|ognizant Communication Corporation|
VOLUME 10, NUMBER 1, 2001
Cell Transplantation, Vol. 10, pp. 3-24, 2001
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Update on Immunoisolation Cell Therapy for CNS Diseases
Dwaine F. Emerich and Heather C. Salzberg
Department of Neuroscience, Alkermes, Inc, 64 Sidney Street, Cambridge MA 02139
Delivery of potentially therapeutic drugs to the brain is hindered by the blood-brain barrier (BBB), which restricts the diffusion of drugs from the vasculature to the brain parenchyma. One means of overcoming the BBB is with cellular implants that produce and deliver therapeutic molecules. Polymer encapsulation, or immunoisolation, provides a means of overcoming the BBB to deliver therapeutic molecules directly into the CNS region of interest. Immunoisolation is based on the observation that xenogeneic cells can be protected from host rejection by encapsulating, or surrounding, them within an immunoisolatory, semipermeable membrane. Cells can be enclosed within a selective, semipermeable membrane barrier that admits oxygen and required nutrients and releases bioactive cell secretions, but restricts passage of larger cytotoxic agents from the host immune defense system. The selective membrane eliminates the need for chronic immunosuppression of the host and allows the implanted cells to be obtained from nonhuman sources. In this review, cell immunoisolation for treating CNS diseases is updated from considerations of device configurations, membrane manufacturing and characterization in preclinical models of Alzheimer's and Huntington's disease.
Key words: Immunoisolation; Blood-brain barrier; CNS diseases
Address correspondence to Dwaine F. Emerich, Ph.D., Department of Neuroscience, Alkermes, Inc., 64 Sidney Street, Cambridge, MA 02139. Tel: (617) 494-0171; Fax: (617) 494-9263.
Neural Xenotransplantation: Pretreatment of Porcine Embryonic Nigral Tissue With Anti-Gal Antibodies and Complement Is Not Toxic for the Dopaminergic Neurons
Thomas Brevig,1,2 Morten Meyer,1 Tom Kristensen,2 and Jens Zimmer1
1Department of Anatomy and Neurobiology, University of Southern
Denmark, DK-5000 Odense C, Denmark
2Department of Clinical Immunology, Odense University Hospital, DK-5000 Odense C, Denmark
The immunogenicity of porcine tissue is a major obstacle to its use as donor material in xenotransplantation for neurodegenerative diseases. We are currently evaluating a novel strategy for reducing the immunogenicity, in which the a-galactosyl epitope (Gala1,3Galb1,4GlcNAc-R) is used as a target for antibody- and complement-mediated removal of microglia. In the present study, our aim was to determine whether a pretreatment with antibodies against the a-galactosyl epitope (anti-Gal) and complement would lyse or otherwise damage dopaminergic neurons in porcine embryonic ventral mesencephalon (VM), the donor tissue for treatment of Parkinson's disease by xenotransplantation. Cell suspensions prepared from VM tissue from 27-day-old pig embryos were incubated with anti-Gal, purified from normal human serum by affinity chromatography, or medium only (control), and subsequently with rabbit complement. After these pretreatments, the cell suspensions were transplanted into the right striatum of 14 adult rats (two groups of 7 animals). The animals were sacrificed 20 days after transplantation, the brains were processed for histology, and the sections were stained for Nissl substance, porcine neurofilament, tyrosine hydroxylase, and rat CD45 to determine graft volume, presence of porcine neurons, content of dopaminergic cells, and leukocyte infiltration, respectively. The VM tissue pretreated with anti-Gal and complement gave rise to dopaminergic grafts that were indistinguishable from those derived from VM tissue given the control pretreatment. In 5 of the 14 animals, the grafts were infiltrated by host leukocytes, but in two of these recipients, the infiltration was only minimal. We conclude that anti-Gal and complement can be applied to porcine embryonic VM tissue without damaging the dopaminergic neurons and their precursors.
Key words: Parkinson's disease; Xenografts; Porcine donor tissue; Ventral mesencephalon; Graft pretreatment; Dopaminergic neurons
Address correspondence to Thomas Brevig, Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Winsløwparken 21, DK-5000 Odense C, Denmark. Tel: +45 65503856; Fax: +45 65906321; E-mail: email@example.com
Adult Bone Marrow Transplantation After Stroke in Adult Rats
Yi Li,1 Jieli Chen,1 and Michael Chopp1,2
1Department of Neurology, Henry Ford Health Sciences Center,
Detroit, MI 48202
2Department of Physics, Oakland University, Rochester, MI 48309
We transplanted adult whole bone marrow prelabeled with bromodeoxyuridine (BrdU) into the ischemic boundary zone of the adult rat brain at 1 day after 2 h of middle cerebral artery occlusion (MCAo). Approximately 3.3% of 106 transplanted bone marrow cells were BrdU reactive at 14 days after MCAo. BrdU-reactive cells expressed neuronal and astrocytic proteins, neuronal nuclei protein (NeuN, 1%), and glial fibrillary acidic protein (GFAP, 5%) immunoreactivities, respectively. In addition, bone marrow transplantation promoted proliferation of ependymal and subependymal cells, identified by nestin (a neuroepithelial stem cell marker), within the ventricular zone and subventricular zone (VZ/SVZ). These data suggest that intracerebral transplantation of bone marrow could potentially be used to induce plasticity in ischemic brain.
Key words: Bone marrow; Transplantation; Stroke; Rat
Address correspondence to Michael Chopp, Ph.D., Henry Ford Hospital, Neurology Department, 2799 West Grand Boulevard, Detroit, MI 48202. Tel: (313) 916-3936; Fax: (313) 916-1318; E-mail: firstname.lastname@example.org
Vandana Zaman, Dennis A. Turner, and Ashok K. Shetty
Departments of Surgery (Neurosurgery) and Neurobiology, Duke University
Medical Center, Durham, NC 27710
Medical Research and Surgery (Neurosurgery) Services, Veterans Affairs Medical Center, Durham, NC 27705
Fetal hippocampal CA3 cell grafts exhibit dramatically enhanced survival when transplanted at an early postlesion delay of 4 days into the lesioned CA3 region of adult hippocampus. However, survival of these homotopic grafts following placement at late postlesion time points when the host milieu is considerably less receptive to grafts is unknown. We hypothesize that an extended postlesion delay at the time of grafting will lead to significant diminution in cell survival of both homotopic and heterotopic fetal transplants grafted to lesioned adult CNS. We quantitatively investigated absolute cell survival of 5´-bromodeoxyuridine-labeled fetal hippocampal CA3 and CA1 cell grafts, following transplantation into the lesioned CA3 region of adult rat hippocampus, at a delay of 45 days after a unilateral intracerebroventricular administration of kainic acid (KA). Survival of these grafts was also analyzed in intact CA3 of the hippocampus contralateral to KA administration for comparison. In lesioned CA3 region, CA3 (homotopic) and CA1 (heterotopic) grafts exhibited comparable but only moderate survival, with a recovery of only 21-31% of injected cells. Cell survival in these grafts into lesioned hippocampus was similar to survival of grafts placed into the contralateral intact CA3 region. These results are in sharp contrast to increased graft survival measured following transplants performed at 4 days postlesion. In such grafts placed early, there was both a significantly higher cell survival than grafts placed into the intact CA3 region and also a characteristic differential survival based on graft cell specificity to the lesioned CA3 region (Zaman et al., Exp. Neurol., 161:535-561, 2000). Thus, the enhanced conduciveness of lesioned CA3 region for survival of homotopic CA3 cell grafts observed at 4 days postlesion wanes by 45 days postlesion to that of intact CA3 region, in spite of residual loss of CA3 neurons with the lesion. Strategies that considerably augment graft cell survival may therefore be critical for optimal integration of fetal grafts into the adult CNS at late postlesion time points.
Key words: 5´-Bromodeoxyuridine; Graft cell survival; Graft integration; Hippocampus; Kainic acid; Neural transplantation
Address correspondence to Ashok K. Shetty, M.Sc., Ph.D., Division of Neurosurgery, Box 3807, Duke University Medical Center, Durham NC 27710. Tel: (919) 286-0411, Ext. 7348; Fax: (919) 286-4662; E-mail: Ashok.Shetty@Duke.Edu
Delta Opioid Peptide Augments Functional Effects and Intrastriatal Graft Survival of Rat Fetal Ventral Mesencephalic Cells
Cesario V. Borlongan, Tsung-Ping Su, and Yun Wang
Cellular Neurobiology, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224
Delta enkephalin analogue [D-Ala(2),D-Leu(5)]enkephalin (DADLE) has been shown to protect dopamine transporters from methamphetamine-induced neurotoxicity. In the present study, we demonstrate that exposure of embryonic ventral mesencephalic cells to DADLE (0.01 g/ml), prior to intrastriatal transplantation, enhanced functional recovery and graft survival in 6-hydroxydopamine-induced hemiparkinsonian rats. At 6 and 8 weeks posttransplantation, animals that received DADLE-treated cell grafts exhibited significantly higher (near normal) spontaneous locomotor behaviors, as well as trends of greater reversal of motor asymmetrical behaviors compared with animals that received nontreated cell grafts. Histological examination revealed that animals transplanted with DADLE-treated cell grafts exhibited about twice the number of surviving tyrosine hydroxylase-immunoreactive grafted neurons compared with those animals that received nontreated cell grafts. These results suggest that DADLE should be considered as an adjunctive agent for neural transplantation therapy in Parkinson's disease.
Key words: Parkinson's disease; Neural transplantation; Fetal mesencephalic cells; Substantia nigra; Striatum; 6-Hydroxydopamine; Tyrosine hydroxylase; Locomotor behavior
Address correspondence to Cesario V. Borlongan, Cellular Neurobiology, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Drive, Baltimore, MD 21224. Tel: (410) 550-2719; Fax: (410) 550-1621; E-mail: email@example.com
Tao Fu,1 Danqing Guo,1 Xuemei Huang,2 Maurice R. G. O'Gorman,3 Lijun Huang,2 Susan E. Crawford,2 and Humberto E. Soriano1
1Department of Pediatrics, 2Department of Pathology, and 3Department of Immunology, Northwestern University Medical School, Children's Memorial Hospital, Chicago, IL 60614
Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.
Key words: Cryostorage; Lactate dehydrogennase release; Cell attachment; Necrosis; Apoptosis
Address correspondence to Humberto E. Soriano, M.D., Department of Pediatrics, Northwestern University Medical School, Children's Memorial Hospital, 2430 N. Halsted Street, Box #221, Chicago, IL 60614. Tel. (773) 880-8308; Fax: (773) 975-8671; E-mail: firstname.lastname@example.org
Pascale David,1 Eliane Alexandre,1 Maxime Audet,2 Marie-Pierre Chenard-Neu,3 Philippe Wolf,1,2 Daniel Jaeck,1,2 Agnès Azimzadeh,1 and Lysiane Richert1,4
1Laboratoire de Chirurgie Expérimentale, Fondation
Transplantation, 5, avenue Molière, 67200 Strasbourg, France
2Centre de Chirurgie Viscérale et de Transplantation, Hôpital de Hautepierre, Avenue Molière, 67098 Strasbourg, France
3Service d'Anatomo-Pathologie, Hôpital de Hautepierre, Avenue Molière, 67098 Strasbourg, France
4Laboratoire de Biologie Cellulaire, Faculté de Pharmacie, 4, Place Saint Jacques, 25030 Besançon, France
Banking of cryopreserved hepatocytes is a prerequisite for large-scale hepatocyte transplantation in the clinic. We compared the efficacy of intrasplenic transplantation into Nagase analbuminemic rats (NAR) of freshly isolated (FIH) and cryopreserved (CH) hepatocytes. Hepatocytes were cryopreserved using a controlled rate freezing protocol. Albumin production of thawed CH and FIH was measured in vitro in culture by ELISA and by Western blot. After in vivo intrasplenic transplantation of NAR with either FIH or CH we assessed 1) albumin in the serum of recipients by ELISA and by Western blotting analysis at different time intervals, and 2) hepatocyte engraftment by albumin immunohistochemical staining into spleens and livers at euthanasia. In vitro, albumin was produced up to day 4 of culture in both CH and FIH. In vivo, no intrasplenic engraftment of hepatocytes occurred. Intrahepatic engraftment of CH (cell number/mm2) was significantly (twofold) lower than that of FIH and appeared only as isolated cells and small (<10 cells) clusters, while bigger clusters (>10 cells) were observed with FIH. In the FIH group, serum albumin production was observed up to 32-49 days posttransplantation while in the CH group no serum albumin production was detected. Our results emphasize the need to improve 1) hepatocyte transplantation procedures either by repeated hepatocytes injections and/or by transplantation under a regeneration response, and 2) the freeze/thaw protocols of hepatocytes.
Key words: Freshly isolated hepatocytes; Cryopreserved hepatocytes; Transplantation; Analbuminemic rats
Address correspondence to Prof. Lysiane Richert, Fondation Transplantation
5, Avenue Molière, 67200 Strasbourg, France. Tel: (33) 3 88 26 06 26; Fax: (33) 3 88 26 12 26; E-mail: email@example.com
Satdarshan P. S. Monga,1 Yi Tang,1 Fabio Candotti,2 Asif Rashid,3 Oliver Wildner,2 Bibhuti Mishra,2 Shareen Iqbal,1 and Lopa Mishra1
1Laboratory of GI Development and Molecular Biology, DVAMC,
Washington, DC 20422, and Fels Cancer Institute, Temple University, Philadelphia,
2Clinical Gene Therapy Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892
3GI Pathology, Johns Hopkins University Hospital, Baltimore, MD 21287
Ex vivo embryonic liver explant culture is a novel and attractive approach to obtain abundant hepatic and hematopoietic stem cells. Gene therapy of autologous hepatic and hematopoietic stem cells represents an alternative therapeutic approach to liver transplantation for genetic and metabolic disorders. In this study we characterize the growth and differentiation of hepatic stem cells utilizing embryonic liver cultures. Day 9.5 liver buds are microdissected and cultured under specific conditions. Modulation of growth conditions by addition of hepatocyte growth factor, Flt-3 ligand, and stem cell factor leads to enrichment of hepatic progenitor cells in embryonic liver explants. Under these conditions, we also demonstrate the role of a novel marker PRAJA-1 to identify hepatic stem cells and transitional hepatocytes. Utilization of dexamethasone enhanced pseudolobule formation with increased hepatocytic and biliary differentiation. Transforming growth factor-b leads to enrichment of biliary cells in the culture. Gut formation is enhanced in the presence of interleukin-3 and blood formation by increasing the mesodermal tissue in these cultures. We also show increased retroviral-mediated expression of the green fluorescent protein expression in the expanded hepatic and hematopoietic stem cells under different culture conditions. Thus, the embryonic liver explant culture is an attractive source for hepatic progenitors and is a possible step towards generating nontumorigenic immortalized hepatocytes with possible transplantation applications.
Key words: Liver; Development; Stem cells; Culture; Explant; Gene therapy
Address correspondence to Lopa Mishra, M.D., 151 DVAMC, 50 Irving street, N.W., Washington, DC 2042. Tel: (202) 745-8326; Fax: (202) 462-2006; E-mail: firstname.lastname@example.org
Ann W. Funkhouser,1 Sabera Vahed,1 and Humberto E. Soriano2
1University of Chicago, Department of Pediatrics, University
of Chicago Children's Hospital, Chicago, IL 60637
2Northwestern University, Department of Pediatrics, Children's Memorial Hospital, Chicago, IL 60614
Xenotransplantation of human liver cells is an expanding field in need of new and precise quantitative techniques. "Real time" PCR is a sensitive and accurate method of quantifying picogram quantities of DNA. We used "real time" PCR with primers complementary to the human a-1-antitrypsin gene to assess the efficiency of engraftment of human liver cells transplanted into immunotolerant RAG-1-/- mice. Standard curves were created by mixing known proportions of human and mouse cells. There was a linear relationship between the PCR cycle at which DNA was amplified [threshold cycle (CT)] and the percent human cells (linear regression, p < 0.00009). Results were reliable, with a maximum 1.27-fold variation in the slopes of repeated standard curves. Linearity was maintained from 100% to as low as 0.01%. Therefore, 1 in 10,000 mouse cells could be detected in a 100 ng DNA sample. We measured the percent engraftment of human liver cells transplanted into the spleen of RAG-1-/- mice. By "real time" PCR assay, 0.23% human cells could be detected at 1 day after human cell transplantation. These results show that "real time" PCR assay is highly sensitive, reproducible, and accurate for detecting human cells in xenotransplanted mice.
Key words: Hepatocyte transplantation; Xenotransplantation; "Real time" RCR; Human liver cells; RAG-1-/- mice
Address correspondence to Ann W. Funkhouser, M.D., Department of Pediatrics, 5841 S. Maryland Ave., MC 6054, Chicago, IL 60637. Tel: (773) 834-3177; Fax: (773) 702-1196; E-mail: email@example.com
Heather A. Clayton,1 Joanna E. Davies,1 Chris D. Sutton,1 Peter R. F. Bell,2 and Ashley R. Dennison1
1Department of General Surgery, Leicester General Hospital,
Leicester, LE5 4PW, UK
2Department of Surgery, University of Leicester, Leicester Royal Infirmary, Leicester, LE2 7LX, UK
Clinical and experimental studies of intrahepatic islet transplantation have allowed histological and systemic observations to be made, but the location of the transplanted islets makes it difficult to assess direct effects on the cells of the liver. An in vitro coculture model of Kupffer cells with islets or pancreatic acinar tissue is described, using porcine tissue and measuring the secretion of thromboxane B2, prostaglandin E2, 6-keto-prostaglandin F1a, and prostaglandin F2a as an indicator of Kupffer cell stimulation. The results have demonstrated activation of Kupffer cells in the presence of acinar or islet tissue, both when the cells were in direct contact and when separated by a membrane. This indicated that the stimulation was due to a soluble factor or factors, and was confirmed by the culture of Kupffer cells with acinar conditioned medium. The degree of stimulation was much greater with acinar tissue than with islets. In subsequent experiments, aprotinin, an enzyme activation inhibitor, was added to the cocultures in an attempt to reduce Kupffer cell activation. This had no effect, possibly due to the fact that the endogenous pancreatic enzymes may already be activated during digestion of the pancreas. Aprotinin alone caused an increase in secretion of eicosanoids from Kupffer cells. The high response to acinar tissue is of particular relevance to islet autotransplantation in which unpurified pancreatic digest is often transplanted. The clinical effectiveness of aprotinin in the light of these results is discussed. In conclusion, although unable to mimic the complex situation following intrahepatic islet transplantation, the coculture model described here allows the opportunity to assess the events relating to specific cell types, and will provide the scope to undertake more detailed studies on the mechanisms involved. The same model could be applied to the coculture of pancreatic tissue with hepatocytes to determine any effects on the normal function of hepatocytes.
Key words: Kupffer cells; Islet autotransplantation; Coculture; Acinar tissue; Aprotinin
Address correspondence to Dr. Heather A. Clayton, Department of General Surgery, Ward 16, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK. Tel: 00 44 116 258 8113; Fax: 00 44 116 258 4708; E-mail: firstname.lastname@example.org
Human Pancreatic Ductal Cells: Large-Scale Isolation and Expansion
Valéry Gmyr,1 Julie Kerr-Conte,1 Brigitte Vandewalle,3 Charles Proye,2 Jean Lefebvre,3 and François Pattou2
1Laboratories of Cell Culture, 2Department of General and Endocrine Surgery, and 3UPRES 1048 University of Lille 2, University Hospital Center of Lille, Lille, France
The in vitro differentiation of pancreatic stem cells has recently been shown to represent a new source of b cells for cell therapy in diabetes. Human ductal cell differentiation, in vitro, has been documented in three-dimensional (3D) culture and recently substantiated. Although encouraging, the optimization of the ductal cell source, expansion and differentiation ex vivo are mandatory for clinical relevance. We compared three sources of human ductal cells (hDC) (method A1-2, B, and C). The classical main duct isolation of hDC by explant (A1), or enzymatic digestion (A2), was compared with two indirect methods: from 3D cultured human islet/duct-enriched fractions (B) and dedifferentiated exocrine fractions (C). Method A: few viable hDC were obtained from the main duct. Method B: embedding islet/duct rich fraction in 3D collagen gels expands the cytokeratin 19 (CK19)-positive ductal component in the form of ductal cysts, as we described previously; monolayers derived from digested cysts were 80% ductal (CK19). Method C: initially adherent amylase-positive exocrine clusters contained 12% (CK19) to 22% (CK7) ductal cells. One-week exocrine cultures were amylase negative and 46% (CK19) to 63% (CK7) ductal. Cell viability varied: <20% (A1), 81 ± 12% (B), 91 ± 2% (C). Extrapolating total yields we obtained (±SEM): 10.5 ± 4.6 x 103 (A1), 36 ± 18 x 103 (A2), 292 ± 50 x 106 (B), 1696 ± 526 x 106 (C) viable hDC per pancreas. A secondary monolayer expansion of cyst-derived hDC (method B) was achieved with NuSerum® (4.2-fold on plastic, 2.6-fold on 804G matrix; p < 0.05 vs. control cells on plastic). First passage exocrine-derived ductal cells also responded to matrix and to growth factors, albeit not significantly. In conclusion, this study demonstrated that an abundant hDC supply can be obtained from islet/duct or exocrine fractions followed by monolayer expansion with NuSerum. If their differentiation capacity is confirmed, in particular exocrine-derived ductal cells may represent a promising abundant source of islets for allogenic and autologous diabetes cell therapy.
Key words: Culture; Human; Ductal cells; Pancreas
Address correspondence to Dr. Julie Kerr-Conte, Laboratory of Cell
Culture, University Hospital Center of Lille, 1 Place de Verdun, Lille
59045 France. Tel: 333 20 62 69 63; Fax: 333 20 62 69 83; E-mail: email@example.com