|ognizant Communication Corporation|
VOLUME 11, NUMBER 1, 2002
Cell Transplantation, Vol. 11, pp. 5-16, 2002
0963-6897/02 $20.00 + 00
Copyright © 2002 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Lone M. Veng,1,3 Kimberly B. Bjugstad,4 Curt R. Freed,1,2,3,4 Philippa Marrack,5 Edward D. Clarkson,4 K. Patricia Bell,4 Cindy Hutt,4 and W. Michael Zawada1,2,4
1Neuroscience Program, Departments of 2Medicine
and 3Pharmacology, 4Division of Clinical Pharmacology,
University of Colorado School of Medicine, Denver, CO 80262
5Howard Hughes Medical Institute, National Jewish Medical and Research Center, Denver, CO 80206
The limited availability of human embryonic tissue for dopamine cell transplants in Parkinson's patients has led to an increased interest in using xenogeneic donor tissue. Unfortunately, without aggressive immunosuppression, such brain xenografts are rejected by the host immune system. Chronic brain xenograft rejection is largely mediated by helper T cells, which require presentation of xenoantigens by major histocompatability complex (MHC) class II for their activation. We examined survival and function of xenografts of E13 mouse mesencephalon deficient in either MHC class I, class II, or both after transplantation into adult hemiparkinsonian rats without immunosuppression. Recipients received grafts from C57BL/6 mice that were either: 1) wild-type (wt), 2) MHC class I knockout (KO), 3) MHC class II KO, 4) MHC class I and II double KO, or 5) saline sham transplants. At 6 weeks after transplantation, recipients of MHC class I KO, class II KO, and double KO xenografts significantly reduced methamphetamine-induced circling rate while rats with wt xenografts and sham-operated rats showed no improvement. MHC class II KO grafts had the greatest number of surviving dopamine neurons. All transplants, including saline sham controls, contained infiltrating host MHC class II-positive cells. Saline sham grafts and MHC class II KO xenografts contained significantly fewer infiltrating host MHC class II-positive cells than did wt grafts. Our results show that MHC class II-deficient xenografts survive transplantation for at least 6 weeks in the absence of immunosuppression, reduce rotational asymmetry, and provoke lesser immune reaction than wt grafts.
Key words: Neural transplantation; Xenotransplantation; Immunosuppression; Major histocompatability complex (MHC); Parkinson's disease; Dopamine
Address correspondence to W. Michael Zawada, Ph.D., Division of Clinical Pharmacology, C-237, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Tel: (303) 315-8455; Fax: (303) 315-3272; E-mail: Mike.Zawada@UCHSC.edu
Intrathecal Spinal Progenitor Cell Transplantation for the Treatment of Neuropathic Pain
Chung-Ren Lin,1,2 Ping-Ching Wu,1 Hsun-Chang Shih,1 Jiin-Tsuey Cheng,2 Cheng-Yuan Lu,1 An-Kuo Chou,1 and Lin-Cheng Yang1
1Department of Anesthesiology, Anesthesiology Research Laboratory,
Kaohsiung Chang Gung Memorial Hospital 833, Taiwan
2Department of Biological Sciences, National Sun-Yat Sen University, Taiwan
Injury to, or dysfunction of, the nervous system can lead to spontaneous pain, hyperalgesia, and/or allodynia. It is believed that the number and activity of GABAergic neurons gradually decreases over the dorsal horn. Glutamic acid decarboxylase (GAD) immunocompetence has been demonstrated on spinal progenitor cells (SPCs) cultivated in vitro. The intrathecal implantation of these cultivated progenitor cells may provide a means of alleviating neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the hind paw of rats. SPCs (1 x 106) were implanted intrathecally on the third day after the CCI surgery. The behavioral response to thermal hyperalgesia was observed and recorded during the 14 days postsurgery. Various techniques were utilized to trace the progenitor cells, confirm the differentiation, and identify the neurotransmitters involved. GAD immunoactivity was revealed for 65% of the cultivated spinal progenitor cells in our study. We also determined that transplanted cells could survive more than 3 weeks postintrathecal implantation. Significant reductions were demonstrated for responses to thermal stimuli for the CCI rats that had received intrathecal SPC transplantation. A novel intrathecal delivery with SPCs reduced CCI-induced neuropathic pain.
Key words: Cell transplantation; Chronic constriction injury; Neuropathic pain; Spinal progenitor cell; Spinal cord; Microdialysis
Address correspondence to Lin-Cheng Yang, M.D., Associate Professor,
Department of Anesthesiology, Chang Gung Memorial Hospital, 123 Ta-Pei
Road, Niao Shung Hsiang, Kaohsiung Hsien, 833,Taiwan. Tel: 886-7-3700726;
Fax: 886-7-3791196; E-mail: firstname.lastname@example.org
Alternative Sources of Neurons and Glia From Somatic Stem Cells
Yvan Torrente,1,3 Marzia Belicchi,1 Federica Pisati,1 Stefano F. Pagano,1,2 Francesco Fortunato,1 Manuela Sironi,4 Maria Grazia D'Angelo,4 Eugenio A. Parati,2 Guglielmo Scarlato,1 and Nereo Bresolin1,4
1IRCCS Ospedale Maggiore Policlinico, Italy
2Laboratory of Neuropharmacology, National Neurological Institute "C. Besta," Milan, Italy
3Centro Dino Ferrari, Institute of Clinical Neurology, Milan, Italy
4IRCCS Eugenio Medea, Bosisio Parini, Italy
Stem cell populations have been shown to be extremely versatile: they can generate differentiated cells specific to the tissue in which they reside and descendents that are of different germ layer origin. This raises the possibility of obtaining neuronal cells from new biological source of the same adult human subjects. In this study, we found that epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cell-like population isolated from several newborn and adult mouse tissues: muscle and hematopoietic tissues. This population, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Brain engraftment of the somatic-derived neural stem cells generated neuronal phenotypes, demonstrating the great plasticity of these cells with potential clinical application.
Key words: Neural stem cells; Transdifferentiation; Multipotent precursor; Gene therapy; Neuronal differentiation
Address correspondence to Prof. Nereo Bresolin, Institute of Clinical Neurology, University of Milan, Padiglione Ponti, Ospedale Policlinico, via Francesco Sforza 35, 20122 Milan, Italy. Tel: 011-39-02-55033843, Fax: 011-39-02-55190392; E-mail: email@example.com
Injection of Chemotherapeutic Microspheres and Glioma III: Parameters to Optimize Efficacy
Dwaine F. Emerich,1 Shelley R. Winn,2 and Raymond T. Bartus1
1Alkermes, Inc., Cambridge, MA 02139
2Department of Surgery, OHSU, Portland, OR 97201
Injectable microspheres may provide a means of providing local, sustained exposure of glioma to chemotherapeutics to improve patient survival. Using a rodent model of surgically resected glioma, we previously demonstrated that direct injections of chemotherapeutic microspheres into the tissue surrounding a resection cavity provide superior survival effects over injections of the same microspheres directly into the surgical cavity. The present experiments extended this novel observation by exploring several parameters related to the use of intraparenchymal injections of chemotherapeutic microspheres to treat glioma. Using a rat model of resected glioma, several principles regarding the use of local sustained release carboplatin microspheres were established. First, an inverted U dose-response was observed, wherein further dose escalation beyond the optimal dose was not efficacious and indeed produced significant local toxicity. Second, it was necessary to expose approximately 40% of the tumor margin to sustained release carboplatin in order to increase survival in this model. Survival was not enhanced when the proportion of the tumor margin exposed to carboplatin was only 20%. Third, the distribution of the chemotherapeutic microsphere injections along the tumor perimeter was shown to be important, requiring that the entire perimeter be proportionately exposed to the chemotherapeutic agent. Together, these data continue to support the development of chemotherapeutic microspheres for treating glioma. However, they also caution that a number of fundamental parameters can profoundly influence the efficacy that might be expected from local sustained delivery. Careful attention to these principles is not only required if chemotherapeutic microspheres are to be used efficaciously, but these principles should provide a foundation to further optimize the potential of this and other polymeric delivery systems under development for local, intraparenchymal drug delivery to glioma.
Key words: Glioma; Sustained release; Microsphere; Carboplatin
Address correspondence to Raymond T. Bartus, Ph.D., Pre-clinical R&D, Alkermes, Inc., 64 Sidney Street, Cambridge, MA 02139. Tel: (617) 494-0171; Fax: (617) 494-9263
Injection of Chemotherapeutic Microspheres and Glioma IV: Eradicating Tumors in Rats
Dwaine F. Emerich,1 Shelley R. Winn,2 and Raymond T. Bartus1
1Alkermes, Inc., Cambridge, MA 02139
2Department of Surgery, OHSU, Portland, OR 97201
Polymer microspheres can be easily injected into the brain to provide a local and sustained delivery of chemotherapeutics to a tumor or surrounding tissue subject to high rates of tumor recurrence following surgery. Building on previous studies that established the clear advantage of local, peritumoral injections of sustained release microspheres, the following experiments utilized two different approaches for maximizing the survival benefit in glioma-bearing rats. In the first experiment, a previously grown cortical tumor was debulked and animals received either one or two treatments with carboplatin-loaded microspheres (either 200 or 800 mg total carboplatin per treatment). In each case, the microspheres were injected along the perimeter of the resection cavity with each treatment separated by 20 days. Survival studies clearly demonstrated that two, temporally spaced injections were superior to a single series of injections. At the lowest dose tested (200 mg), median survival was increased an additional 40% over that in animals receiving one treatment. At the higher dose (800 mg), one third of the animals receiving two separate treatments were long-term survivors (>150 days) and showed complete eradication of the tumor on histological examination. In the second experiment, we directly compared the efficacy produced by sustained release carboplatin or 1,3-bis[2-chloroethyl]-1-nitrourea (BCNU) alone versus injecting carboplatin and BCNU-loaded microspheres blended together as a single suspension. Carboplatin and BCNU both enhanced survival, with BCNU being significantly less effective than carboplatin. However, the greatest improvements in survival were seen when a blended suspension of carboplatin and BCNU microspheres was injected around the surgical cavity. In contrast, spatially alternating injections of BCNU and carboplatin microspheres was significantly less effective and the increase in survival was no greater than that achieved with BCNU alone. These data offer further support for the potential utility of local, sustained release chemotherapeutic microspheres for treating glioma. Moreover, they suggest that injectable chemotherapeutic microspheres may offer important advantages by (a) permitting multiple, temporally spaced injections to be made, as needed, and (b) providing the opportunity to deliver combinations of several different efficacious drugs directly to the tumor site to enhance survival beyond what can be achieved with delivery of any single chemotherapeutic agent.
Key words: Glioma; Sustained release; Microsphere; Carboplatin; BCNU
Address correspondence to Raymond T. Bartus, Ph.D., Pre-clinical
R&D, Alkermes, Inc., 64 Sidney Street, Cambridge, MA 02139. Tel: (617)
494-0171; Fax: (617) 494-9263
Long-Term Insulin Independence Following Repeated Islet Transplantation in Totally Pancreatectomized Diabetic Pigs
Eugenio Morsiani,1 Luciano Fogli,1 Giovanni Lanza, Jr.,2 Laura T. Lebow,1 Achilles A. Demetriou,1 and Jacek Rozga1
1Division of Surgical Research, Department of Surgery, Cedars-Sinai
Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048
2Department of Pathology, University of Ferrara School of Medicine, Corso Giovecca, 203, 44100 Ferrara, Italy
Clinical islet transplantation (Tx) in type I diabetic patients has been successful so far only in a minority of cases, probably because of multiple factors, partly immunologic and partly nonimmunologic in nature. Preclinical studies of islet Tx in large animals are still needed to clarify the reasons and find possible solutions. In this study, we tested the feasibility of noninvasive, repeated intrahepatic allo-Tx of porcine pancreatic islets obtained from multiple donors, in pigs rendered diabetic by total pancreatectomy (Pct). In group I Yucatan miniature swine (n = 6), after induction of diabetes by Pct, repeated islet allo-Tx of >80% pure islets was performed. Islets obtained from two pigs of the Hanford breed were injected twice a week, half freshly isolated and half 48-h cultured, over a period of 11 days, for a total of 23,647 ± 1617 islet equivalents (IE)/kg recipient body weight (BW). In group II Yucatan miniature swine (n = 3), after Pct, a single allo-Tx of >80% pure islets, previously obtained from two donors of the Hanford breed, was performed, using a total of 22,416 ± 1124 IE/kg BW. In group III Yucatan miniature swine (n = 3), auto-Tx of 60-75% pure islets, averaging 2980 ± 424 IE/kg BW, was performed a few hours after Pct. Group IV Yucatan mini pigs (n = 3) underwent Pct and were used as diabetic controls. Group V animals (n = 3) were normal control Yucatan mini pigs. Porcine islets were isolated by a modification of the standard collagenase digestion and Ficoll gradient purification method. Donors and recipients were chosen on the basis of moderate to high mutual alloreactivity in mixed lymphocyte culture (MLC). In groups I and II, cyclosporine A (CsA) was started 4 days before allo-Tx, at the dose of 15 mg/kg IM, and then gradually reduced to 4 mg/kg IM. In all group I animals, normal fasting blood glucose (FBG) was restored within 2-3 weeks. Two normoglycemic pigs died of acute pneumonia at 33 and 112 days, respectively, and one animal became progressively hyperglycemic at 100 days. After 3 months, discontinuation of CsA treatment resulted in FBG increase in two group I animals. In one pig, CsA was stopped after 151 days, and normoglycemia persisted until euthanasia, after 8 months. In group II pigs, normoglycemia lasted 4-20 days, with a progressive increase of insulin requirement thereafter. In group III animals, after islet auto-Tx, normoglycemia lasted 7-10 days, while insulin daily requirement progressively increased thereafter, stabilizing at 0.4 IU/kg/day, corresponding to about one third of the amount required in diabetic controls. The single most important result in this series of experiments is that intraportal allo-Tx of a sufficient islet mass, divided in multiple subtherapeutic doses, produced a better metabolic long-term control in comparison to a single injection of the same amount of islets. The technique of multiple-donor repeated islet Tx may prove useful to overcome the problem of primary nonfunction or early graft failure, currently limiting the success of clinical islet Tx in most cases.
Key words: Repeated islet transplantation; Experimental diabetes; Total pancreatectomy
Address correspondence to Luciano Fogli, M.D., Divisione di Chirurgia
Generale, Ospedale Bellaria, Via Altura, 3, 40139 Bologna, Italy. Tel:
0039 051 6225445; Fax: 0039 051 6225706; E-mail: Luciano.Fogli@ausl.bo.it
Takehisa Matsuda and Hongbing He
Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565-8565, Japan
The object of this study was to develop a compliant hybrid vascular graft minimally supported by an elastomeric scaffold for arterial replacement. The hybrid vascular grafts designed were composed of three layers: an inner surface lined with endothelial cells (ECs); a hybrid medial tissue composed of a collagenous gel embedded with smooth muscle cells (SMCs); and an outer layer made of a laser-processed micropored segmented polyurethane (SPU) film with the circular pore size (diameter 150 mm) but different film thickness (50-200 mm) and different pore-to-pore distances (1 or 4 mm). The approximate dimensions of the hybrid vascular graft without the SPU film were as follows: inner diameter, 5 mm; length, 5 cm; thickness, 50 mm. The intraluminal pressure-external diameter relationship was measured by infusion of a phosphate buffer solution into the hybrid vascular graft. Canine carotid arteries and commercially available ePTFE grafts served as controls. Decrease in the thickness of the SPU film and increase in the pore density of the SPU film increased the pressure-dependent distensibility of the hybrid vascular grafts. The thinner the film and higher the pore density, the more compliant was the hybrid graft. The pressure-induced distensibility of the designed hybrid graft was found to be close to that of native carotid arteries.
Key words: Hybrid vascular graft; Endothelial cell; Smooth muscle cell; Compliance; Segmented polyurethane
Address correspondence to Takehisa Matsuda at his present address: Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi, Higashiku, Fukuoka 812-8582, Japan. Tel: 81-92-642-6210; Fax: 81-92-642-6212; E-mail: firstname.lastname@example.org
Newly Designed Compliant Hierarchic Hybrid Vascular Graft Wrapped With Microprocessed Elastomeric Film--II: Morphogenesis and Compliance Change Upon Implantation
Hongbing He1 and Takehisa Matsuda2
1Department of Bioengineering, National Cardiovascular Center
Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565-8565, Japan
2Department of Biomedical Engineering, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Hierarchic structured hybrid tubular vascular media composed of endothelial cells (ECs), which covered the luminal surface, and smooth muscle cells (SMCs), which resided in the tubular collagen gel, were wrapped with thin segmented polyurethane elastomeric films designed to provide compliance matching with native arteries and transmural tissue permeability using a laser-directed ablation technique to provide different pore densities. Two hybrid grafts with high and low pore densities (inner diameter: 150 mm and length: 4 cm), and exhibiting pressure-dependent distensibility in response to pulsatile pressure, were bilaterally implanted into canine common arteries for up to 6 months. Irrespective of the pore density, high patency was achieved and no dilation and bursting occurred. Maintenance of full endothelialization during the entire course of implantation period was observed for the graft wrapped with the film with higher pore density. On the other hand, the graft wrapped with the film with lower pore density exhibited markedly reduced endothelialization at a later period of implantation, probably due to delamination of neoarterial tissue from the segmented polyurethane (SPU) surface. There were some differences in transmural tissue ingrowth between the two grafts. At anastomotic sites, neoarterial thickness for type A graft was smaller than that for type B graft regardless of the implantation period. Slightly reduced compliance was observed for both types of grafts at the sixth month of the implantation period. This study indicates that a hybrid vascular graft minimally supported with a thin elastomeric film can be used to replace diseased arteries if micropores are well designed for tissue permeability and anchoring.
Key words: Hybrid vascular grafts; Segmented polyurethane; Compliance matching; Endothelial cell; Smooth muscle cell; Tissue permeability
Address correspondence to Takehisa Matsuda, Department of Biomedical
Engineering, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan. Tel: 81-92-642-6210; Fax: 81-92-642-6212;