ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 15, NUMBER 3, 2006

Cell Transplantation, Vol. 15, pp. 279-294, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Neural Transplantation in Huntington's Disease: The NEST-UK Donor Tissue Microbiological Screening Program and Review of the Literature*
M. Farrington,1 T. G. Wreghitt,1 A. M. L. Lever,2 S. B. Dunnett,3 A. E. Rosser,3 and R. A. Barker4

1Clinical Microbiology and Public Health Laboratory, Health Protection Agency & Addenbrooke's Hospital, Cambridge, UK
2Department of Medicine, University of Cambridge, Cambridge, UK
3School of Biosciences, Cardiff University, Cardiff, UK
4Department of Neurology, University of Cambridge, Cambridge, UK

Neural transplantation of human fetal tissue for Huntington's disease (HD) is now entering the clinical arena. The safety of the procedure has now been demonstrated in a number of studies, although the efficacy of such an approach is still being investigated. Stringent but practicable screening of the donor tissue for potential pathogens is an essential prerequisite for successful implementation of any novel transplant program that uses human fetal tissue. In this article we summarize the UK-NEST protocol for the screening of human fetal tissue being grafted to patients with mild to moderate HD. We describe the results of microbiological screening of 87 potential tissue donors in a pilot study, and of the first four donor-recipient patients included in the UK-NEST series. The rationale for the adoption and interpretation of the various tests is described and our methodology is compared with those previously used by other centers. This article therefore presents a comprehensive, logical yet pragmatic screening program that could be employed in any clinical studies that use human fetal tissue for neurotransplantation.

Key words: Tissue transplantation; Fetal tissue transplantation; Brain tissue transplantation; Huntington's disease; Microbiological techniques

Address correspondence to Dr. M. Farrington, Clinical Microbiology and Public Health Laboratory, Box 236 Addenbrooke's Hospital, Cambridge CB2 2QW, UK. Tel: +44-1223-216825; Fax: +44-1223-242775; E-mail: mark.farrington@addenbrookes.nhs.uk

*Contributing authors on behalf of the NEST-UK consortium.

Cell Transplantation, Vol. 15, pp. 295-309, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Chemokines and Their Receptors in Islet Allograft Rejection and as Targets for Tolerance Induction
Shaheed Merani,1 Wayne Truong,1 Wayne W. Hancock,2 Colin C. Anderson,1 and A. M. James Shapiro1

1Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton AB, Canada
2Department of Pathology and Laboratory Medicine, Joseph Stokes, Jr. Research Institute and Biesecker Pediatric Liver Center, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA, USA

Graft rejection is a major barrier to successful outcome of transplantation surgery. Islet transplantation introduces insulin secreting tissue into type 1 diabetes mellitus recipients, relieving patients from exogenous insulin injection. However, insulitis of grafted tissue and allograft rejection prevent long-term insulin independence. Leukocyte trafficking is necessary for the launch of successful immune responses to pathogen or allograft. Chemokines, small chemotactic cytokines, direct the migration of leukocytes through their interaction with chemokine receptors found on cell surfaces of immune cells. Unique receptor expression of leukocytes, and the specificity of chemokine secretion during various states of immune response, suggest that the extracellular chemokine milieu specifically homes certain leukocyte subsets. Thus, only those leukocytes required for the current immune task are attracted to the inflammatory site. Chemokine blockade, using antagonists and monoclonal antibodies directed against chemokine receptors, is an emerging and specific immunosuppressive strategy. Importantly, chemokine blockade may potentiate tolerance induction regimens to be used following transplantation surgery, and prevent the need for life-long immunosuppression of islet transplant recipients. Here, the role for chemokine blockade in islet transplant rejection and tolerance is reviewed.

Key words: Chemokine; Islet; Allograft; Transplant; Tolerance; Review

Address correspondence to Dr. A. M. James Shapiro, M.D., Ph.D., FRCS(Eng), FRCSC, Wyeth-Ayerst Canada/CIHR Clinical Research Chair in Transplantation, Director, Clinical Islet Transplant Program, University of Alberta Roberts Centre, 2000 College Plaza, Edmonton, Alberta, Canada T6G 2C8. Tel: (780) 407 7330; Fax: (780) 407 6933; E-mail: amjs@islet.ca

Cell Transplantation, Vol. 15, pp. 311-317, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Porcine Islet Graft Function Is Affected by Pretreatment With a Caspase-3 Inhibitor

Daniel Brandhorst,1 Vidya Kumarasamy,2 Adel Maatoui,2 Alexandra Alt,2 Reinhard G. Bretzel,2 and Heide Brandhorst1

1Department of Clinical Immunology, Rudbeck Laboratory, Uppsala University Hospital, 75185 Uppsala, Sweden
2Third Medical Department, University Hospital, 35385 Giessen, Germany

During the isolation procedure and after transplantation islets are subjected to numerous variables associated with the induction of apoptosis. The present study investigated the effect of transient pretreatment with caspase inhibitors on function and survival of transplanted pig islets. Isolated porcine islets (3000 IEQ) were incubated overnight in 200 mM of the caspase-3 inhibitor DEVD-CMK prior to transplantation into diabetic nude mice. Glucose-stimulated insulin release of pretreated islets was assessed during static incubation. DEVD-CMK successfully prevented the expression of capase-3 and DFF as demonstrated in heat-shocked pig islets. Nevertheless, transient pretreatment of freshly isolated pig islets with DEVD-CMK resulted in a significantly decreased final graft function of 50.0% (n = 16) compared to 85.7% (n = 14) in control islets (p < 0.05). Glucose-stimulated insulin release of porcine islets (n = 6) was not significantly effected by overnight culture with DEVD-CMK. Morphological assessment revealed that this caspase-3 inhibitor significantly increased the percentage of necrosis to a small, but nevertheless significant, extent in comparison to control islets (p < 0.05). The study demonstrates that short-time pretreatment with the caspase-3 inhibitor DEVD-CMK reduces the capacity of transplanted porcine islets to restore normoglycemia in diabetic nude mice.

Key words: Porcine islets; Islet transplantation; Apoptosis; Caspase-3 inhibitors

Address correspondence to Daniel Brandhorst, Department of Clinical Immunology, Rudbeck Laboratory, C11, Uppsala University Hospital, Dag Hammerskölds väg 20, 75185 Uppsala, Sweden. Tel: +46-70-4250636; Fax: +46-18-611-0222; E-mail: Daniel.Brandhorst@klinimm.uu.se

Cell Transplantation, Vol. 15, pp. 319-324, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Survival and Function of Transplanted Islet Cells on an In Vivo, Vascularized Tissue Engineering Platform in the Rat: A Pilot Study*
David L. Brown,1,2 Peter J. Meagher,1,3 Kenneth R. Knight,1 Effie Keramidaris,1 Rosalind Romeo-Meeuw,1 Anthony J. Penington,1 and Wayne A. Morrison1

1Bernard O'Brien Institute of Microsurgery and the Department of Surgery, University of Melbourne, St. Vincent's Hospital, Melbourne, Australia
2Division of Plastic Surgery, University of Michigan, Ann Arbor, MI, USA
3Department of Plastic Surgery, St George's Hospital Medical School, University of London, London, UK

As in vivo tissue engineering of complex tissues and organs progresses, there is a need for an independently vascularized, alterable, and recoverable model. Current models of islet cell transplantation (release into the portal venous system, placement under the renal capsule, and microencapsulation) lack these qualities. We have developed a model of angiogenesis and spontaneous tissue generation in the rat that lends itself as a potential platform for tissue engineering. In this experiment, we examined the effectiveness of such a model in addressing some of the shortcomings of endocrine pancreatic transplantation. An arteriovenous loop was created in the groins of five adult inbred Sprague-Dawley rats, and placed within polycarbonate chambers. Isolated pancreatic islet cell clusters were placed within the chambers, suspended in a matrix of Matrigel®. The chambers were recovered at 3 weeks, and the newly generated tissue was processed for histologic and immunohistochemical analysis. By 3 weeks, spontaneous generation of angiogenesis and collagen matrix and deposition of a collagen matrix was observed. Surviving islet cells were identified by histology and their viability was confirmed via immunohistochemistry for insulin and glucagon. This study demonstrates the ability to maintain viability and functionality of transplanted islet cells on a tissue-engineered platform with an independent vascular supply. The model provides the ability to alter the graft environment via matrix substitution, cellular coculture, and administration of growth factors. The transplanted tissues are recoverable without animal sacrifice and are microsurgically transferable. This model may provide an in vivo culture platform for the study of islet transplantation.

Key words: Islet transplantation; Tissue engineering; In vivo vascularization

Address correspondence to David L. Brown, M.D., 2130 Taubman Center, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA. Tel: 734-615-3435; Fax: 734-763-5354; E-mail: davbrown@umich.edu

*Presented at Annual Meeting of the Plastic Surgery Research Council, Boston, MA, April 19, 2002.

Cell Transplantation, Vol. 15, pp. 325-334, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Maintenance of Mouse, Rat, and Pig Pancreatic Islet Functions by Coculture With Human Islet-Derived Fibroblasts
Atsushi Miki,1 Michiki Narushima,1 Teru Okitsu,2 Yuichi Takeno,3 Alejandro Soto-Gutierrez,1 Jorge David Rivas-Carrillo,1 Nalú Navarro-Alvarez,1 Yong Chen,1 Kimiaki Tanaka,1 Hirofumi Noguchi,2 Shinichi Matsumoto,2 Michinori Kohara,4 Jonathan R. T. Lakey,5 Eiji Kobayashi,3 Noriaki Tanaka,1 and Naoya Kobayashi1

1Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2Department of Transplant Surgery, Kyoto University Hospital, 54 Seigoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan
3Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Tochigi 329-0498, Japan
4Department of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo113-8613 Japan
5Human Pancreatic Islet Transplant Program, University of Alberta, Alberta T2N 4N1, Canada
Development of an efficient preculture system of islets is ideal. Toward that goal, we constructed a human pancreatic islet-derived fibroblast cell line MNNK-1 for a source as a coculture system for freshly isolated islets to maintain islet functions. Human pancreatic islet cells were nucleofected with a plasmid vector pYK-1 expressing simian virus 40 large T antigen gene (SV40T) and hygromycin resistance gene (HygroR). One of the transduced cell lines, MNNK-1, was established and served as a feeder cell in the coculture for freshly isolated mouse, rat, and pig islets. Morphology, viability, and glucose-responding insulin secretion were analyzed in the coculture system. MNNK-1 cells were morphologically spindle shaped and were negative for pancreatic endocrine markers. MNNK-1 cells were positive for &alpha;-smooth muscle actin and collagen type I and produced fibroblast growth factor. Coculture of the mouse, rat, and pig islets with MNNK-1 cells maintained their viability and insulin secretion with glucose responsiveness. A human pancreatic islet-derived fibroblast cell line MNNK-1 was established. MNNK-1 cells were a useful means for maintaining morphology and insulin secretion of islets in the coculture system.

Key words: Islets; Simian virus 40 large T antigen; Coculture

Address correspondence to Naoya Kobayashi, M.D., Ph.D. Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel/Fax: (+81) 86-235-7485; E-mail: immortal@md.okayama-u.ac.jp

Cell Transplantation, Vol. 15, pp. 335-342, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Differentiation of Human Embryonic Stem Cells to Hepatocytes Using Deleted Variant of HGF and Poly-amino-urethane-Coated Nonwoven Polytetrafluoroethylene Fabric
Alejandro Soto-Gutierrez, Nalú Navarro-Alvarez, Jorge David Rivas-Carrillo, Yong Chen, Tomoki Yamatsuji, Noriaki Tanaka, and Naoya Kobayashi

Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
Human embryonic stem (hES) cells have recently been studied as an attractive source for the development of a bioartificial liver (BAL). Here we evaluate the differentiation capacity of hES cells into hepatocytes. hES cells were subjected to suspension culture for 5 days, and then cultured onto poly-amino-urethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric in the presence of fibroblast growth factor-2 (bFGF) (100 ng/ml) for 3 days, then with deleted variant of hepatocyte growth factor (dHGF) (100 ng/ml) and 1% dimethyl sulfoxide (DMSO) for 8 days, and finally with dexamethasone (10-7 M) for 3 days. The hES cells showed gene expression of albumin in a time-dependent manner of the hepatic differentiation process. The resultant hES-derived hepatocytes metabolized the loaded ammonia and lidocaine at 7.8% and 23.6%, respectively. A million of such hepatocytes produced albumin and urea at 351.2 ng and urea at 7.0 mg. Scanning electron microscopy showed good attachment of the cells on the surface of the PTFE fabric and well-developed glycogen rosettes and Gap junction. In the present work we have demonstrated the efficient differentiation of hES cells to functional hepatocytes. The findings are useful to develop a BAL.

Key words: Human ES cells; Hepatocyte; Hepatocyte growth factor; Differentiation

Address correspondence to Naoya Kobayashi, M.D., Ph.D., Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel/Fax: (+81) 86-235-7485; E-mail: immortal@md.okayama-u.ac.jp

Cell Transplantation, Vol. 15, pp. 343-350, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Development and Characterization of an HEK293-Derived Cell Line for Use in an Intratumoral Cytokine Delivery System

M. J. Hamilton,1 Q. X. Huang,1 C. L. Li,2 and K. A. O. Ellem1

1Queensland Institute of Medical Research, Brisbane, Queensland, Australia
2Stem Cell Program, Institute of Zoology/Genomics Research Center, Academia Sinica, 129 Academia Rd Sec 2, Nankang, Taipei 11529, Taiwan

As part of ongoing work to develop a method of cytokine delivery for use as an intratumoral depot, we noted that HEK293 cells, encapsulated in alginate, died within 24-48 h after in vivo, intratumoral implantation. We hypothesized that the highly hypoxic and acidic conditions found inside the tumor was the cause of the cells' premature demise. Therefore, we set out to develop a cell line, derived from HEK293, that would survive these hostile conditions. The HEK293 line was selected in 0.3-0.5% oxygen conditions over several weeks, followed by a further 6-week period of culture in alternating hypoxic and normoxic conditions. The most rapidly growing clones were selected and grown in normoxic conditions for several weeks to ensure their stability. The clones were then compared to the original line in terms of cell proliferation in normoxia and hypoxia, colony-forming efficiency, and morphological characteristics. The resulting line was able to proliferate in the harshest of conditions and continues to release its biological payload after alginate microencapsulation.

Key words: HEK293 cells; Hypoxic cell selection; Intratumoral environment; Mouse; Cytokines

Address correspondence to Dr. Michael J. Hamilton, Cancer Immunotherapy Lab, I Floor CBCRC, Queensland Institute of Medical Research, 300 Herston Road, Herston, Queensland 4006, Australia. Tel: +61 7 33620181; Fax: +61 7 33620111; E-mail: Michael.Hamilton@qimr.edu.au

Cell Transplantation, Vol. 15, pp. 351-358, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Short-Term Heart Retention and Distribution of Intramyocardial Delivered Mesenchymal Cells Within Necrotic or Intact Myocardium
Nguyen Tran,1,2 Yan Li,1 Fatiha Maskali,3 Laurent Antunes,4 Pablo Maureira,1 Marie-Helene Laurens,3 Pierre-Yves Marie,3 Gilles Karcher,3 Frederique Groubatch,1 Jean-François Stoltz,1 and Jean-Pierre Villemot1

1Laboratory of Surgery School, Faculty of Medicine-Nancy, Avenue de la forêt de Haye, BP184, 54505 Vandoeuvre-lès-Nancy Cedex, France
2Department of Cell Therapy and Tissue Engineering, UMR7563 CNRS, Faculty of Medicine-Nancy, Avenue de la forêt de Haye, BP184, 54505 Vandoeuvre-lès-Nancy Cedex, France
3Department of Nuclear Medicine, INSERM U684, CHU-Nancy, Rue du Morvan, 54500 Vandoeuvre-lès-Nancy, France
4Laboratory of Pathology, CHU-Nancy, Rue du Morvan, 54500 Vandoeuvre-lès-Nancy, France

Cell therapy with bone marrow mesenchymal stem cells (BMSCs) is a new strategy for treating ischemic heart failure, but data concerning the distribution and retention of transplanted cells remain poor. We investigated the short-term myocardial retention of BMSCs when these cells are directly injected within necrotic or intact myocardium. 111Indium-oxine-labeled autologous BMSCs were injected within either 1-month-old infarction (n = 6) or normal myocardium (n = 6) from rats. Serial in vivo pinhole scintigraphy was scheduled during 1 week in order to track the implanted cells. The myocardial retention of BMSCs was definitely higher in myocardial infarction than in normal myocardial area (estimated percent retention at 2 h: 63 ± 3% vs. 25 ± 4%, p < 0.001) and the estimated cardiac retention values were unchanged in both groups along the 7 days of follow-up. On heart sections at day 7, labeled BMSCs were still around the injection site and appeared confined to the scarred tissue corresponding either to the infarct area or to the myocardium damaged by needle insertion. BMSCs have a higher retention when they are injected in necrotic than in normal myocardial areas and these cells appear to stay around the injection site for at least a 7-day period.

Key words: Myocardial infarction; Cell transplantation; Mesenchymal cells; Radioisotopes; Imaging

Address correspondence to Nguyen Tran, Ph.D., Laboratory of Surgery School, Faculty of Medicine, 9, Avenue de la Forêt de Haye, 54500 Vandoeuvre-lès-Nancy, France. Tel: (+33); Fax: (+33); E-mail: Nguyen.Tran@medecine.uhp-nancy.fr

Cell Transplantation, Vol. 15, pp. 359-365, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Survival of Microencapsulated Islets at 400 Days Posttransplantation in the Omental Pouch of NOD Mice

Tsunehiro Kobayashi,1,2 Yukio Aomatsu,3 Hiroo Iwata,4 Tatsuya Kin,2 Hiromichi Kanehiro,1 Michiyoshi Hisanga,1 Saiho Ko,1 Mitsuo Nagao,1 George Harb,2 and Yoshiyuki Nakajima1

1First Department of Surgery, Nara Medical University, Nara, 634-8522, Japan
2Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada
3Department of Surgery, Matsubara Municipal Hospital, Osaka, 580-0044, Japan
4Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan
The long-term durability of agarose microencapsulated islets against autoimmunity was evaluated in NOD mice. Islets were isolated from 6-8-week-old prediabetic male NOD mice and microencapsulated in 5% agarose hydrogel. Microencapsulated or nonencapsulated islets were transplanted into the omental pouch of spontaneously diabetic NOD mice. Although the diabetic NOD mice that received nonencapsulated islets experienced a temporary reversal of their hyperglycemic condition, all 10 of these mice returned to hyperglycemia within 3 weeks. In contrast, 9 of 10 mice transplanted with microencapsulated islets maintained normoglycemia for more than 100 days. Islet grafts were removed at 100, 150, 200, 300, and 400 days posttransplantation. A prompt return to hyperglycemia was observed in the mice after graft removal, indicating that the encapsulated islet grafts were responsible for maintaining euglycemia. Histological examination revealed viable islets in the capsules at all time points of graft removal. In addition, b-cells within the capsules remained well granulated as revealed by the immunohistochemical detection of insulin. No immune cells were detected inside the microcapsules and no morphological irregularities of the microcapsules were observed at any time point, suggesting that the microcapsules successfully protected the islets from cellular immunity. Sufficient vascularization was evident close to the microcapsules. Considerable numbers of islets showed central necrosis at 400 days posttransplantation, although the necrotic islets made up only a small percentage of the islet grafts. Islets with central necrosis also showed abundant insulin production throughout the entire islets, except for the necrotic part. These results demonstrate the long-term durability of agarose microcapsules against autoimmunity in a syngeneic islet transplantation model in NOD mice.

Key words: Islet transplantation; Autoimmunity; Microencapsulation; Agarose

Address correspondence to Tsunehiro Kobayashi M.D., Ph.D., Surgical-Medical Research Institute, 1074 Dentistry Pharmacy Centre, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada. Tel: (780) 492-3386; Fax: (780) 492-1627; E-mail: tsunehirokobayashi@hotmail.com