Establishment and characterization of conditionally immortalized organ of corti cell lines

A culture of cells was isolated from the organ of Corti of 2-week-old H-2Kb-tsA58 (Immortomouse) transgenic mice. All cells of these mice harbor a mutant of the simian virus 40 A-gene, encoding a thermolabile large T-antigen (Tag) protein. At 33 degrees C the Tag protein is functional and induces cell proliferation, but at 39 degrees C it is rapidly denatured and inactivated. Isolated organ of Corti cells growing at 33 degrees C were predominantly small, rounded or fusiform and proliferated rapidly. When moved to 39 degrees C, the cells reduced their rate of proliferation and differentiated into specific morphological phenotypes. Four cell lines were cloned by limiting dilution and characterized by immunofluorescence microscopy and Western blot. The cell lines, named OC-k1, OC-k2, OC-k3 and OC-k4, have been passaged at least 50 times with retention of a stable phenotype. These cell lines were all positive for the neuroepithelial precursor cell marker nestin and for the inner ear cell marker OCP2. In addition, the cells showed reactivity to epithelial and neuronal cell markers, but with a pattern of protein expression different for each clone and different between cells of the same clone growing at 33 degrees C or 39 degrees C. Some of the clones exhibited asymmetric cell division which is a characteristic commonly ascribed to stem cells. These cell lines can be used advantageously to study mechanisms and signals involved in the control of cell differentiation and morphogenesis of the mammalian inner ear and to isolate inner ear specific proteins.     

Establishment of immortalized rat Kupffer cell lines

BACKGROUND: Kupffer cells have been implicated in the pathogenesis of various liver diseases. Primary cultures of Kupffer cells have a very limited life span, tend to de-differentiate and become senescent, and therefore are not suitable for cell signaling studies. AIM: To establish immortalized rat Kupffer cell lines that facilitate mechanistic studies of cell signaling and signal transduction. METHODS: Rat Kupffer cells were sub-cultured with EGF to obtain rat Kupffer Cell line 1 (RKC1), and subsequently transfected with Simian Virus 40 Large T-antigen expression vector to obtain rat Kupffer Cell line 2 (RKC2). RESULTS: RKC1 and RKC2 are similar to primary Kupffer cells as they express the molecular markers ED1, ED2, ED3, and F4/80, and upregulate TNF-alpha, IL-6, IL-1beta, Fas /FasL, and NF-kappaB, as well as TLR4 in response to LPS or pancreatic elastase. Additionally, RKC1 and RKC2 maintain phagocytic properties of latex beads and exhibit increased telomerase and stabilized p53 activity. CONCLUSION: Immortalized RKC1 and RKC2 cells maintain properties of primary Kupffer cells and can be valuable tools in evaluating the role of Kupffer cells in immune diseases and in liver-cell based drug discovery.     

Acidic amino acid transport characteristics of a newly developed conditionally immortalized rat type 2 astrocyte cell line (TR-AST).

To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibited positive immunostaining for anti-GFAP antibody and A2B5 antibody, characteristics associated with type 2 astrocytes, and expressed glutamine synthetase. Acidic amino acid transporters, GLT-1 and system xc-, which consists of xCT and 4F2hc, were expressed in all TR-ASTs by RT-PCR. On the other hand, GLAST expression was found in TR-AST3 and 5. The characteristics of [3H]L-glutamic acid (L-Glu) uptake by TR-AST5 include an Na+-dependent and Na+-independent manner, concentration-dependence, and inhibition by L-aspartic acid (L-Asp) and D-aspartic acid (D-Asp). The corresponding Michaelis-Menten constants for the Na+-dependent and Na+-independent process were 36.3 microM and 155 microM, respectively. [3H]L-Asp and [3H]D-Asp uptake by TR-AST5 had an Na+-dependent and Na+-independent manner. This study demonstrated that GLT-1, system xc-, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.     

Establishment of SV40-tsA58 transgenic rats as a source of conditionally immortalized cell lines.

To isolate a variety of rat cell lines with differentiated functions, we established transgenic rat lines expressing the temperature-sensitive large T-antigen of simian virus 40 (SV40) tsA58 mutant under the control of the SV40 large T-antigen itself. We microinjected the DNA into 564 eggs of Wistar rat and 23 independent transgenic candidates were obtained. Ten pups died before weaning and eight transgenic rats could not transmit the transgene to the progeny. Finally, five lines of the transgenic rat were established. Although one line (#1511-6) had low reproductivity, the other four lines reproduced normally. Three out of the four lines (#1507-2, #1509-7, #1519-8) appeared normal but the other line had tumors in the brain and subcutaneous tissue at 3 weeks of age (#1511-6), and in the kidneys and subcutaneous tissue at 18 to 19-weeks of age (#1507-5). Fibroblast cells prepared from transgenic fetuses of lines #1507-5 and #1519-8 expressed the transgene and exhibited temperature-dependent growth. Both of the lines (#1507-5 and #1519-8) were successfully generated to be homozygous by sibling mating of transgenic offspring. These transgenic rat lines have bred through many generations and have been established to be a ready source of novel conditionally immortalized cell lines.     

Establishment and characterization of immortalized cell lines from rat parotid glands

Summary This study reports for the first time the establishment of immortalized cell lines from normal adult rat parotid glands. The freshly prepared cellular clumps obtained from parotid glands of isoproterenol-treated rats were incubated in 0.2% trypsin solution without EDTA. These clumps were transfected with plasmid vectors pSV ₃ ^neo and pSV ₅ ^neo by electroporation and calcium phosphate-Co-DNA-precipitation techniques. The untransfected and transfected cellular clumps were plated in precoated dishes containing modified MCDB-153 medium. Epithelial cells grew from the clumps that were attached. All epithelial cells from untransfected culture died within 6 to 8 wk. Two cell lines which were isolated from transfected cultures subsequently grew on regular tissue culture dishes. One of them, which was isolated from pSV ₅ ^neo transfected cultures, exhibited non-epithelial cell morphology, but at confluency, many cells mature to acinar-like cells containing numerous granules. The other cell line (2RS), which was isolated from pSV ₃ ^neo transfected culture, contained cells of non-epithelial and epithelial morphology. During the initial phase of the growth, MCDB-153 medium was essential; however, at a later time, RPMI medium was better than MCDB-153 or F12 medium for maintaining morphology and growth of these cells. The immortalized cells grew in RPMI with a doubling time of about 25 h, synthesize T-antigen,α-amylase mRNAs of 1176 and 702 bp, andα-amylase and were non-tumorigenic. These amylase-producing cells can be a useful model to study the mechanisms of regulation of growth and differentiation in these cells.     

Establishment of tsA58 transgenic rats as a source of conditionally immortalized cell lines with differentiated functions.

To isolate a variety of rat cell lines with differentiated functions, we developed transgenic rat lines that ubiquitously express the temperature-sensitive large T-antigen gene of the simian virus 40 (SV40) tsA58 mutant under the control of the SV40 large T-antigen promoter. These rats might be advantageous for simultaneously establishing cell lines from different tissues of rats with the same genetic origin. The transgenic rat lines transmit a functional copy of the transgene and were bred with sib mating to generate the homozygous transgene. The established cell lines from this transgenic rat had temperature dependent growth and retained some of the differentiated functions of each particular tissue, and were useful as a ready source of novel conditionally immortalized cell lines. The possible use and perspectives of these transgenic cell lines are discussed.     

Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.M. Matsuo and K. Koizumi contributed equally to this work. This study was supported in part by Grants-in-Aid for the 21st Century COE Program and for CLUSTER (Cooperative Link of Unique Science and Technology for Economy Revitalization) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.     

Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue

Summary This investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique. Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum. The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400μg/ml geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines, 1RB₃ derived from cells transfected with pSV_3neo, and 2RB₅ derived from cells transfected with pSV_3neo, revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones. Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells. These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies.     

Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line

Tooth development was cooperatively regulated by the epithelial ameloblasts and mesenchymal odontoblasts. Ameloblasts secrete enamel matrix, critical for enamel formation. While there are several reports about establishment of immortalized ameloblast-like cells by introducing viral oncogene, we tried to establish a spontaneously immortalized ameloblast-lineage cell line, maintaining the cell type specific character, including the ability to induce in vitro bio-mineralization. The established cell line (ameloblast-lineage cell; ALC) maintained the expression of several ameloblast specific genes (Amelogenin, Tuftelin, and Enamelin) in long-term culture. They formed calcified nodules after the induction by medium switching from SMEM to DMEM, having high-level alkaline-phosphatase activity. The size and number of calcified nodule formation were enhanced by TGF-beta treatment. Six weeks after sub-cutaneous implantation of ALC to athymic nude mice, we ectopically observed enamel epithelium like structure formation, chondrogenesis, and calcification. These data indicate that ALC is a useful experimental tool to analyze ameloblast character.     

Generation and characterization of a conditionally immortalized lung clara cell line from the H-2Kb-tsA58 transgenic mouse

The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.     

Establishment and characterization of immortalized hippocampal neural precursor cell lines

In the mammalian central nervous system, a complexcircuit of neurons contributes to higher behaviors.Each region of the brain has a unique function derivedfrom various types of neurons. Several neuralprecursor cell lines have been established from basalganglia of fetal brain. In this study, hippocampalneural precursor cell lines were established from thehippocampus of p53^-/- embryos. By means ofintegration of a MycER regulatable oncoprotein intop53^-/- neural precursor cells, several immortallines were established from embryonic hippocampalprimordium, with bFGF and estrogen continuouslysupplied for activation of the MycER protein. A dualluciferase study demonstrated that the MycER proteinblocked the expression of a glial cell marker protein,GFAP, probably contributing to the persistent celldivision of the immortalized neural precursor cells.These cell lines differentiate into neuronal and glialcell types after withdrawal of bFGF. The phenotype ofthe hippocampal cell lines differed from that of thebasal ganglia cell lines as observed in a clonaldensity culture. This result implies that each regionof the brain has a unique developmental program, thatmay be imprinted in each of the neural precursor cells.     

Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

Background  

The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.     

Establishment and characterization of a tamoxifen-mediated reversible immortalized mouse dental papilla cell line

Odontoblasts are a type of non-proliferating and terminally differentiated cells that play an important role in the pulpo–dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited life span. We combined the traditional strategy of “Cre/LoxP-based reversible immortalization” with a tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line, mDPCET. mDPCs were sequentially transduced with a floxed SV40 T antigen-TK (SV40Tag-TK) and an ^ERT2Cre^ERT2-expressing plasmid. Clonal-isolated SV40Tag- and Cre-positive cells showed modified growth characteristics and a significantly extended life span. When mDPCET cells were treated with 4-hydroxytamoxifen, ^ERT2Cre^ERT2 translocated from the cytoplasm to the nucleus and caused the excision of SV40Tag-TK, which led to the reversion of mDPCETs. After the immortalization was reversed, the cells underwent replicative senescence and transitioned into a more differentiated state. Tamoxifen-mediated reversible immortalization, therefore, allows for the expansion of primary mDPCs, leads to the production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent a safe and ready-to-use method due to its simple manipulation.     

Functional characterization of a conditionally immortalized mouse epididymis caput epithelial cell line MEPC5 using temperature-sensitive simian virus 40 large T-antigen

A conditionally immortalized epididymis caput cell line, MEPC5, was established by infecting primary cultured mouse epididymis caput cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and the cells grew continuously. However, the downregulation of T-antigen at a nonpermissive temperature of 39 degrees C and the upregulation of cell density at 33 degrees C were associated with growth arrest and the increased protein expression of p21(waf1), a cell cycle inhibitor. The cells expressed epididymal caput-expressed genes such as phosphatidylethanolamine binding protein, polyoma enhancer activator 3, ME1, sulfated glycoprotein-2 (SGP-2), androgen receptor, and retinoic acid receptor alpha. Interestingly, the expression levels of ME1 and SGP-2 were significantly elevated under the cell growth-restricted conditions. The established mouse epididymis caput epithelial cell line MEPC5 retains some characteristics of differentiated epididymis epithelial cells, and should prove an excellent model for studies of gene expression and the physiological functions of epididymis caput epithelial cells.