ognizant Communication Corporation

CELL TRANSPLANTATION

ABSTRACTS
VOLUME 9, NUMBER 5, 2000

Cell Transplantation, Vol. 9, pp. 567-576, 2000
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The Localization and Functional Contribution of Striatal Aromatic L-Amino Acid Decarboxylase to L-3,4-Dihydroxyphenylalanine Decarboxylation in Rodent Parkinsonian Models

Ken Nakamura,1 Maqbool Ahmed,1 Eliav Barr,2* Jeffrey M. Leiden,2** and Un Jung Kang1

Committee on Neurobiology and 1Departments of Neurology, Pharmacological & Physiological Sciences, and 2Department of Medicine, The University of Chicago, Chicago, IL 60637

L-3,4-Dihydroxyphenylalanine (L-dopa) is the mainstay of therapy for patients with Parkinson's disease (PD), and mediates its primary effects through conversion into dopamine by aromatic L-amino acid decarboxylase (AADC). Given the loss of AADC-containing nigrostriatal dopaminergic neurons in PD, however, the location of residual AADC that converts L-dopa into dopamine remains controversial. The first objective of this study was to establish the presence of AADC expression in striatal neurons and glia using reverse transcriptase and PCR. Transcripts for the neuronal but not nonneuronal forms of AADC were detected in striatal tissue, cultured striatal neurons, and glia. We then examined whether this striatal AADC expression represents a physiologically significant source of dopamine production. No dopamine release was detected following incubation of striatal cultures with L-dopa or transduction with adenovirus expressing tyrosine hydroxylase. Our data establish the presence of AADC expression in the striatum both in vivo and in vitro, but suggest that striatal components do not represent a primary source of L-dopa decarboxylation following nigrostriatal denervation in rats. Understanding the source and localization of AADC is important in understanding the complications of L-dopa therapy and in designing rational therapeutic strategies for PD, including cellular transplantation and gene therapy.

Key words: Aromatic L-amino acid decarboxylase; L-3,4-Dihydroxyphenylalanine; Parkinson's disease; Striatum; Glia; Decarboxylation

Address correspondence to Un Jung Kang, M.D., Department of Neurology, MC 2030, University of Chicago, 5841 S. Maryland Ave, Chicago, IL 60637. Tel: (773) 702-6389; Fax: (773) 702-9060; E-mail: u-kang@uchicago.edu

*Current address: Merck Research Laboratories, West Point, PA 19486.
**Current address: Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115.




Cell Transplantation, Vol. 9, pp. 577-584, 2000
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Grafting of Nigral Tissue Hibernated With Tirilazad Mesylate and Glial Cell Line-Derived Neurotrophic Factor

Åsa Petersén, Oskar Hansson, Mia Emgård, and Patrik Brundin

Section for Neuronal Survival, Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, Sölvegatan 17, 22362 Lund, Sweden

Transplantation of embryonic ventral mesencephalon is a potential therapy for patients with Parkinson's disease. As only around 5-10% of embryonic dopaminergic neurons survive grafting into the adult striatum, it is considered necessary to use multiple donor embryos. To increase the survival of the grafted dopaminergic neurons, the clinical transplantation program in Lund currently employs the lipid peroxidation inhibitor, tirilazad mesylate, in all solutions used during tissue storage, preparation, and transplantation. However, the difficulty in obtaining a sufficient number of donor embryos still remains an important limiting factor for the clinical application of neural transplantation. In many clinical transplantation programs, it would be a great advantage if human nigral donor tissue could be stored for at least 1 week. This study was performed in order to investigate whether storage of embryonic tissue at 4°C for 8 days can be applied clinically without creating a need to increase the number of donors. We compared the survival of freshly grafted rat nigral tissue, prepared according to the clinical protocol, with tissue transplanted after hibernation. Thus, in all groups tirilazad mesylate was omnipresent. One group of rats was implanted with fresh tissue and three groups with hibernated tissue with or without addition of glial cell line-derived neurotrophic factor (GDNF) in the hibernation medium and/or the final cell suspension. Earlier studies have suggested that GDNF improves the survival of hibernated nigral transplants. We found no statistically significant difference between the groups regarding graft survival after 3 weeks. However, there was a nonsignificant trend for fewer surviving dopaminergic neurons in grafts from hibernated tissue compared to fresh controls. Furthermore, we show that the addition of GDNF to the hibernation medium and/or to the final cell suspension does not significantly increase the survival of the dopaminergic neurons.

Key words: Embryonic tissue; Hibernation; Tirilazad mesylate; Glial cell line-derived neurotrophic factor (GDNF); Parkinson's disease

Address correspondence to Åsa Petersén, Section for Neuronal Survival, Wallenberg Neuroscience Center, Department of Physiological Sciences, Sölvegatan 17, S-22362 Lund, Sweden. Tel: +46-46-2220563; Fax: +46-46-2220531; E-mail: asa.petersen@mphy.lu.se




Cell Transplantation, Vol. 9, pp. 585-594, 2000
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Distribution of AAV-TK Following Intracranial Convection-Enhanced Delivery Into Rats

Janet Cunningham,1 Yoshitsugu Oiwa,3 Dea Nagy,2 Greg Podsakoff,1 Peter Colosi,1 and Krys S. Bankiewicz2

1Avigen, Inc., Alameda, CA
2Center for Functional Imaging, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA
3Molecular Therapeutics Section, LMMN, NINDS, National Institutes of Health, Bethesda, MD

Adeno-associated virus (AAV)-based vectors are being tested in animal models as viable treatments for glioma and neurodegenerative disease and could potentially be employed to target a variety of central nervous system disorders. The relationship between dose of injected vector and its resulting distribution in brain tissue has not been previously reported nor has the most efficient method of delivery been determined. Here we report that convection-enhanced delivery (CED) of 2.5 x 108, 2.5 x 109, or 2.5 x 1010 particles of AAV-thymidine kinase (AAV-TK) into rat brain revealed a clear dose response. In the high-dose group, a volume of 300 mm3 of brain tissue was partially transduced. Results showed that infusion pump and subcutaneous osmotic pumps were both capable of delivering vector via CED and that total particle number was the most important determining factor in obtaining efficient expression. Results further showed differences in histopathology between the delivery groups. While administration of vector using infusion pump had relatively benign effects, the use of osmotic pumps resulted in notable toxicity to the surrounding brain tissue. To determine tissue distribution of vector following intracranial delivery, PCR analysis was performed on tissues from rats that received high doses of AAV-TK. Three weeks following CED, vector could be detected in both hemispheres of the brain, spinal cord, spleen, and kidney.

Key words: Adeno-associated virus; Thymidine kinase (ACC-TK); Intracranial delivery; Convection-enhanced delivery

Address correspondence to Krys S. Bankiewicz, M.D., Ph.D., Molecular Therapeutics Section, LMMN, NINDS, National Institutes of Health, 36 Convent Drive, Bldg. 36 Room 5W21, MSC 4164, Bethesda, MD 20892. Tel: (301) 435-6834; Fax: (301) 594-5799; E-mail: kbank@codon.nih.gov




Cell Transplantation, Vol. 9, pp. 595-607, 2000
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Technique for Bilateral Intracranial Implantation of Cells in Monkeys Using an Automated Delivery System

Krys S. Bankiewicz,1 John Bringas,2 Phillip Pivirotto,1 Ethan Kutzscher,3 Dea Nagy,4 and Marina E. Emborg5

1Molecular Therapeutics Section, LMMN, NINDS, National Institutes of Health, Bethesda, MD
2Cromedica Inc., Victoria, BC, Canada
3Pacific Neuroscience Institute, Orinda, CA
4Center for Functional Imaging, LBNL, UC Berkeley, Berkeley, CA
5Research Center for Brain Repair, Rush Presbyterian Medical Center, Chicago, IL

Intracerebral grafting combined with gene transfer may provide a powerful technique for local delivery of therapeutic agents into the CNS. The present study was undertaken to: (i) develop a reliable and reproducible automated cell implantation system, (ii) determine optimal implantation parameters of cells into the striatum, (iii) determine upper safe limits of cellular implantation into the neostriatum of monkeys. Autologous fibroblasts were infused into six sites of the striatum in nonhuman primates (Macaca mulatta, n = 11). Twenty-six-gauge cannulae were inserted vertically through cortical entry sites into the striatum (two sites in the caudate nucleus and four sites in the putamen) at predefined coordinates based on magnetic resonance imaging (MRI). The cannulae were guided by an electronically operated, hydraulic micropositioner and withdrawn at controlled rates, while cells (5, 10, 20, 40, or 80 ml/site) were infused simultaneously. Varying infusion rates and cell concentrations were also evaluated. Visualization and evaluation of graft placement were performed using contrast MRI at 3-5 days postsurgery. Animals were monitored for signs of clinical complications and sacrificed 2 weeks following surgery. Postimplantation MRI revealed a tissue mass effect of the implant with shifting of midline, edema, and infiltration of the white tracts at 40 and 80 ml/site. In addition, these animals developed transient hemiparesis contralateral to the implant site. MRI of animals grafted with 20 ml/site exhibited columnar-shaped implants and evidence of infiltration into white matter tracts possibly due to a volume effect. No clinical side effects were seen in this group. At 14 days postsurgery, MRI scans showed consistent columnar grafts (measuring approximately 5 mm in height) throughout the striatum in animals implanted with 5 or 10 ml/site. No signs of clinical side effects were associated with these volumes and postmortem histological examination confirmed MRI observations. Optimal surgical parameters for delivery of cells into the striatum consist of a graft volume of 10 ml/site, an infusion rate of 1.6 ml/min, a cell concentration of 2.0 x 105 cells/ml, and a cannula withdrawal rate of 0.75 mm/min. These results show that infusion of cells into the striatum can be done in a safe and routine manner.

Key words: Cell therapy; Primates; Grafting; CNS; Magnetic resonance imaging

Address correspondence to Krys S. Bankiewicz, M.D., Ph.D., Molecular Therapeutics Section, LMMN, NINDS, National Institutes of Health, 36 Convent Drive, Bldg. 36 Room 5W21, MSC 4164, Bethesda, MD 20892. Tel: (301) 435-6834; Fax: (301) 594-5799; E-mail: kbank@codon.nih.gov




Cell Transplantation, Vol. 9, pp. 609-622, 2000
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Behavioral Evaluation of Hemiparkinsonian MPTP Monkeys Following Dopamine Pharmacological Manipulation and Adrenal Co-Graft Transplantation

Leonard L. Howell,1,2,3 Larry D. Byrd,1,3 Ann M. McDonough,1 P. Michael Iuvone,3 and Roy A. E. Bakay4

1Yerkes Regional Primate Research Center and Departments of 2Psychiatry and Behavioral Sciences, 3Pharmacology, and 4Neurosurgery, Emory University, Atlanta, GA 30322

Bradykinesia and rigidity are the symptoms that most directly correlate with loss of striatal dopamine in Parkinson's disease. In the hemiparkinsonian (HP) monkey, this is represented by paucity of movement as measured by computerized movement analysis, diminished manual dexterity on clinical examination, and diminished performance on operant behavioral tasks. The present study used an MPTP-induced HP model in rhesus monkeys to evaluate the effectiveness of adrenal medullary and peripheral nerve co-grafts in diminishing parkinsonian symptoms. Unoperated controls (N = 4), surgical controls with caudate lesioning (N = 4), and caudate co-grafted (N = 4) HP monkeys demonstrated diminished movement in the home cage following MPTP. This behavior persisted in unoperated controls, but improved in both surgical control and co-grafted monkeys. Functional hand dexterity evaluations demonstrated similar impairment in all three groups, but only surgical controls and co-grafted monkeys demonstrated improvement. In general, rotational behavior in response to apomorphine was consistent with recovery of function in surgical controls and co-grafted monkeys, but marked between-subject variability precluded group statistical analyses. None of the monkeys could perform the operant task using the affected limb following MPTP. However, the performance of two co-grafted animals demonstrated partial recovery. L-dopa improved operant performance, demonstrating a dopaminergic component to the task. The results demonstrate recovery of behavioral function after surgical treatment, with adrenal co-grafted monkeys showing the greatest degree of improvement.

Key words: MPTP-induced parkinsonism; Adrenal co-graft; Dopamine; Nonhuman primate

Address correspondence to Leonard L. Howell, Ph.D., Yerkes Regional Primate Research Center, Emory University, Atlanta, GA 30322. Tel: (404) 727-7786; Fax: (404) 727-1266; E-mail: leonard@rmy.emory.edu




Cell Transplantation, Vol. 9, pp. 623-627, 2000
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Neurotrophic Factors NGF and FGF-2 Alter Levels of Huntingtin (IT15) in Striatal Neuronal Cell Cultures

Nadia S. K. Haque* and Ole Isacson

Program in Neuroscience, Harvard Medical School; Department of Neurology, Massachusetts General Hospital; Neuroregeneration Laboratories, McLean Hospital, Belmont, MA 02178

A mutation of the human IT15 gene is responsible for Huntington's disease (HD) and the causative factor in the major neuronal loss observed in the striatum. The growth factors basic fibroblast growth factor (FGF-2), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) improve survival and promote differentiation of striatal neurons, as well as exert a neuroprotective effect when such neurons are challenged with metabolic toxins or excitatory amino acids. Using Western blotting and striatal cell cultures, we found that FGF-2 increased the level of huntingtin in a dose-dependent fashion, whereas NGF decreased huntingtin expression. The neurotrophic factor-specific, dose-dependent effect on striatal levels of huntingtin may be relevant to understanding the normal role of IT15 and developing new therapies against the disease provoking mutated IT15.

Key words: Huntington's disease; Western blotting; Striatum; Cell cultures; Basic fibroblast growth factor (FGF-2); Brain-derived neurotrophic factor (BDNF); Nerve growth factor (NGF)

Address correspondence to Dr. Ole Isacson, Neuroregeneration Laboratories, Harvard Medical School, McLean Hospital, MRC 119, 115 Mill St., Belmont, MA 02178. Tel: (617) 855-3283; Fax: (617) 855-3284; E-mail: isacson@helix.mgh.harvard.edu

*Current address: Dr. Nadia Haque, Geron Corporation, San Francisco, CA 94080.




Cell Transplantation, Vol. 9, pp. 629-636, 2000
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Intraparenchymal NGF Infusions Rescue Degenerating Cholinergic Neurons

Mark H. Tuszynski

Department of Neurosciences, University of California-San Diego, La Jolla, CA 92093-0626
Veterans Affairs Medical Center, San Diego, CA 92161

Nerve growth factor (NGF) exerts both trophic (cell survival) and tropic (axonal growth-promoting) effects on several neuronal populations. In particular, its robust ability to prevent lesion-induced and spontaneous age-related basal forebrain cholinergic neuronal degeneration, and to promote mnemonic recovery, has suggested its potential use as a therapeutic agent in Alzheimer's disease. When infused intracerebroventricularly, however, NGF is associated with several adverse effects that make this delivery route impractical. The present study examined whether intraparenchymal infusions of NGF adjacent to cholinergic neuronal soma are an effective and well-tolerated means of providing NGF to degenerating cholinergic neurons. Cholinergic neuronal rescue together with axonal sprouting responses and local tissue damage in the brain were assessed in adult rats that underwent complete unilateral fornix transections, followed by intraparenchymal infusions of recombinant human NGF for a 2-week period. Intraparenchymal NGF infusions prevented the degeneration of 94.7 ± 6.6% of basal forebrain cholinergic neurons compared to 21.7 ± 2.6% in vehicle-infused animals (p < 0.0001). Cholinergic axons sprouted toward the intraparenchymal NGF source in an apparent gradient-dependent manner. Glial responses to intraparenchymal infusions were minimal, and no apparent toxic effects of the infusions were observed. Thus, when infused intraparenchymally, NGF rescues basal forebrain cholinergic neurons, alters the topography of axonal sprouting responses, and does not induce adverse affects over a 2-week infusion period. Intraparenchymal NGF delivery merits further study at longer term time points as a means of treating the cholinergic component of neuronal loss in Alzheimer's disease.

Key words: Nerve growth factor (NGF); Cholinergic system; Memory; Neurotrophic factors: Alzheimer's disease; Aging; Drug delivery; Regeneration

Address correspondence to Mark H. Tuszynski, M.D., Ph.D., Department of Neurosciences-0626, University of California-San Diego, La Jolla, CA 92093. Tel: (619) 534-8857; Fax: (619) 534-5220; E-mail: mtuszyns@ucsd.edu




Cell Transplantation, Vol. 9, pp. 637-656, 2000
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Initial Characterization of the Transplant of Immortalized Chromaffin Cells for the Attenuation of Chronic Neuropathic Pain

M. J. Eaton,1,2 M. Martinez,1 S. Karmally,1 T. Lopez,1 and J. Sagen2

1The Miami Project To Cure Paralysis, University of Miami School of Medicine, Miami, FL 33136
2Department of Neurological Surgery, University of Miami School of Medicine, Miami, FL 33136

Cultures of embryonic day 17 (E17) rat adrenal and neonatal bovine adrenal cells were conditionally immortalized with the temperature-sensitive allele of SV40 large T antigen (tsTag) and chromaffin cell lines established. Indicative of the adrenal chromaffin phenotype, these cells expressed immunoreactivity (ir) for tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines. At permissive temperature in vitro (33°C), these chromaffin cells are proliferative, have a typical rounded chromaffin-like morphology, and contain detectable TH-ir. At nonpermissive temperature in vitro (39°C), these cells stop proliferating and express increased TH-ir. When these immortalized chromaffin cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after a unilateral chronic constriction injury (CCI) of the rat sciatic nerve, they survived longer than 7 weeks on the pia mater around the spinal cord and continued to express TH-ir. Conversely, grafted chromaffin cells lost Tag-ir after transplant and Tag-ir was undetectable in the grafts after 7 weeks in the subarachnoid space. At no time did the grafts form tumors after transplant into the host animals. These grafted chromaffin cells also expressed immunoreactivities for the other catecholamine-synthesizing enzymes 7 weeks after grafting, including: dopamine-b-hydroxylase (DbH) and phenylethanolamine-N-methyltransferase (PNMT). The grafted cells also expressed detectable immunoreactivities for the opioid met-enkephalin (ENK), the peptide galanin (GAL), and the neurotransmitters g-aminobutyric acid (GABA) and serotonin (5-HT). Furthermore, after transplantation, tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI were significantly reduced during a 2-8-week period, related to the chromaffin cell transplants. The maximal antinociceptive effect occurred 1-3 weeks after grafting. Control adrenal fibroblasts, similarly immortalized and similarly transplanted after CCI, did not express any of the chromaffin antigenic markers, and fibroblast grafts had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that embryonic and neonatal chromaffin cells can be conditionally immortalized and will continue to express the phenotype of primary chromaffin cells in vitro and in vivo; grafted cells will ameliorate neuropathic pain after nerve injury and can be used as a homogeneous source to examine the mechanisms by which chromaffin transplants reverse chronic pain. The use of such chromaffin cell lines that are able to deliver antinociceptive molecules in models of chronic pain after nerve and spinal cord injury (SCI) offers a novel approach to pain management.

Key words: Chromaffin cells; Cell lines; Transplantation; Chronic constriction injury

Address correspondence to M. J. Eaton, Ph.D., The Miami Project To Cure Paralysis, University of Miami School of Medicine, 1600 N.W. 10th Avenue (R-48), Miami, FL 33136. Tel: (305) 243-6226; Fax: (305) 243-4427; E-mail: meaton@miami.edu




Cell Transplantation, Vol. 9, pp. 657-667 2000
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The X-gal Caution in Neural Transplantation Studies

J. Sanchez-Ramos,1,5,6 S. Song,1,5,6 M. Dailey,1,5 F. Cardozo-Pelaez,1,5,6 C. Hazzi,3,5 T. Stedeford,1,5 A. Willing,2,5 T. B. Freeman,2,5 S. Saporta,4,5 T. Zigova,2,5 P. R. Sanberg,2,5 and E. Y. Snyder7

Departments of 1Neurology, 2Neurosurgery, 3Pharmacology, 4Anatomy, and the 5Neuroscience Program, University of South Florida, Tampa FL
6James Haley VA Hospital, Tampa FL
7Departments of Neurology, Pediatrics, & Neurosurgery, Children's Hospital, Harvard Medical School, Boston MA

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme b-galactosidase (b-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X-gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully--with adequate optimization and individualization of various parameters (e.g., pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial b-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have emphasized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli b-gal activity.

Key words: lacZ; b-Galactosidase; X-gal cell tracking & identification; Neural transplantation; Histology

Address correspondence to Juan Sanchez-Ramos, Ph.D., M.D., Department of Neurology MDC 55, University of South Florida, 12901 Bruce B Downs Blvd., Tampa, FL 33612. Tel: (813) 974-5841; Fax: (813) 974-7200; E-mail: jsramos@com1.med.usf.edu




Cell Transplantation, Vol. 9, pp. 671-673 2000
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Treatment of Carbon Tetrachloride and Phenobarbital-Induced Chronic Liver Failure With Intrasplenic Hepatocyte Transplantation*

Naoya Kobayashi,1,2 Masahiro Ito,1 Junta Nakamura,1 Jin Cai,1 James M. Hammel,1 and Ira J. Fox1

1Department of Surgery, University of Nebraska Medical Center, Omaha, NE 68198
2First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan

Hepatocyte transplantation (HTx) has been shown to improve the survival of laboratory animals with experimentally induced acute liver failure and to ameliorate the physiologic abnormalities associated with liver-based metabolic deficiencies. However, the role of HTx in the treatment of liver cirrhosis (LC) has not been adequately studied. In order to address this issue, HTx was performed in rats following induction of stable LC using phenobarbital (PhB) and carbon tetrachloride (CCl4). Intrasplenic transplantation of 50 x 106 primary hepatocytes could significantly improve liver functions and prolong the survival of rats with irreversible, decompensated LC.

Key words: Hepatocyte transplantation; Liver cirrhosis; Carbon tetrachloride; Phenobarbital

Address correspondence to Ira J. Fox, M.D., Department of Surgery, University of Nebraska Medical Center, Omaha, NE 68198. Tel: (402) 559-4076; Fax: (402) 559-6107, or to Naoya Kobayashi, M.D., Ph.D., First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (+81) 86-235-7259; Fax: (+81) 86-235-8775.

*A more comprehensive review of the work can be found in Hepatology 31:851-857; 2000.




Cell Transplantation, Vol. 9, pp. 675-680, 2000
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Strong, Long-Term Transgene Expression in Rat Liver Using Chicken b-Actin Promoter Associated With Cytomegalovirus Immediate-Early Enhancer (CAG Promoter)

Motomichi Kosuga,1,3 Shin Enosawa,2 Xiao-Kang Li,2 Seiichi Suzuki,2 Nobutake Matsuo,3 Masao Yamada,1 Jayanta Roy-Chowdhury,4 Osamu Koiwai,5 and Torayuki Okuyama1,3

Departments of 1Genetics and 2Experimental Surgery, National Children's Medical Research Center, Tokyo, Japan
3Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan
4Departments of Medicine and Molecular Genetics, and the Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, NY
5Department of Science and Technology, Science University of Tokyo, Tokyo, Japan

For successful gene therapy in hepatic enzyme deficiencies, it is essential to use promoters that can maintain strong transcriptional activity for the long term in the liver. Using Gunn rats, a model animal for Crigler-Najjar syndrome type I, the long-term transcriptional function of the CAG promoter (a combination of chicken b-actin promoter and cytomegalovirus immediate-early enhancer) was evaluated in the rat liver. We constructed a plasmid pCAGGHUGT, containing expression cassettes of human bilirubin UDP-glucuronosyltransferase (BUGT) and hygromycin phosphotransferase, under the control of the CAG promoter and murine phosphoglycerate kinase promoter, respectively. Conditionally immortalized Gunn rat hepatocytes (IGRH), which had been established using mutant SV40 large T antigen (TST), were transfected with pCAGGHUGT. A stably transfected clone IGRHUGT, expressing a high level of BUGT, was obtained after selection with hygromycin. At 33°C, the cells doubled in number in approximately 72 h; however, at 37°C, cell proliferation stopped, indicating that the characteristic of temperature-dependent proliferation was retained in this clone. Ten million cells were injected into the spleen of syngeneic Gunn rats five times at 10-day intervals. Serum bilirubin levels were reduced by 45-50% at 70 days after the first transplantation and remained so throughout the duration of the study (120 days). These results suggested that the CAG promoter was able to maintain strong transcriptional activity in rat liver for at least 120 days.

Key words: Gunn rats; Immortalized hepatocytes; CAG promoter

Address correspondence to Torayuki Okuyama, Department of Genetics, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku Tokyo 154-8509, Japan. Tel: +81-3-3414-8121, ext. 2752; Fax: +81-3-3414-3208; E-mail: tora@nch.go.jp




Cell Transplantation, Vol. 9, pp. 681-686, 2000
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Establishment of Fluorescein Diacetate and Ethidium Bromide (FDAEB) Assay for Quality Assessment of Isolated Islets

Masaaki Miyamoto,1 Yoshihiko Morimoto,1 Yuka Nozawa,1 A. N. Balamurugan,1 Baoyou Xu,2 and Kazutomo Inoue1

1Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Japan

One of the most important factors in clinical islet transplantation is isolation of a great number of islets with good viability. According to viability assessments of isolated islets, the static incubation test and the perifusion test of islets, which are used retrospectively, take much time and need various apparatus. But viability assessments of isolated islets for clinical islet transplantation require a simple, rapid, sensitive, and prospective method. We have developed a microfluorometric viability assay for isolated human, porcine, and dog islets of pancreata using fluorescein diacetate and ethidium bromide (FDAEB). Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation (490 nm) and ethidium bromide (EB) causes dead cells to fluoresce red. In this study, we investigated the applicability of FDAEB staining to quality assessment of isolated islets for clinical use by correlation with the counting method with insulin secretion of islets. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDAEB staining. The proportion of living islets within isolated canine islets, as measured by microfluorometric counting, was found to correlate highly significantly on low-temperature (24°C) culture (R = 0.831, p < 0.001) and on 37°C culture (R = 0.553, p < 0.05) with the insulin contents of the same islets. Therefore, it is possible to differentiate degrees of viability, and a scoring system is described for this purpose. The FDAEB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation.

Key words: Islet transplantation; Fluorescein diacetate (FDA); Ethidium bromide (EB); Viability assay

Address correspondence to Prof. Kazutomo Inoue, Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin Sakyo-ku, Kyoto 606-8507, Japan. Tel: (81)75-751-4848; Fax: (81)75-751-4145; E-mail: K-inoue@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 9, pp. 687-692, 2000
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Phenotype Correction in Murine Mucopolysaccharidosis Type VII by Transplantation of Human Amniotic Epithelial Cells After Adenovirus-Mediated Gene Transfer

Motomichi Kosuga,1,3 Satori Takahashi,1,4 Kyoko Sasaki,1 Shin Enosawa,2 Xiao-Kang Li,2 Sanae Okuyama,2 Masayuki Fujino,2 Seiichi Suzuki,2 Masao Yamada,1 Nobutake Matsuo,3 Norio Sakuragawa,5 and Torayuki Okuyama1,3

Departments of 1Genetics and 2Experimental Surgery, National Children's Medical Research Center, Tokyo, Japan
3Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan
4Department of Science and Technology, Science University of Tokyo, Tokyo, Japan
5Department of Inherited and Metabolic Diseases, National Institute of Neuroscience, Tokyo, Japan

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human b-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10-15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.

Key words: Human amniotic cells; Cell therapy; Gene transfer; Mucopolysaccharidosis type VII

Address correspondence to Torayuki Okuyama, Department of Genetics, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan. Tel: +81-3-3414-8121, ext. 2752; Fax: +81-3-3414-3208; E-mail: tora@nch.go.jp




Cell Transplantation, Vol. 9, pp. 693-695, 2000
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Grafting of Mitomycin C-Treated Islet Xenograft Under the Kidney Capsule Produces a Clear Bleb

Yoshihiro Sato,1 Tadeusz R. Grochowiecki,2 Yutaka Takeda,2 Keizo Dono,2 Kazuya Ise,1 Yukio Kanazawa,1 Takuro Saito,1 Tsuyoshi Abe,1 and Mitsukazu Gotoh1

1Department of Surgery I, Fukushima Medical University, 1 Hikarigaoka Fukushima, 960-1295, Japan
2Department of Surgery II, Osaka University Medical School, 2-2 Yamadaoka, Suita 565-0871, Japan

We have previously shown that mitomycin C (MMC) treatment of donor tissue resulted in significant prolongation of graft survival in allo- and xenotransplantation models. However, the mechanisms involved in this prolongation are not clearly understood. This study aims to shed light on the immune responses to MMC-treated islet xenografting under the kidney capsule. Collagenase-digested WS (RT1k) rat islets incubated for 30 min with MMC and subsequently cultured for 20 h were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (B6;H-2b) mice. The grafts were harvested on postgrafting day 7 and sections were prepared and stained by hematoxylin and eosin (H&E). Histological study of the grafts in a group not treated with MMC showed marked cellular infiltration and destruction of islet clusters, whereas that of MMC-treated grafts demonstrated a bleb formation under the kidney capsule, in which islet cell clusters were reorganized, creating a layer of cells fixed to the interior of the bleb. Minimal invasion by inflammatory cells was observed only at the edge of the bleb, and most islet cells were protected from these infiltrating cells. In conclusion, MMC treatment induces remodeling of islet structure and forms a bleb under the kidney capsule, where no inflammatory cell infiltration occurs, suggesting that this site is a kind of immunologically privileged environment for xenografted islets.

Key words: Transplantation; Islet xenograft; Mitomycin; Remodeling

Address correspondence to Mitsukazu Gotoh, M.D., Ph.D., Professor & Chairman, Department of Surgery I, Fukushima Medical University, 1 Hikarigaoka Fukushima, 960-1295, Japan. Tel: +81-245-48-2111, ext. 2330; Fax: +81-245-48-2735; E-mail: mgotoh@cc.fmu.ac.jp




Cell Transplantation, Vol. 9, pp. 697-700, 2000
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Clonogenic Colony-Forming Ability of Flow cytometrically Isolated Hepatic Progenitor Cells in the Murine Fetal Liver

Hideki Taniguchi,1 Reika Kondo,1 Atsushi Suzuki,1 Yun-wen Zheng,1 Yasutsugu Takada,1 Kiyoshi Fukunaga,1 Ken-ichiro Seino,1 Kenji Yuzawa,1 Masaaki Otsuka,1 Katashi Fukao,1 and Hiromitsu Nakauchi2

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

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45-TER119- cells (the nonblood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45-TER119- cells in the murine fetal liver.

Key words: Liver; Stem cell; Flow cytometry; Cell isolation

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




Cell Transplantation, Vol. 9, pp. 701-704, 2000
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Inducing Proliferation of Human Amniotic Epithelial (HAE) Cells for Cell Therapy

Satoshi Terada,1 Keiko Matsuura,2 Shin Enosawa,3 Masao Miki,1 Akinori Hoshika,2 Seiichi Suzuki,3 and Norio Sakuragawa4

1Department of Applied Chemistry & Biotechnology, Faculty of Engineering, Fukui University, 3-9-1, Bunkyo, Fukui 910-8507, Japan
2Department of Pediatrics, Tokyo Medical University, 6-1-1, Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
3National Children's Medical Research Center, 3-35-31, Taishidou, Setagaya-ku, Tokyo, 154-8509, Japan
4Department of Inherited Metabolic Disorders, National Institute of Neuroscience, NCNP, 4-1-1, Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan

Probably because amnion is derived from the fetus and is exposed to the maternal immune system, human amniotic epithelial (HAE) cells do not express the HLA-A, -B, -C, or -DR antigens on their surfaces, suggesting that HAE cells do not induce rejection (immune reaction) after allotransplantation. And the amnion, like the placenta, is useless to the mother and child after birth. Therefore, HAE cells or tissues were expected to be suitable for allotransplantation. Because HAE cells produce large amounts of enzymes, amnion transplantation has been carried out in order to correct inborn errors of metabolism by supplementing lysosomal enzyme deficiencies. However, several problems remain before amnion allotransplantation can be accepted as effective. The HAE cell population is limited, because the maximum number of HAE cells obtainable from one donor is about 2 x 108 cells, and HAE cells proliferate poorly in in vitro culture. In this study, we aimed at increasing the HAE cell population in vitro. First, we investigated the effect of several cytokines on HAE cell proliferation and found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), and transforming growth factor-b stimulated it, whereas IL-6 and LIF inhibited it. Second, we investigated the effects of amniotic fluid on HAE cell proliferation and observed that IL-6 in amniotic fluid inhibits it. Then, to inhibit the dying of cells, we attempted to inhibit apoptosis (one mode of cell death). Treatment with caspase III inhibitor increased the cell viability of HAE cells by 20%.

Key words: Amnion; Hepatocyte growth factor; IL-6; Caspase inhibitor

Address correspondence to Satoshi Terada, Department of Applied Chemistry & Biotechnology, Faculty of Engineering, Fukui University, 3-9-1, Bunkyo, Fukui 910-8507, Japan. Tel: +81-776-27-8645; Fax: +81-776-27-8747; terada@acbio.fukui-u.ac.jp




Cell Transplantation, Vol. 9, pp. 705-709, 2000
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Grafting of Encapsulated Dopamine-Secreting Cells in Parkinson's Disease: Long-Term Primate Study

Isao Date, Tetsuro Shingo, Hideyuki Yoshida, Kenjiro Fujiwara, Kazuki Kobayashi, and Takashi Ohmoto

Department of Neurological Surgery, Okayama University Medical School, Okayama, Japan

The transplantation of encapsulated dopamine-secreting cells into the striatum represents one potential means of treating Parkinson's disease. The present study investigated the ability of encapsulated PC12 cells, which are derived from rat pheochromocytoma, to supply L-dopa and dopamine into the primate brain in the long term and to effect functional improvement in the animals. Following polymer encapsulation, PC12 cells were transplanted into the striatum of hemiparkinsonian monkeys. The secretion of L-dopa and dopamine from the encapsulated cells, the morphology of these cells, the histology of the host striatum surrounding the capsule, and functional changes in the host animals were examined 1, 6, and 12 months after transplantation. Analysis of retrieved capsules revealed that the PC12 cells survived and continued to release L-dopa and dopamine even 12 months after transplantation. The histological response of the host brain surrounding the capsules was minimal and there were no signs of immunological rejection or tumor formation. The physical condition of the host animals was good for 12 months, and hematologic and cerebrospinal fluid analysis revealed that no animals suffered from infection or immunological reaction. These PC12 cell-grafted monkeys showed improvements in hand movements after transplantation, effects that lasted for at least 12 months. These results further support the potential use of this approach for the treatment of Parkinson's disease.

Key words: Neural transplantation; Encapsulation; PC12 cells; Parkinson's disease

Address correspondence to Isao Date, M.D., Department of Neurological Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558 Japan. Tel: 086-235-7336; Fax: 086-227-0191; E-mail: idate333@med.okayama-u.ac.jp




Cell Transplantation, Vol. 9, pp. 711-715, 2000
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Long-Term Culture of Glutamine Synthetase-Transfected HepG2 Cells in Circulatory Flow Bioreactor for Development of a Bioartificial Liver

Shin Enosawa,1 Tomoyuki Miyashita,1 Seiichi Suzuki,1 Xiao-Kang Li,1 Miyuki Tsunoda,1 Hiroshi Amemiya,1 Mitsugu Yamanaka,2 Shinya Hiramatsu,2 Naoko Tanimura,2 Takeshi Omasa,2 Kenichi Suga,2 and Toshiharu Matsumura3

1Department of Experimental Surgery and Bioengineering, National Children's Medical Research Center, Tokyo, Japan
2Graduate School of Technology, University of Osaka, Osaka, Japan
3Meiji Institute of Health Science, Tokyo, Japan

Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity. After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes. The cells were cultured in a circulatory flow bioreactor for 109 days, while they multiplied from 5 x 107 to 4 x 109 cells. Three days after inoculation, the ammonia level of the culture medium was lowered to a level maintained thereafter, suggesting that using recombinant cell lines for bioartificial livers enables long-term repeated treatment for hepatic failure patient. Judging from the rate of decrease in the amount of the added ammonia, the ammonia removal capability of 4 x 109 GS-HepG2 cells was almost equivalent to 5 x 108 porcine hepatocytes inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-a (TNF-a). Cytokine removal therefore promises to be another useful property of bioreactor cells.

Key words: Bioartificial liver; HepG2; Glutamine synthetase; Ammonia

Address correspondence to Dr. S. Enosawa, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509 Japan. Tel: +81-3-3414-8121, ext. 2776; Fax: +81-3-3411-7309; E-mail: senosawa@nch.go.jp




Cell Transplantation, Vol. 9, pp. 717-724, 2000
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Neurite Growth Capability of Rat Fetal Neuronal Cells Against Matured CNS Myelin In Vitro

Koichi Hara,1 Koichi Uchida,2 Atsushi Fukunaga,2 Yoshiaki Kuroshima,2 Motoyuki Yamada,2 and Takeshi Kawase2

1Department of Neurosurgery, Ootawara Red Cross Hospital, Tochigi, Japan
2Department of Neurosurgery, Keio University School of Medicine, Tokyo, Japan

Reconstruction of neurocircuits by transplanted cells is expected to become an effective therapy for brain damage. In order to establish the transplantation therapy, it is necessary to find transplantable cells capable of reconstructing the lesioned neurocircuitry. We have reported that the younger neuronal cells such as neural stem cells are useful transplant materials because of their vigorous capacity for forming abundant neurites. On the other hand, it was reported that myelin-associated neurite growth inhibitor prevents neurite regeneration. In this study, we used rat fetal neuronal cells to examine the neurite growth capacity in the presence of mature CNS myelin. Crude CNS myelin was prepared from the brains of adult Wistar rats using previously described procedures. Testing wells were precoated with poly-L-lysine and additionally by overnight drying of a suspension containing 0, 5, 10, 15, or 20 mg/cm2 of the crude myelin protein. On embryonic days 10, 12, 15, and 17 (E10, E12, E15, and E17) embryos were surgically removed, mesencephalic neural plates were dissected out from the E10 embryos, and midbrain cells were taken from the E12, E15, and E17 embryos. The neural plates and midbrain cells were placed on the myelin-coated wells. After 24 h of culture (72 h in the case of neural plates), the number of surviving cells and the length of the neurites were examined immunocytochemically using anti-neurofilament (NF) antibody. Neurite length was measured by image analyzer Luzex-F. The mesencephalic neural plate was able to grow neurites even on 20 mg/cm2 central myelin. Almost the same number of midbrain cells attached themselves to the wells without myelin in every culture obtained from various stages of embryos. The number of cells attached on the myelin-coated wells decreased with the concentration of myelin. The number of NF-positive cells was higher in cultures of materials obtained from older embryos than in cultures obtained from younger embryos. The younger cells grew longer neurites than the older cells in the myelin noncoated wells. Neurite growth was inhibited strongly when the concentration of the central myelin was 10 mg/cm2 or greater, but on the 5 mg/cm2 myelin, the younger the cells were, the longer neurites they had. When the length of the longest neurites in one field of the image analyzer was further examined in the same way, the younger the cells were, the longer their axons grew on 0 and 5 mg/cm2 myelin. Thus, CNS myelin was seen to be a significant inhibitor of the recovery of injured neural tissue of the adult CNS. Younger cells grew longer neurites than older cells on CNS myelin, and so it was suggested that neural stem cells or younger neurons may serve as tissue for transplantation therapy.

Key words: Neurite growth; Fetal neuronal cells; Myelin-associated neurite growth inhibitor; Reconstruction of neurocircuits

Address correspondence to K. Hara, M.D., Department of Neurosurgery, Ootawara Red Cross Hospital, 2-7-3 Sumiyoshi-cho, Ootawara, Tochigi 324-8686, Japan. Tel: (+81) 287-23-1122, ext. 2567; Fax: (+81) 287-23-3004.




Cell Transplantation, Vol. 9, pp. 725-728, 2000
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Role of the Liver in Alloimmune Response Following Inoculation of Donor Spleen Cells

Li He,1 Keizo Dono,1 Mitsukazu Gotoh,2 Masaki Okumura,1 Yutaka Takeda,1 Junzo Shimizu,1 Hiroaki Nagano,1 Shoji Nakamori,1 Koji Umeshita,1 Masato Sakon,1 and Morito Monden1

1Department of Surgery II, Osaka University Medical School, Osaka 565-0871, Japan
2Department of Surgery I, Fukushima Municipal Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan

The liver is thought to be an immunologically privileged organ in the response to inoculated antigens. We previously demonstrated that it is possible to localize inoculated antigens to the liver alone or only to extrahepatic tissue using orthotopic syngeneic liver transplantation (OSLT). In this study, we analyzed more detailed mechanisms of the anti-alloimmune response in the liver. DA rat spleen cells were systemically injected into WS rats (donor spleen cell inoculation, DSI). In the sensitized liver-grafted (SLG) group, after DSI, liver grafts were retrieved from sensitized WS rats, then transplanted into naive WS rats. In the sensitized liver-removed (SLR) group, after DSI, WS rats were totally hepatectomized and given livers transplanted from naive WS rats. All the rats were challenged with heterotopic heart grafts 10 days after DSI. Mean heart graft survival in the control, DSI, SLG, and SLR groups were 11.6 ± 1.6, 10.7 ± 2.4, 4.4 ± 1.0, and 24.6 ± 6.3 days, respectively. Accelerated rejection in the SLG group as well as graft prolongation in the SLR group disappeared when OSLT was performed 2 days after DSI or later. Irradiation of DA splenocytes before inoculation did not alter graft survival in SLG. However, pretreatment with gadolinium chloride prior to DSI attenuated the antidonor response in the SLG group. In conclusion, a vigorous antidonor response occurred in the liver after systemic inoculation of spleen cells. It peaked 1 day after DSI and disappeared rapidly. Kupffer cells seemed to play an important role in this phenomenon.

Key words: Rat orthotopic syngeneic liver transplantation; Alloimmune response; Kupffer cell; Donor antigen inoculation

Address correspondence to Morito Monden, M.D., Ph.D., Department of Surgery II, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel: +81 (6) 6879-3251; Fax: +81 (6) 6879-3259; E-mail: monden@surg2.med.osaka-u.ac.jp




Cell Transplantation, Vol. 9, pp. 729-732, 2000
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Modified Subcutaneous Tissue With Neovascularization Is Useful as the Site for Pancreatic Islet Transplantation

Yoshiyuki Kawakami,1 Hiroo Iwata,3 Yuanjung Gu,2 Masaaki Miyamoto,2 Yoshinobu Murakami,3 Toru Yamasaki,1 Wanxing Cui,1 Yoshito Ikada,3 Masayuki Imamura,1 and Kazutomo Inoue2

1Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, 54-Shogoin Kawara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
2Department of Organ Reconstruction and 3Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, 53-Shogoin Kawara-cho, Sakyo-ku, Kyoto, 606-8507, Japan

The success rate of subcutaneous transplantation of pancreatic islets has been extremely low. Insufficient oxygen supply to the grafted islets is one possible major obstacle to the preservation of graft function. This study attempted to use basic fibroblast growth factor (bFGF) in subcutaneous transplantation to induce neovascularization and a sufficient blood flow around the space formed for grafted islets in the subcutaneous tissues. A bFGF-releasing device was designed enclosing bFGF in a polyethylene terephthalate mesh bag coated with polyvinylalcohol hydrogel. In the vascularized group (n = 5), two bFGF-releasing devices were implanted bilaterally into the subcutaneous tissue of the back of streptozotocin-induced diabetic Lewis rats. One week after implantation, isolated rat islets (5000) were syngeneically transplanted subcutaneously after the removal of the devices. In the control group (n = 5), no devices were implanted and the same number of rat islets was transplanted directly. One week after the implantation of the devices into the test animals, a thick, well-vascularized capsule was observed in the subcutaneous site. All vascularized recipient rats showed significant decreases in nonfasting blood glucose and maintained normoglycemia for more than 1 month after islet transplantation. However, in the control group, all rats failed to achieve normoglycemia after transplantation. This study provides evidence that the subcutaneous tissue is a promising site for pancreatic islet transplantation, offering convincing advantages in acceptability for diabetic recipients. Establishment of this subcutaneous islet transplantation technique will afford some new perspectives on successful clinical islet transplantation.

Key words: Subcutaneous pancreatic islet transplantation; Basic fibroblast growth factor; Angiogenesis; Transplantation site

Address correspondence to Kazutomo Inoue, Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53-Shogoin Kawara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. Tel: (81)75-751-4856; Fax: (81)75-751-4145; E-mail: K-inoue@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 9, pp. 733-735, 2000
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Treatment of Surgically Induced Acute Liver Failure With Transplantation of Highly Differentiated Immortalized Human Hepatocytes

Naoya Kobayashi,1,2 Masahiro Miyazaki,1 Kenichi Fukaya,1 Yusuke Inoue,1 Masakiyo Sakaguchi,1 Hirofumi Noguchi,2 Toshihisa Matsumura,2 Takamasa Watanabe,2 Toshinori Totsugawa,2 Noriaki Tanaka,2 and Masayoshi Namba1

1Department of Cell Biology, Institute of Molecular and Cellular Biology, and 2First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan

Primary human hepatocytes are an ideal source of hepatic function in bioartficial liver (BAL), but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, primary human fetal hepatocytes were immortalized using simian virus 40 large T antigen. One of the immortal cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplantation of OUMS-29 cells protected 90% hepatectomized rats from hyperammonemia and significantly prolonged their survival. Essentially unlimited availability of OUMS-29 cells supports their clinical use for BAL treatment.

Key words: Immoralized human hepatocytes; Hepatocyte transplantation; Simian virus 40 large T antigen; Acute liver failure

Address correspondence to Masayoshi Namba, M.D., Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: (+81) 86-235-7393; Fax: (+81) 86-235-7400; E-mail: mnamba@med.okayama-u.ac.jp or to Naoya Kobayashi, M.D., Ph.D., First Department of Surgery, Okayama University Medical School, 2-5-1-Shikata-cho, Okayama, 700-8558, Japan. Tel: (81)86-235-7259; Fax: (81)86-235-8775; E-mail: ntanaka@med.okayama-u.ac.jp




Cell Transplantation, Vol. 9, pp. 737-742, 2000
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Efficient Cre/loxP Site-Specific Recombination in a HepG2 Human Liver Cell Line

Naoya Kobayashi,1 Hirofumi Noguchi,1 Karen A. Westerman,2 Toshihisa Matsumura,1 Takamasa Watanabe,1 Toshinori Totsugawa,1 Toshiyoshi Fujiwara,1 Philippe Leboulch,2 and Noriaki Tanaka1

1First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2Harvard-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, MA 02139

A worldwide shortage of donor livers is a limiting factor of the clinical application of hepatocyte transplantation (HTX). To resolve this issue, we focused on a reversible immortalization system that allows temporary expansion of primary hepatocyte populations by transfer of an oncogene that can be subsequently excised. As a preliminary test toward this goal, we examined the efficacy of Cre/loxP site-specific recombination in a transformed human liver cell line, HepG2. The present study utilized retroviral transfer of a prototypical immortalizing gene, simian virus 40 large T antigen (SV40Tag), flanked by a pair of loxP recombination targets and adenovirus-mediated Cre/loxP recombination. Here we report that complete elimination of the retroviral transferred oncogene was achieved by site-specific recombination using a replication-deficient recombinant adenovirus vector producing Cre recombinase (Ad-Cre).

Key words: HepG2 cells; Reversible immortalization; Cre/loxP site-specific recombination

Address correspondence to Naoya Kobayashi, M.D., Ph.D., First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (+81) 86-235-7259; Fax: (+81) 86-235-8775; E-mail: ntanaka@med.okayama-u.ac.jp