ognizant Communication Corporation


VOLUME 11, NUMBER 8, 2002

Cell Transplantation, Vol. 11, pp. 733-746, 2002
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Quantitative [18F]Fluorodopa/PET and Histology of Fetal Mesencephalic Dopaminergic Grafts to the Striatum of MPTP-Poisoned Minipigs

Annette Møller Dall,1 Erik Hvid Danielsen,2 Jens Christian Sørensen,3 Flemming Andersen,2 Arne Møller,4 Jens Zimmer,1 Albert H. Gjedde,2,5 the DaNeX Group, and Paul Cumming2

1Department of Anatomy and Neurobiology, University of Southern Denmark, 5000 Odense C, Denmark
2PET Centre, Aarhus General Hospital, 8000 Aarhus C, Denmark
3Department of Neurobiology, University of Aarhus, 8000 Aarhus C, Denmark
4NsGene, 2750 Ballerup, Denmark
5McGill University, Montreal, Quebec, Canada

The functional restoration of the dopamine innervation of striatum in MPTP-poisoned Göttingen minipigs was assessed for 6 months following grafting of fetal pig mesencephalic neurons. Pigs were assigned to a normal control group and a MPTP-poisoned group, members of which received no further treatment, or which received bilateral grafts to the striatum of tissue blocks harvested from E28 fetal pig mesencephalon with and without immunosuppressive treatment after grafting, or with additional co-grafting with immortalized rat neural cells transfected to produce GDNF. In the baseline condition, and again at 3 and 6 months postsurgery, all animals were subjected to quantitative [18F]fluorodopa PET scans and testing for motor impairment. At the end of 6 months, tyrosine hydroxylase (TH)-containing neurons were counted in the grafts by stereological methods. The MPTP poisoning persistently reduced the magnitude of k3D, the relative activity of DOPA decarboxylase in striatum, by 60%. Grafting restored the rate of [18F]fluorodopa decarboxylation to the normal range, and normalized the scores in motor function. The biochemical and functional recovery was associated with survival of approximately 100,000 TH-positive graft neurons in each hemisphere. Immunosuppression did not impart a greater recovery of [18F]fluorodopa uptake, nor were the number of TH-positive graft neurons or the volumes of the grafts increased in the immunosuppressed group. Contrary to expectation, co-grafting of transfected GDNF-expressing HiB5 cells, a rat-derived neural cell line, tended to impair the survival of the grafts with the lowest values for graft volumes, TH-positive cell numbers, behavioral scores, and relative DOPA decarboxylase activity. From the results we conclude that pig ventral mesencephalic allografts can restore functional dopamine innervation in adult MPTP-lesioned minipigs.

Key words: Minipig; MPTP; Parkinsonism; [18F]Fluorodopa; DOPA decarboxylase; PET; Grafting; Stereology

Address correspondence to Paul Cumming, PET Centre, Aarhus General Hospital, Norrebrogade 44, Aarhus C, Denmark DK-8000. Tel: +45 8949-3334; E-mail: paul@pet.auh.dk

*Contributing authors are from the Danish Neuronal Xenografting (DaNeX) Group.

Cell Transplantation, Vol. 11, pp. 747-752, 2002
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Neovascularization Induced by Autologous Bone Marrow Cell Implantation in Peripheral Arterial Disease

Kensuke Esato, Kimikazu Hamano, Tao-Sheng Li, Akira Furutani, Atsushi Seyama, Hiroaki Takenaka, and Nobuya Zempo

Division of Cardiovascular Surgery, Department of Bioregulation, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, Japan 755-8505

Neovascularization has recently been used as a new treatment for severe ischemic disease. We tried to induce therapeutic neovascularization by autologous bone marrow cell implantation (BMCI) in eight selected patients with chronic peripheral arterial disease (PAD), in whom traditional treatments had failed. Improvement of subjective symptoms was seen in seven patients after treatment. Of three limbs with toe or finger ulceration, complete healing was achieved in two, while the other one became less severe after treatment. No relative toxicity was observed in any of the patients. BMCI might be a feasible treatment for selected patients with chronic PAD.

Key words: Neovascularization; Ischemia; Bone marrow cell implantation; Peripheral arterial disease

Address correspondence to Kimikazu Hamano, M.D., Division of Cardiovascular Surgery, Department of Bioregulation, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi, Japan 755-8505. Tel: +81-836-22-2261; Fax: +81-836-22-2260; E-mail: kimikazu@po.cc.yamaguchi-u.ac.jp

Cell Transplantation, Vol. 11, pp. 753-758, 2002
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Embryonic Stem Cells Attenuate Viral Myocarditis in Murine Model

Ju-Feng Wang,1 Yingke Yang,1 Guangwu Wang,2 Jiangyong Min,1 Matthew F. Sullivan,1 Peipei Ping,2 Yong-Fu Xiao,1 and James P. Morgan1

1Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
2Cardiology Division, Department of Medicine, University of Louisville, Kentucky, KY 40202

We used mice to test our hypothesis that in response to viral invasion, stem cells may migrate into the heart and attenuate the effect of viral myocarditis. Male BALB/c mice were divided into three groups: mouse embryonic stem (ES) cell control, encephalomyocarditis virus (EMCV), and EMCV + ES cells. After administration of ES cells via tail vein, mice were immediately inoculated with EMCV. Mice were sacrificed at different days after EMCV inoculation. Mortality was recorded. Inflammatory cell infiltration and necrosis (major pathological changes of viral myocarditis) were evaluated by hematoxylin-eosin staining. ES cell migration and differentiation were identified by immunofluorescence. The survival rate in the EMCV + ES cell group (80%) was significantly increased (p < 0.05) over the EMCV-alone group (64%). Also, the incidence of inflammatory cell infiltration and myocardial lesions was lower in the EMCV + ES cell mice. Furthermore, the result of green fluorescent protein (GFP) and a-actinin analysis indicated that ES cells migrated into the heart and differentiated into myocytes after virus inoculation. In conclusion, ES cells significantly increased the survival of viral myocarditis mice and also decreased the necrosis and infiltration of inflammatory cells. These results demonstrated the ability of stem cells to mitigate the effects of viral infection on the heart and illustrated their potential therapeutic application to other mammalian species, including humans.

Key words: Myocarditis; Embryonic stem cells; Mouse; Encephalomyocarditis virus

Address correspondence to James P. Morgan, M.D., Ph.D., Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215. Tel: (617) 667-2191; Fax: (617) 667-1615; E-mail: jmorgan@caregroup.harvard.edu

Cell Transplantation, Vol. 11, pp. 759-767, 2002
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Autotransplantation in mdx Mice of mdx Myoblasts Genetically Corrected by an HSV-1 Amplicon Vector

Mathieu Bujold,1 Nicolas Caron,1 Goeffrey Camiran,1 Santwana Mukherjee,2 Paul. D. Allen,2 Jacques P. Tremblay,1 and Yaming Wang2

1Laboratoire de Génétique Humaine, Centre de Recherche du Centre Hospitalier de l'Universit, Laval (CHUL), Ste-Foy (Qc), Canada, G1V 4G2
2Department of Anesthesia, Brigham & Women's Hospital, Boston, MA

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, characterized by a lack of dystrophin. To eliminate the need for immunosuppressive drugs, transplantation of genetically modified autologous myoblasts has been proposed as a possible therapy for this myopathy. An HSV-1 amplicon vector (HSVDGN), containing a 17.3-kb full-length MCK-driven mouse dystrophin cDNA, an eGFP gene, and a neomycin resistance gene driven by CMV or SV40 promoters, respectively, was constructed and used to transduce mdx primary myoblasts. The presence of the eGFP and neomycin resistance genes facilitated the evaluation of the initial transduction efficiency and the permanent transduction frequency. At low multiplicities of infection (MOI 1-5), the majority of myoblasts (60-90%) expressed GFP. The GFP-positive mdx myoblasts were sorted by FACS and selected with neomycin (300 mg/ml) for 2 weeks. Up to 2% of initially infected mdx myoblasts stably expressed the three transgenes without further selection at that time. These altered cells were grafted into the tibialis anterior muscles of 18 mdx mice. Some of the mice were immunosuppressed with FK506 due to the anticipation that eGFP and the product of neomycin resistance gene might be immunogenic. One month after transplantation, numerous muscle fibers expressing mouse dystrophin were detected by immunohistochemistry, in both immunosuppressed (10-50%) and nonimmunosuppressed (5-25%) mdx mice. Our results demonstrated the capability of permanently expressing a full-length dystrophin in dystrophic myoblasts with HSV-1 amplicon vector and raised the possibility of an eventual treatment of DMD based on the transplantation of genetically modified autologous myoblasts.

Key words: Autologous transplantation; Duchene muscular dystrophy (DMD); HSV-1 amplicons; Full-length dystrophin cDNA; Dystrophin-positive fibers

Address correspondence to Yaming Wang, M.D., Department of Anesthesia, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. Tel: (617) 732-6879; Fax: (617) 732-6927; E-mail address: yaming@zeus.bwh.harvard.edu

Cell Transplantation, Vol. 11, pp. 769-777, 2002
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Efficacy of the Oxygen-Charged Static Two-Layer Method for Short-Term Pancreas Preservation and Islet Isolation From Nonhuman Primate and Human Pancreata*

Shinichi Matsumoto,1,2 Theodore H. Rigley,3 Sabrina A. Qualley,1 Yoshikazu Kuroda,4 Jo Anna Reems,1,5 and R. Brian Stevens3

1Puget Sound Blood Center/Northwest Tissue Center, 921 Terry Avenue, Seattle, WA 98104
2University of Washington Medical Center, Department of Surgery, Division of Transplantation, 1959 N.E. Pacific Street, Seattle, WA 98195
3University of Nebraska Medical Center, Department of Surgery, Section of Transplantation, 983285 Nebraska Medical Center, Omaha, NE 68498-3285
4Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kobe University, 7-5-2 Kusunoki-Cho Chuo-Ku Kobe, Japan 650
5University of Washington Medical Center, Department of Hematology, Division of Medicine, 1959 N.E. Pacific Street, Seattle, WA 98195

Previous reports indicate that the two-layer method (TLM) of human pancreas preservation is superior to University of Wisconsin solution (UW) when pancreata are preserved for extended periods (i.e., >24 h) prior to islet isolation. In this study, the efficacy of using the TLM for preserving pancreata for short periods (i.e., <13 h) was evaluated using both nonhuman primate and human pancreata preserved with a TLM kit precharged with oxygen. An oxygen precharged TLM (static TLM) was established and compared with the original TLM with continuous oxygen supply. For the static TLM, the perfluorochemical was fully oxygenated and the oxygen supply removed prior to pancreas preservation. In the primate model, pancreata were preserved by the static TLM, the original TLM, and UW for 5 h prior to islet isolation. In the human model, pancreata were preserved with the static TLM or the original TLM or UW for 4-13 h. Both primate and human pancreata were processed by intraductal collagenase injection and digestion followed by continuous density gradient purification to isolate islets. Islets were assessed for islet yield, purity, viability, and in vitro functionality. In the primate model, islet yield, viability, and in vitro functionality were significantly improved by both the static TLM and the original TLM with similar results. Postculture islet yields were 23,877 ± 3619 IE/g in the static TLM, 21,895 ± 3742 IE/g in the original TLM, and 6773 ± 735 IE/g in UW. In the human model, both the static TLM and the original TLM significantly increased islet yield compared with UW with postculture islet yields of 2659 ± 549 IE/g in the static TLM, 2244 ± 557 IE/g in the original TLM, and 1293 ± 451 IE/g in UW. Nonhuman primate and human pancreata stored in the static TLM, immediately upon procurement, yield isolated islets of a substantially higher quantity than when pancreata are stored in UW. Thus, the use of the static TLM should replace the use of UW for storage of pancreata during transport prior to islet isolation.

Key words: Nonhuman primate islet isolation (Macaca nemestrina); Human islet isolation; Pancreas preservation and transportation; Two-layer method (TLM)

Address correspondence to Shinichi Matsumoto, M.D., Ph.D., Puget Sound Blood Center, 921 Terry Avenue, Seattle, WA 98104. E-mail: shinichim@mac.com or Brian Stevens, M.D., Ph.D., University of Nebraska Medical Center, 983285 Nebraska Medical Center, Omaha, NE 68498-3285. E-mail: rbstevens@surgery.unmc.edu

*This article was partially presented at the Annual Meeting of International Pancreas and Islet Transplantation Association (IPITA) in Innsbruck (June 2001).

Cell Transplantation, Vol. 11, pp. 779-785, 2002
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Induction of Chimerism in Mice Using Human MHC Class I-Mismatched Hoechst 33342 Side Population Donor Stem Cells

John D. Jackson,2 Guimei Zhou,1 Charles A. Kuszynski,2 Jin Cai,1 and Ira J. Fox1

1Department of Surgery and 2Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 68198

A population of Hoechst 33342-stained cells, termed side population (SP) cells, can reconstitute the hematopoietic system of syngeneic mice. This study examined whether limiting numbers of SP cells can repopulate mice across a xenogeneic MHC class I barrier. SP cells were isolated from HLA.B7 and HLA.A2.1 transgenic mice by FACS and placed in colony assays or transplanted into irradiated C57BL/6 (B/6) recipients. SP cells contained few colony-forming cells when placed directly in culture. The number of GM-CFC and HPP-CFC increased up to 3000- and 300-fold, respectively, after 7 days in IL-3- and SCF-stimulated liquid culture. BMC-derived GM-CFC increased up to only 12-fold and HPP-CFC decreased after 7 days in culture. HLA-B7 SP cells (2500-5000) were transplanted into lethal-irradiated B/6 mice. Two-color flow analysis, 4-6 weeks after transplantation, showed that HLA-B7 expression in granulocyte-, macrophage-, and lymphocyte-specific lineages from reconstituted mice was similar to that in B7 transgenic mice. Secondary transplanted B/6 mice also showed a pattern of HLA-B7 expression similar to that in transgenic mice and were followed for longer than 16 weeks with stable chimerism. When HLA-A2.1 SP cells were transplanted into sublethally irradiated mice, 50% of the mice expressed HLA-A2 by PCR analysis in short-term repopulation studies. These data confirm that limiting numbers of SP cells can repopulate the major hematopoietic lineages in lethal and sublethally irradiated mice across a human MHC class I barrier. Therefore, SP cells may be useful for establishing mixed chimerism, which may induce immunologic nonresponsiveness to donor antigens in solid organ transplantation.

Key words: Chimerism; Side population; Transplantation; MHC class I

Address correspondence to Ira J. Fox, M.D., University of Nebraska Medical Center, Department of Surgery, 983285 Nebraska Medical Center, Omaha, NE 68198-3285. Tel: (402) 559-8859; Fax: (402) 559-6749; E-Mail: ijfox@unmc.edu

Cell Transplantation, Vol. 11, pp. 787-798, 2002
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Development of a Novel Cytomedical Treatment That Can Protect Entrapped Cells From Host Humoral Immunity

Ryo Suzuki,1 Yasuo Yoshioka,1 Etsuko Kitano,2 Tatsunobu Yoshioka,1 Hiroaki Oka,1 Takayuki Okamoto,1 Naoki Okada,3 Yasuo Tsutsumi,1 Shinsaku Nakagawa,1 Jun-ichi Miyazaki,4 Hajime Kitamura,2 and Tadanori Mayumi1

1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
2Department of Medical Technology, Osaka Prefectural College of Health Sciences, Osaka 538-8555, Japan
3Department of Biopharmaceutics, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan
4Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka 565-0871, Japan

Cell therapy is expected to relieve the shortage of donors needed for organ transplantation. When patients are treated with allogeneic or xenogeneic cells, it is necessary to develop a means by which to isolate administered cells from an immune attack by the host. We have developed "cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that alginate-poly-L-lysine-alginate microcapsules and agarose microbeads could protect the entrapped cells from injury by cellular immunity. However, their ability to isolate from humoral immunity was insufficient. It is well known that the complement system plays an essential role in rejection of transplanted cells by host humoral immunity. Therefore, the goal of the present study was to develop a novel cytomedical device containing a polymer capable of inactivating complement. In the screening of various polymers, polyvinyl sulfate (PVS) exhibited high anticomplement activity and low cytotoxicity. Murine pancreatic b-cell line (MIN6 cell) entrapped in agarose microbeads containing PVS maintained viability and physiological insulin secretion, replying in response to glucose concentration, and resisted rabbit antisera in vitro. PVS inhibited hemolysis of sensitized sheep erythrocytes (EAs) and rabbit erythrocytes by the complement system. This result suggests that PVS inhibits both the classical and alternative complement pathways of the complement system. Next, the manner in which PVS exerts its effects on complement components was examined. PVS was found to inhibit generation of C4a and Ba generation in activation of the classical and alternative pathways, respectively. Moreover, when the EAC1 cells, which were carrying C1 on the EAs, treated with PVS were exposed to C1-deficient serum, hemolysis decreased in a PVS dose-dependent manner. These results suggest that PVS inhibits C1 in the classical pathway and C3 convertase formation in the alternative pathway. Therefore, PVS may be a useful polymer for developing an anticomplement device for cytomedical therapy.

Key words: Complement; Polyvinyl sulfate (PVS); Cytomedicine; Cell therapy; Humoral immunity; Agarose microbeads

Address correspondence to Tadanori Mayumi, Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel: +81-6-6879-8175; Fax: +81-6-6879-8179; E-mail: mayumi@phs.osaka-u.ac.jp

Cell Transplantation, Vol. 11, pp. 799-801, 2002
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The Immunoprotective Effect of Sertoli Cells Coencapsulated With Islet Xenografts Is Not Dependent Upon Fas Ligand Expression

Hua Yang, Ayman Al-Jazaeri, and James R. Wright, Jr.

Departments of Pathology and Surgery, IWK Health Centre and Dalhousie University Faculty of Medicine and School of Biomedical Engineering, Dalhousie University Faculties of Medicine and Engineering, Halifax, Nova Scotia, Canada B3H 1V7

Coencapsulation with Sertoli-enriched testicular cell fractions prolongs islet graft survival time compared with islet encapsulation alone in a highly discordant tilapia (fish)-to-mouse xenotransplantation model. Here we investigate whether Fas ligand (Fas-L) expression by testicular Sertoli cells is responsible for this additional protective effect. Sertoli-enriched testicular cell fractions (7 x 106 cells) harvested from either Fas-L-defective (group I) or Fas-L-positive (group II) mice were coencapsulated in alginate gel spheres with fish islets and then transplanted into streptozotocin-diabetic Balb/c recipients. Group III mice received encapsulated islets without coencapsulated Sertoli cells. After transplantation, blood glucose levels were monitored three times per week. Mean graft survival times for the three groups were: group I = 35.6 ± 10.2 days (n = 9), group II = 31.3 ± 9.4 days (n = 7), and group III = 23.3 ± 2.2 days (n = 6) (ANOVA, p = 0.043). Coencapsulation, regardless of the Fas-L status of the Sertoli cell donors, modestly prolonged graft survival. There was no significant difference between Fas-L-deficient and Fas-L-positive donors. Our results suggest that Fas/Fas-L interaction is not responsible for the additional protection afforded to encapsulated discordant islet xenografts by coencapsulation with Sertoli cells.

Key words: Islet xenotransplantation; Sertoli cells; Alginate encapsulation; Fas ligand

Address correspondence to James R. Wright, Jr., M.D., Ph.D., Department of Pathology & Laboratory Medicine, IWK Health Centre, 5850 University Ave., Halifax, Nova Scotia, Canada B3H 1V7. Tel: (902) 428-8185; Fax: (902) 428-3215; E-mail: jim.wright@iwk.nshealth.ca

Cell Transplantation, Vol. 11, pp. 803-811, 2002
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Effects of Carboxypeptidase E Overexpression on Insulin mRNA Levels, Regulated Insulin Secretion, and Proinsulin Processing of Pituitary GH3 Cells Transfected With a Furin-Cleavable Human Proinsulin cDNA

Luca Polastri,1 Francesca Galbiati,1 Franco Folli,2 and Alberto M. Davalli1

1Department of Medicine and 2Unit for Metabolic Diseases Scientific Institute San Raffaele, Milan, Italy

We recently developed two rat pituitary GH3 cell clones engineered to secrete human insulin (InsGH3). InsGH3 cells convert proinsulin into mature insulin, which is partially stored into a readily releasable pool of secretory granules. The efficiency of these processes, however, is relatively low in these cells, either in vitro or in vivo. This study was aimed at determining whether carboxypeptidase E (Cpe) overexpression can increase proinsulin processing and regulated secretion by InsGH3 clones. Indeed, in its membrane-bound form Cpe works as sorting receptor for the regulated secretory pathway of many hormones while, in its soluble form, Cpe takes part to the late step of insulin maturation. We obtained two Cpe-overexpressing cell lines from two different InsGH3 clones (InsGH3/C1 and C7). In the Cpe-overexpressing cell lines, derived from InsGH3 of clone 1 (InsGH3/C1-HACpe), in which the membrane-bound form of exogenous Cpe is accounted for by 90% of total Cpe immunoreactivity, we observed an increase in proinsulin gene expression, and in basal and stimulated insulin secretion compared with the original clone. In contrast, in the Cpe-overexpressing cell line derived from InsGH3 of clone 7 (InsGH3/C7-HACpe), where the exogenous membrane-bound form was only 60% of total Cpe, we detected a decrease in basal insulin release and a modest, albeit significant, increase in intracellular proinsulin processing. In conclusion, Cpe overexpression can increase regulated insulin secretion and proinsulin processing in InsGH3 cells; however, such improvements appear quantitatively and qualitatively modest.

Key words: Carboxypeptidase E; Insulin secretion; Bioenegineering; Pituitary cells

Address correspondence to Alberto M. Davalli, M.D., Department of Medicine, San Raffaele Scientific Institute, Milan, Italy, Via Olgettina 60, 20132 Milan, Italy. E-mail: alberto.davalli@hsr.it

Cell Transplantation, Vol. 11, pp. 813-820, 2002
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Oxygen Tension and Blood Flow in Relation to Revascularization in Transplanted Adult and Fetal Rat Pancreatic Islets

Per-Ola Carlsson and Göran Mattsson

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden

We have previously recorded a decreased oxygen tension and blood flow in syngeneically transplanted rat pancreatic islets. The present study related measurements of oxygen tension and blood flow to the vascular density in such grafts implanted beneath the renal capsule. We also evaluated whether transplanted fetal islets are better revascularized than adult islets, and if the degree of revascularization is directly related to the islet vascular endothelial growth factor (VEGF) production. Tissue pO2 was measured using Clark microelectrodes, whereas islet graft blood flow was measured with laser-Doppler flowmetry. The vascular density of endogenous and transplanted islets was quantified in histological specimens stained with the lectin Bandeiraea simplicifolia (BS-1). Tissue pO2 in the transplanted adult and fetal islet grafts was similar and markedly lower than in the endogenous islets. The blood perfusion of both the adult and fetal islet grafts was 60-65% of that in the renal cortex. Administration of D-glucose did not affect tissue pO2 in either the endogenous or transplanted islets, nor graft blood perfusion. The number of capillaries found in the transplanted adult and fetal islets was similar and markedly lower than in endogenous islets. However, in the connective tissue stroma, which constituted ~20% of all islet grafts, the vascular density was higher than in the corresponding endocrine parts of these grafts. Incubated adult islets released higher amounts of VEGF than fetal islets. In conclusion, the previously described low oxygen tension of syngeneically transplanted adult rat islets is related to a low vascular density. Similar low oxygen tension and vascular density are seen in grafted fetal islets. The amount of VEGF production does not correlate to the degree of revascularization of the grafts.

Key words: Angiogenesis; Cell transplantation; Experimental; Islet graft

Address correspondence to Per-Ola Carlsson, M.D., Ph.D., Department of Medical Cell Biology, Biomedical Center, Husargatan 3, Box 571, SE-751 23 Uppsala, Sweden. Tel: +46 18 471 4425; Fax: +46 18 556401; E-mail: Per-Ola.Carlsson@medcellbiol.uu.se

Cell Transplantation, Vol. 11, pp. 821-826, 2002
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Evaluating the Effect of Serine Proteases on Collagenase Activity During Human Islet Isolation

Natisha L. Rose,1,2 Monica M. Palcic,1 and Jonathan R. T. Lakey2

1Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada
2Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada

Inconsistencies in human islet yields after collagenase digestion have been attributed to the activation of endogenous enzymes of the donor pancreas. It has been suggested that pancreatic serine proteases contribute to the proteolysis of collagenase. This study defined the effects of endogenous enzymes within the pancreas on pancreas dissociation during collagenase digestion. Levels of collagenase activity from samples taken throughout several steps in islet isolation procedures, both with and without the addition of the serine protease inhibitor Pefabloc, were determined by a spectrophotometric assay using N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala as the substrate. Results clearly demonstrated that the level of collagenase activity remains stable throughout the isolation procedure despite differences in the donor factors from several cadaveric donor pancreases. This was further demonstrated by observing no difference in activity levels after incubating commercial collagenase preparations with serine proteases and analyzing by means of collagenase activity and SDS-PAGE. These data show that the presence of serine proteases does not affect the level of collagenase activity; however, they likely damage the islet cells upon prolonged digestion of the pancreatic tissue. Further efforts at examining exogenous and endogenous enzyme levels may result in the development of an enzyme cocktail that is both stable and effective for digesting the human pancreas while preserving islet function and viability.

Key words: Pancreas; Serine proteases; Collagenase; Islet isolation

Address correspondence to Jonathan R. T. Lakey, Ph.D., Director Clinical Islet Laboratory, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, Department of Surgery, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada. Tel: (780) 492-3077; Fax: (780) 492-6335; E-mail: jonathan.lakey@ualberta.ca

Cell Transplantation, Vol. 11, pp. 827-838, 2002
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The Morphology of Islets Within the Porcine Donor Pancreas Determines the Isolation Result: Successful Isolation of Pancreatic Islets Can Now Be Achieved From Young Market Pigs

Mareike Krickhahn, Christoph Bühler, Thomas Meyer, Arnulf Thiede, and Karin Ulrichs

Experimental Transplantation Immunology (ETI), Department of Surgery, University of Wuerzburg Hospital, Josef-Schneider-Strasse 2, D-97080 Wuerzburg, Germany

Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 ± 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 ± 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter >200 mm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RBLarge Islets: 5680 ± 3,318 IEQ/g (n = 20); RBSmall Islets: 1353 ± 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrepT density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrepT was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.

Key words: Xenotransplantation; Islets of Langerhans; Pig; Enzymatic digestion; Islet morphology; Density gradient purification

Address correspondence to Professor Dr. Karin Ulrichs, Experimental Transplantation Immunology (ETI), Department of Surgery, University of Wuerzburg Hospital, Josef-Schneider-Strasse 2, D-97980 Wuerzburg, Germany. Tel: +49-931-201-3355; Fax: +49-931-201-2249; E-mail: ulrichs@chirurgie.uni-wuerzburg.de