ognizant Communication Corporation

Cell Transplantation, Vol. 11, pp. 399-402, 2002
0963-6897/02 $20.00 + 00
Copyright © 2002 Cognizant Comm. Corp.
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Department of Surgery II, Nagasaki University School of Medicine, Nagasaki, 852-8501, Japan

We studied the effect of preoperative hepatocyte transplantation on the prevention of liver failure in cirrhotic rats after hepatic resection. Two groups of Lewis rats were rendered cirrhotic by IP injection of 1% dimethylnitrosamine and were subjected to 33% hepatectomy. Two days before the resection, 36 rats in group I received intrasplenic hepatocyte transplantation, and 25 rats in group II were given intrasplenic injection of normal saline as a control. By the end of the third postoperative day, the rats in group I had better survival and a better biochemical profile than those in group II. The liver growth rate and the labeling index of proliferating cell nuclear antigen (PCNA-LI) showed a steady rise in group I. Compared with group II, group I had a significantly lower transforming growth factor (TGF-b1) level (p < 0.05). We conclude that preoperative intrasplenic hepatocyte transplantation improves survival and facilitates regeneration in cirrhotic rats after hepatic resection.

Key words: Hepatocyte transplantation; Cirrhosis; Hepatic resection

Address correspondence to Tarik A. Ahmad, M.D., Department of Surgery II, Nagasaki University School of Medicine, Nagasaki, 852-8501, Japan. Tel: +81 95-849-7316; Fax: +81 95-849-7319.

¹Institute of Biological Engineering, Jilin University, 8 Xinmin Street, Changchun City, Jilin Province 130021, China
²Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
³Institute of Molecular and Cellular Bioscience, University of Tokyo, Bunkyoo-ku, Tokyo 113-0032, Japan

To investigate the feasibility of fetal liver cells for liver tissue engineering, the supporting function of poly-L-lactic acid (PLLA) for fetal liver cells and the effects of oncostatin M (OSM) on hepatic differentiation were studied. After preparing three-dimensional biodegradable PLLA scaffold having a well-developed open-pore structure by a gas-forming method with ammonium chloride particles as a porogen and a gas-forming reagent, fetal liver cells separated from E14.5-C57BL/6CrSlc murine embryos were inoculated in the PLLA scaffolds. Cells were cultured in Williams' E medium with or without OSM (10 ng/ml) for 30 days with a medium change every 2 days. Results showed that there were significant increases in the number of cells and in albumin secretion in PLLA culture compared within monolayer culture on day 15. In addition, a significant increase in albumin secretion was observed in OSM-added PLLA culture compared with OSM-free culture, and there was only a slightly enhanced albumin secretion in monolayer cultures with OSM. These results suggest that PLLA may enhance the biological activity of OSM for inducing maturation of fetal liver cells. Interestingly, the number of cells in PLLA culture with OSM decreased compared with OSM-free PLLA culture at day 15. This may be because promotion of hepatic development by OSM simultaneously suppressed in vitro hematopoiesis (i.e., blood cell production). In summary, our results indicate that the three-dimensional PLLA scaffold is a good support material for the cultivation of fetal liver cells and that OSM is capable of not only terminating hematopoiesis of the fetal liver but also stimulating the maturation of hepatic parenchymal cells in vitro.

Key words: Poly-L-lactic acid scaffolds; Fetal mouse liver cells; Oncostatin M; Liver tissue engineering

Address correspondence to Jinlan Jiang, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan. Tel: +813-5452-6348; Fax: +813-5452-6348; E-mail: jiangjl@iis.u-tokyo.ac.jp

¹Department of Organ Regeneration, Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan
²Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
³Second Department of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan

Cell death is thought to take place through at least two distinct processes: apoptosis and necrosis. There is increasing evidence that dysregulation of the apoptotic program is involved in liver diseases. However, there is no method to simply evaluate apoptosis in the liver tissue at present. It has been reported that the expression of asialoglycoprotein receptors (AGPRs) increases with apoptosis, but there is no report until now that investigates the influence of soluble AGPRs on apoptosis of hepatocytes. Soluble AGPRs have been reported to be present in human serum under physiological conditions. In the present study, in order to investigate the correlation between apoptosis of hepatocytes and soluble AGPR, mouse soluble AGPRs were detected using SDS-PAGE and Western blot analysis was conducted using anti-extracellular mouse hepatic lectin-1 (Ex-MHL-1) antiserum (polyclonal rabbit serum). The mouse soluble AGPRs were present in culture medium and mouse serum when hepatocytes were damaged. The soluble AGPRs increased proportionately, as the number of dead hepatocytes increased. In addition, soluble AGPRs existed more when apoptotic cell death was observed in in vitro and in vivo than when necrotic cell death was observed. The extracellular moiety of MHL-1 exists in the culture medium and mouse serum as a soluble AGPR, but the detailed mechanism of releasing soluble AGPR from hepatocytes has not been revealed yet. We described the first evidence for the relation between quantity of soluble AGPRs with two kinds of cell death: necrosis and apoptosis. Based on the results of our study, soluble AGPRs might become a new marker of apoptosis in the liver tissue and be useful for clinical diagnosis and treatment for liver diseases.

Key words: Liver; Apoptosis; Soluble asialoglycoprotein receptor; Extracellular mouse hepatic lectin-1

Address correspondence to Toshihiro Akaike, Department of Organ Regeneration, Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. Tel: (+81) 263-37-3191; Fax: (+81) 263-37-3195; E-mail: akaike@sch.md.shinshu-u.ac.jp

¹Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
²The Japan Health Sciences Foundation

Hepatocyte-based biological therapies are increasingly envisioned for temporary support in acute liver failure and provision of specific-liver functions in liver-based metabolic deficiency. One of the hurdles to develop such therapies is severe shortage of human livers for hepatocyte isolation. To address the issue, we have focused on reversible immortalization of human hepatocytes. Such technology can allow rapid preparation of functional and uniform human hepatocytes. Here we present our strategy to construct transplantable human hepatocyte cell lines.

Key words: Immortalized hepatocytes; Cre/loxP system; Cell therapies

Address correspondence to Naoya Kobayashi, M.D., Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: (+81) 86-235-7255; Fax: (+81) 86-221-8775; E-mail: immortal@md.okayama-u.ac.jp

¹Division of Gastroenterology I, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 Japan
²Department of Surgery, and ³Department of Cell Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
⁴The Japan Health Sciences Foundation

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G₁ phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirus-based p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immunofluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G₁ checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.

Key words: p21; Protein transduction; Immortalized hepatocytes

Address correspondence to Naoya Kobayashi, M.D., Ph.D., Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: (+81) 86-235-7255; Fax: (+81) 86-221-8775; E-mail: immortal@md.okayama-u.ac.jp

¹Department of Surgery, Institute of Clinical Medicine, and ²Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nodai, Tsukuba, Ibaraki 305-8575, Japan

We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.

Key words: Embryonic stem cell; Embryoid bodies; Hepatocyte; Definitive endoderm

Address correspondence to Hideki Taniguchi, M.D., Ph.D., Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Tel: 81-298-53-3221; Fax: 81-298-53-3222; E-mail: rtanigu@md.tsukuba.ac.jp

¹Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan
²Institute of Biological Engineering, Jinlin University, 8 Xinmin Street, Changchung, Jilin 130021, China
³Institute of Molecular and Cellular Bioscience, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Although cells isolated from fetal liver are one of the major sources for liver tissue engineering, it is still very difficult to induce them to fully differentiate in vitro into mature hepatocytes. We therefore investigated the effects of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) on differentiation in terms of the expression of various liver-specific functions, because these factors have been reported to induce the emergence of possible hepatocyte progenitor cells (small hepatocytes) in adult rat hepatocyte culture or maturation of fetal mouse liver cells in culture. Fetal liver cells isolated from mouse embryos were cultured for 5 weeks in collagen-precoated plates. NA (10 mM) and DMSO (1%) remarkably enhanced the emergence of small hepatocytes, and OSM also synergistically enhanced the selective growth of small hepatocytes and inhibited the growth of blood cell populations. In the presence of these three factors, such small hepatocytes became dominant in culture, so that they covered almost 60-70% of confluence after week 2. In addition, some of them piled up over the small hepatocyte monolayer and displayed distinctively differentiated morphology, such as the emergence of binucleated cells, formation of tight gap junctions, and possible bile duct structures. Although OSM alone had very weak effects on hepatocyte functions, albumin secretion and cytochrome P450IA1/2 capacity were greatly enhanced when combined with NA or DMSO. This functional observation closely agreed with the emergence of small hepatocytes. In contrast, ammonium removal was strongly dependent on DMSO alone. DNA amount basis functions of fetal cells with three factors at week 5 were 1/7 for albumin secretion, 3 times higher for ammonium removal, and 1/10 for P450 capacity, compared with those of cultured adult mouse hepatocytes. These results show that inclusion of NA, DMSO, and OSM in the culture medium significantly enhances in vitro maturation of fetal liver cells when compared with conventional culture conditions.

Key words: Fetal mouse liver cell; In vitro maturation; Adult mouse hepatocytes; Liver-specific function

Address correspondence to Y Sakai, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan. Tel: +81-3-5452-6352; Fax: +81-3-5452-6353; E-mail: sakaiyas@iis.u-tokyo.ac.jp

¹National Research Institute for Child Health and Development
²Tokyo University of Agriculture
³National Institute of Neuroscience

It has been hoped that amniotic epithelial cells would be a gene carrier to neural and hepatic tissue, because of 1) the presence of neural and hepatic stem-like cells, 2) the ability to cryopreserve them, 3) long-term survival in the transplanted site, and 4) few ethical problems concerning procurement. But transplantation of a sufficient number of cells to adult tissue needs large-scale cell supply and may lead to vascular embolism. We attempted transplantation of amniotic epithelial cells into fetal liver, because 1) the fetal liver is at the proliferative stage, 2) the number of cells required is small, and 3) the fetal stage is advantageous for the induction of immunological tolerance. Amniotic epithelial cells from day 18.5-20.5 fetuses were transfected with adenoviral AdlacZ and harvested to inject into fetal rat liver of the syngeneic strain (day 18.5-20.5). The efficacy of cell transplantation into the liver increased in the order: intraplacental < intraumbilical vein < intrahepatic route. LacZ-transfected amniotic cells (1-8 x 10⁵ cells), hepatocytes (5 x 10⁵ cells), or AdlacZ vector solution (1.7 x 10⁷ pfu) were injected through the uterine membrane into the liver. Transplanted cells formed a cellular mass and survived for up to 14 days after birth, whereas lacZ-transfected cells were rapidly decreased after the injection of AdlacZ vector or rat hepatocytes as a gene carrier so that the use of amniotic epithelial cells as a gene carrier will result in long-term expression of exogenous genes in the liver.

Key words: Amniotic epithelial cells; In utero manipulation; Rat fetus; Liver; Gene carrier

Address correspondence to Dr. S. Enosawa, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567 Japan. Tel: +81-3-3416-0181, ext. 8776; Fax: +81-3-3411-7309; E-mail: enosawa@nch.go.jp

¹Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
²Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, and CREST (Japan Science and Technology Corporation), 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as a and b cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.

Key words: Pancreas; Stem cell; Colony; Flow cytometry

Address correspondence to Hideki Taniguchi, M.D., Ph.D., Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Tel: 81-298-53-3221; Fax: 81-298-53-3222; E-mail: rtanigu@igaku.md.tsukuba.ac.jp

¹Department of Metabolism, Kobe Mahoshi Hospital, Kobe 651-1242, Japan
²Department of Geriatric Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan
³Department of Metabolism and Community Health Science, Kobe University School of Medicine, Kobe 654-0142, Japan

A fetus in the uterus is not rejected at any time during the entire gestational period, even without the administration of immunosuppressive agents, though fetus is a kind of allograft. This prevention of rejection is considered to be associated with the presence of placental tissues. This hypothesis was tested by the allografting of islets together with placental tissues (trophoblasts) in streptozotocin (STZ)-induced diabetic mice. Placentae were harvested from the mice at the 14th postgestation day by being peeled off carefully and with the maternal decidua left behind, and cut into small pieces. Five hundred freshly isolated islets together with placental tissues obtained from ICR mice were placed under the left kidney capsule of STZ-induced diabetic C57BL/6J mice. The nonfasting blood glucose level was reduced from 477 ± 41 mg/dl at the time of pretransplant to that of the intact normal mice (161 ± 18 mg/dl) soon after the cografting, and did not return to the pretransplant level before the 14th posttransplant day. The grafting of the same number of islets alone and/or liver tissues dropped the blood glucose level, but not to that of the intact normal mice. It returned to the pretransplant level within 1 week. This is the first successful prolongation of survival of allografted islets without immunosuppressive agents through cotransplantation of allogenic placental tissues. The underlying mechanism remains to be clarified.

Key words: Diabetes; Islet allotransplantation; Placenta; Trophoblast; Immunoisolation; Immunosuppressive agent

Address correspondence to Kazuhisa Suzuki, M.D., Ph.D., Department of Metabolism, Kobe Mahoshi Hospital, 12-3 Kokotani, Kami-Tanigami, Yamada-Cho, Kita-Ku, Kobe 651-1242, Japan. Tel: +81-78-582-0111; Fax: +81-78-581-8464; E-mail: kazukobe@venus.dti.ne.jp

¹Institute of Life Science, Soka University, Tangi-cho 1-236, Hachioji, Tokyo 192-8577, Japan
²Laboratory of Molecular Biology, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
³Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan

In order to invent a screening system to check in vivo gene function and the efficiency of gene transfer mediated by a retroviral vector system, we established a novel packaging cell, PacC6/A8, that is transplantable to rat brains. The packaging cell is based on the gene of the neuropatogenic retrovirus, A8-V. For expression in the brain, a vector that expresses brain-derived neurotrophic factor (BDNF) tagged by c-Myc-His₆ (LxA/bdmh) was constructed. After transfection of LxA/bdmh to PacC6/A8, a cloned cell line, PacC6/A8/bmh, was established. PacC6/A8/bmh cells stably produced pseudotyped retroviruses carrying LxA/bdmh. For a control, a retroviral vector that bears the gene that codes enhanced green fluorescent protein (EGFP) tagged by C-Mic-His₆ was also created and used for the establishment of PacC6/A8/gfmh cells that produce pseudotyped retroviruses carrying LxA/gfmh. PacC6/A8/bmh and PacC6/A8/gfmh cells were injected to the brain of newborn rats. A tumor was formed in all the rats injected that did not exhibit any symptoms until 3-4 weeks after the injection. A histological study of the injected rats revealed that the transferred BDNF gene was expressed in the brain of rats injected with PacC6/A8/bmh cells, but not in rats with PacC6/A8/gfmh cells. Interestingly, many activated microglia had migrated into the tumor induced by PacC6/A8/bmh cells, and expressed a high amount of BDNF.

Key words: Retroviral vector; Ecotropic; Gene therapy, CNS

Address correspondence to Prof. Rihito Watanabe, M.D., Institute of Life Science, Soka University, Tangi-cho 1-236, Hachioji, Tokyo 192-8577, Japan. Tel: + 81-426-91-9465; Fax: + 81-426-91-9315; E-mail: rihito@t.soka.acjp

Department of Neurosurgery, School of Medicine, Keio University, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan

The purpose of this study was to elucidate the biological significance and the possibility of intracerebral grafting of neuroepithelial stem cells derived from the mesencephalic neural plate. Immunohistological studies of embryonic day 10.5 (E10.5) Wister rats revealed strong nestin expression in the mesencephalic part of the neural plate. Mesencephalic neural plates removed from E10.5 rats were processed to either tissue or cell dissociation culture. They were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, cell proliferation and differentiation into neurons extending long neurites were obvious in a serum-free medium, in a medium containing 3% serum, and in a medium containing 20 ng/ml epidermal growth factor. On the other hand, in a medium containing 10 ng/ml basic fibroblast growth factor (bFGF), both vigorous cell proliferation and sphere formation were recognized. Furthermore, marked neurite growth was rarely seen in this culture. When they were plated in a dissociation culture, cell proliferation and neurosphere generation were also recognized only in a medium containing bFGF, depending on the initial cell concentration. The spheres, generated 7 days after the primary cell culture, were positively stained by nestin. These data suggested that bFGF was able to amplify the stem cell population present in the mesencephalic neural plate derived from early embryos. This might make it possible to obtain a large number of stem cells as donor material for neural transplantation on demand.

Key words: Mesencephalic neural plate; Neuroepithelial stem cell; Neural transplantation; Basic fibroblast growth factor

Address correspondence to Koichi Uchida, M.D., Department of Neurosurgery, School of Medicine, Keio University, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan. Tel: 81 3 5363 3808; Fax: 81 3 3354 8053; E-mail: koichi@sc.itc.keio.ac.jp

Institute of Life Science, Soka University, Tangi-cho 1-236, Hachioji, Tokyo, 192-8577, Japan

A8 virus (A8-V) is a molecular clone of the neuropathogenic FrC6 virus derived from the Friend murine leukemia virus (F-MuLV). The A8-V infects endothelia and microglia in the brain. We constructed a gene transfer system with the A8-V gene. Pseudotyped virus carrying the surface protein of A8-V (A8-SU) transduced the b-glactosidase gene incorporated in the retroviral vector efficiently to cultured microglial cells derived from newborn rats. Ex vivo gene transferred microglial cells were then injected into the right hemisphere of 3-day-old and 3-week-old rat brains. All of the rats examined at 4 weeks after the injection contained the labeled microglial cells in the brain (7/7 and 5/5 of the rats injected at 3 days and 3 weeks, respectively). None of the rats showed pathological changes in the whole body investigated, including the central nervous system, 4 weeks after transplantation of the labeled microglial cells.

Key words: Retroviral vector; Ecotropic; Ex vivo; CNS

Address correspondence to Prof. Rihito Watanabe, M.D., Institute of Life Science, Soka University, Tangi-cho 1-236, Hachioji, Tokyo. Tel: + 81-426-91-9465; Fax.: + 81-426-91-9315; E-mail: rihito@t.soka.ac.jp

¹Tissue Engineering Laboratory, Department of Mechanical Engineering, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan
²Institute of Industrial Science, University of Tokyo, Minato-ku, Tokyo, Japan

Although rapid formation of a smooth inner surface is important in constructing an artificial vascular graft, a conventional model that uses a biodegradable polymer such as poly-glycolic acid needs long-term culture to form it. In another model, which uses collagen gel, it is reported that prompt formation of the smooth inner surface was achieved. But the mechanical properties were not suitable, resulting in rupture under high pressure at the arterial level. Therefore, we propose a new artificial vascular graft model made of biodegradable polymer, gel, and cells. At first we manufactured an artificial vascular graft (i.d. 5 mm, o.d.7 mm) consisting of poly-L-lactic acid (PLLA) with open pore structures by using gas-forming methods. After mixing human normal aortic smooth muscle cells (SMCs) with type I collagen solution, pores of the PLLA scaffold were filled with the mixture. The collagen mixture was made into gel in the pores of the PLLA scaffold, incubating at 37°C. WET-SEM analysis showed that the prompt formation of a smooth inner surface was achieved in the new model. The ratio of incorporation of SMCs into the artificial vascular graft became approximately 100% by using the cell-collagen mixture, whereas only 40% of SMCs were trapped in the conventional model where SMCs were inoculated as a cell-medium suspension. Therefore, it was suggested that the new artificial vascular graft model was superior in smooth inner surface formation and cell inoculation, compared with conventional models using either biodegradable polymer or gel.

Key words: Biodegradable polymer; Gel; Artificial blood vessel; Cells; Collagen

Address correspondence to Katsuko S. Furukawa, Department of Mechanical Engineering, Graduate School of Engineering, University of Tokyo, 713, 8th building, 7-3-1 Hongo 7-3-1; Bunkyo-ku, Tokyo 113-8656, Japan. Tel: +81-3-5841-6375; Fax: +81-3-5841-6442; E-mail: furukawa@tissue.t.u-tokyo.ac.jp

¹Department of Surgery and 2Department of Cell Biology, Okayama University Graduate School of Medicine and Dentistry, ²-5-1 Shikata-cho, Okayama 700-8558, Japan
³The Japan Health Sciences Foundation

Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells. The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro. For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E. coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system. The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs. Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity. There was no significant difference in transduction efficacy between early and late-passage phases in both cells. LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay. These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.

Key words: Lentivirus; LacZ gene; Endothelial cells; Mesenchymal stem cells

Address correspondence to Naoya Kobayashi, M.D., Department of Surgery, Okayama University Graduate School of Medicine and Dentistry. 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (+81) 86-235-7259; Fax: (+81) 86-221-8775; E-mail: immortal@md.okayama-u.ac.jp

Biomedical Engineering Laboratory, Graduate School of Engineering, The University of Tokyo, 7-3-1 Bunkyo, 113-8656 Tokyo, Japan

Tissue engineering approaches have been clinically tried to repair damaged articular cartilages. It is an essential step to uniformly seed chondrocytes into 3D scaffolds in order to reconstruct tissue-engineered cartilages in vitro, but the tissue engineering could not have been provided with efficient cell seeding methods. Type I collagen is clinically used and known as a cytocompatible material, having recognition sites for integrins. Collagen gel encapsulating chondrocytes has been tried for making regenerated cartilages, but it is found difficult to have the gel keep its original shape after long-term culture, because of shrinking. On the other hand, 3D scaffolds, either of a nonwoven structure or a sponge-like structure, involve difficulty in that chondrocytes could not be uniformly seeded, although they have adequate initial mechanical properties. In this study, by combining collagen gelation with a nonwoven PLLA scaffold, we achieved uniform cell seeding into the 3D scaffold. Bovine articular chondrocytes were mixed with type I collagen solution, and the solution was poured into the nonwoven PLLA scaffold (1.5 mm thick, f 15 mm). The collagen-chondrocyte mixture was made into gel at 37°C for 1 h. The 0.39% collagen mixture was viscous enough to prevent cells from precipitating during gelation. Almost all chondrocytes were able to be incorporated into the PLLA scaffolds by mixing with collagen solution and subsequently making into gel, while 30-40% of the chondrocytes seeded as a cell suspension were not trapped into the PLLA scaffolds. The method presented, where chondrocytes were mixed with collagen solution, and the mixture was incorporated into a 3D scaffold, then made into gel in the scaffold, could serve as an alternative for in vitro cartilage regeneration, also simultaneously having the advantages of both materials.

Key words: Chondrocyte; Collagen; Gelation; PLLA; Cartilage; Regeneration

Address correspondence to Takashi Ushida, Biomedical Engineering Laboratory, Graduate School of Engineering, The University of Tokyo, 7-3-1 Bunkyo, 113-8656 Tokyo, Japan. Tel: +81-3-5841-6374; Fax: +81-3-5841-6374; E-mail: ushida@ingram.t.u-tokyo.ac.jp