If all distinct cell types of the body could be clonally isolated and stocked, similar to cDNA or genomic DNA libraries, they would be very invaluable for studying the tissue and cellular functions. We developed new method to establish conditionally immortalized cell lines that retain the differentiated cell functions similar to the original tissues, using the temperature-sensitive simian virus 40 large tumor antigen gene transgenic mouse. We successfully established many functional cell lines including hepatocyte, renal tubule cell, vascular smooth muscle cell, gastric surface mucous cell and various bone marrow stromal cells. We developed in vitro hematopoietic microenvironment to support growth and differentiation of hematopoietic stem cells and their progenitors by coculture with bone marrow stromal cells. In addition, we showed that bone marrow stromal cell lines have properties of mesenchymal stem cells that differentiate towards osteoblast, skeletal muscle cell, endothelial cell, adipocyte, smooth muscle cell and cardiomyocyte. Using newly developed transgenic rat, we established various endothelial cell lines from brain capillary, retinal capillary, bone marrow, and choroid plexus, which can be used for a novel in vitro system to investigate transport functions at the blood-tissue fluid barrier and to develop high throughput screening systems. Our transgenic mouse and rat will be useful to develop new types of cell lines with differentiation potentials. The established cell lines will be useful to study tissue functions at cellular and molecular levels and may be applicable to high throughput drug screening, toxicity test, and to develop the alternatives to animal experiments.

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Retinal degenerative disease such as retinitis pigmentosa and age-related macular degeneration causes visual defects with the progress in their diseases. In case of retinitis pigmentosa a large number of mutations in genes that involved in phototransduction pathway have been identified. However pharmacological treatment is presently unavailable for these diseases. Several strategies such as retinal pigment epithelial cell transplantation, growth factor application, vitamin supplementation or gene therapy are under examination for preventing the photoreceptor cell death. Among these strategies, some growth factors are commonly effective on preventing photoreceptor cell death even the mutated genes cause that photoreceptor cell death is different. Is it possible to prevent photoreceptor cell death and to maintain the visual function by continuously supplying growth factors to photoreceptor cells? Which methodology is better to supply growth factor continuously, gene transfer to retina or transplantation? We describe here the approach to prevent photoreceptor cell death by using gene transfected iris pigment epithelial cell transplantation.

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After the gut endoderm is formed, subsequent permissive induction signals from notochord allows the pancreas to merge and outgrows from the specific site of the embryonic gut epithelium. The first pancreas specific gene, pdx-1, is expressed around this stage. Interaction between pancreatic epithelium and surrounding mesenchyme allows the pancreas bud to further grow and differentiate to form ductal, exocrine or endocrine lineages. Several lines of evidence from gene knockout mouse and cell lineage studies suggest that a common pancreas stem cell first give rise to exocrine and endocrine progenitor cells. The endocrine progenitor then give rise to four different endocrine cells, α, β, δ, and PP cells, although it is not known how and when these cells arise. However, knowledge on the molecular mechanism how the genes define the pancreatic cell lineage is very limited. To date, pancreas remains to be one of the least understood organ in terms of the molecular mechanism of development. Identification of essential genes that regulate pancreas differentiation is indispensable for lineage specific differentiation of stem cells in culture. Here, I reviewed the literatures on development and regeneration of the Pancreas. Some recent results on growing pancreatic beta cells from ES (embryonic stem) cells are also introduced.

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Now that the human genome project has been completed, the rationale of genome-oriented development of medicine has been stressed. Conditionally immortalized cell lines could play an important role in “post-genome” projects, as they are applicable to high throughput screening system, which enables the prompt and massive screening of lead compounds and their optimization. In this regard, clonal cells, derived from temperature-sensitive simian virus 40 large tumor antigen gene transgenic mice, are interesting. They grew at the permissive temperature (33°C), but their growth was arrested at the non-permissive temperature (39°C). Therefore, first, under the permissive condition, we could establish cell lines, which had been impossible to establish because of their character and the small quantity. Second, through a simple method like a temperature shift from 33°C to 39°C, we can also analyze gene expression profiles and their functions during the cell maturation. Finally, clonal cells derived from a T-antigen transgenic mouse, after being genetically crossed with a mutant mouse, would be utilized as the tools to examine the targeted gene function. Actually, by using vitamin D receptor gene-disrupted mice as mutants, we have established clonal bone marrow stromal cell lines and have clearly shown that 1, 25(OH)₂D₃ suppresses differentiation of stromal cells into adipocytes in a VDR-dependent manner.

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Elucidating the molecular mechanism of the brain harrier transport is a key step toward successful drug delivery to the brain. In this review, we focused on the usefulness and the limitations of the newly developed in vitro model. To overcome several limitations of classical in vitro model, conditionally immortalized mouse (TM-BBB) and rat (TR-BBB) brain capillary endothelial cell lines and a choroid plexus epithelial cell line (TR-CSFB) were established from the temperature sensitive SU 40 large T antigen gene transgenic animals. mRNA and protein expression of GLUT1 in TR-BBB was 100 folds greater than that of primary culture, Several known transporter genes were identified for TR-BBB or TM-BBB, including organic anion transporter 3 (OAT 3) and organic anion transporting polypeptide 2 (oatp 2). Employing TM-BBB, new brain barrier functions have been clarified such as creatine transporter (CRT) and GABA transporter (GAT 2/BGT-1). TR-BBB has been applied for the study of expressional regulation of transporters at the BBB, e. g., L-proline and glycine transporter (ATA 2) and taurine transporter (TAUT). Moreover, one of the most important subjects for the in vitro BBB model is to clarify the expression and regulation of the tight junction proteins at the BBB. Co-culture systems among TR-BBB, conditionally immortalized astrocyte (TR-AST) and pericyte (TR-PCT) cell lines were developed and revealed that the expression of occludin, a tight-junction component, was induced by soluble factors secreted from TR-AST and TR-PCT. For the brain drug delivery research, the new in vitro model will provide rational and efficient strategies.

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Syncytiotrophoblasts, which form a continuous barrier between the maternal and fetal circulation, play an essential role in restriction of drug delivery through the blood-placental barrier (BPB). In the BPB many influx and efflux transporters for substrates such as neurotransmitters, neuroactive steroids, and nucleosides may play in the development of the fetus and fetal brain. However, the functions of these transporters in BPB, and the molecular and cellular aspects of these transporters remain unclear. The human term placenta and human chorionic carcinoma cell lines, such as BeWo and JAR cells have been mainly used for analysis of transporter and hormone secretion in the placenta. However it is difficult to confirm in vivo evidence using these conventional cell lines. In order to investigate the relationship between in vitro and in vivo results, cell lines from animals as an in vitro model are needed. We have established new syncytiotrophoblast cell lines (TR-TBTs) from the recently developed transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg-rat). TR-TBTs have temperature-sensitive cell growth due to the expression of is SV 40 large T-antigen. These cell lines had a syncytium-like morphology, could be prepared as monolayers, expressed cytokeratins, rat syncytiotrophoblast markers, and in vivo influx and efflux transporters such as oatp2, TauT, GAT-2/BGT-1, and mdr 1 a. Moreover, TR-TBTs exhibited apical or basal GLUT1. localizations and apical GLUT3 localizations. Thus, these results suggest that TR-TBTs could be very useful tool to clarify the BPB functions, including transporter genes and their regulation systems.

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