultures of human melanocytes and intestinal epithelial cells, as well as a mouse mesenchymal non-neoplastic cell line and a malignant subline of the same have been quantitatively studied in tissue culture for their sensitivity to thymidine. All three tumor lines produced solid tumors when injected into nude thymus-deficient mice. No tumors were obtained by injecting cells of the human normal long-term cultures or of the non-neoplastic mouse line. The tumor-producing lines showed a greater sensitivity to the lethal effects of high concentrations of thymidine than their non-tumor-producing counterparts. Less than 23% of the tumor cells survived 72 hours in the presence of 1 mg/ml of thymidine, in contrast to 60% or more of the non-tumor cells. Colony formation was much more inhibited by thymidine and the differential between normal and tumor cells was even more pronounced. Tumor cells which also were treated for 72 hours with 1 mg/ml of thymidine and then plated in fresh medium formed very few colonies. If the plating efficiency of the untreated controls is considered as 100%, 4.3% or less of the treated tumor cells formed colonies, in contrast to 33% or more of the non-tumor cells.     

Mesenchymal stem cells: a promising candidate in regenerative medicine

Mesenchymal stem cells were initially characterized as plastic adherent, fibroblastoid cells. In recent years, there has been an increasing focus on mesenchymal stem cells since they have great plasticity and are potential for therapeutic applications. Mesenchymal stem cells or mesenchymal stem cell-like cells have been shown to reside within the connective tissues of most organs. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. A number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm, and in addition, mesenchymal stem cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine and may also serve as efficient delivery vehicles in site-specific therapy.     

Tumorigenicity of Vero cells

One of the current criteria for evaluating the acceptability of cell lines for use in vaccine production is lack of tumorigenicity. Vero cells represent an example of a class of cells known as continuous cell lines. They were derived from African green monkey kidney, and their growth properties and culture characteristics have many advantages over other cell substrates for use in vaccine production. We have tested Vero cells for tumorigenicity in nude mice and in a human muscle organ culture system, and found a significant increase in their tumorigenic potential with increasing passage numbers. Cells at passage 232 and higher produced nodules in all nude mice inoculated. Histologically the nodules were well defined, anaplastic tumors, which exhibited some of the characteristics of renal adenocarcinomas. In about 6 to 8 days all of the nodules began to regress. Data were obtained that suggested an immune mechanism was the basis for the regression phenomenon.     

Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells.

Mesenchymal progenitor cells derived from cord blood (unrestringated somatic stem cells, USSC) and bone marrow (mesenchymal stem cells, MSC) are able to differentiate under defined culture conditions into at least bone, cartilage, adipose and muscle cells in vitro. The culture media and other in vitro conditions influence the osteogenic differentiation potency of both cell types. To increase and expand the number of osteoblasts in vitro an optimization of culture conditions is required. The aim of this study was to evaluate different culture media toward their osteogenic promoting capacity on human USSCs and MSCs in vitro. Immunohistochemical stainings against osteonectin (ON), osteopontin (OP) served as markers for an osteoblastic differentiation. Cellular morphology was analysed by light microscopy technique. We found significant differences between bone marrow and cord blood derived stem cells towards an osteoblastic differentiation. Considering the number of osteoblasts MesenCult seems to have advantages in bone marrow progenitor cells, whereas low glucose DMEM and HAMS-F12 promoted an osteoblastic differentiation in cord blood derived cells more than other tested media.