ognizant Communication Corporation

Cell Transplantation, Vol. 16, pp. 1-8, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

¹Department of Surgery, University of Alberta, Edmonton, AB Canada
²Clinical Islet Transplant Program, University of Alberta, Edmonton, AB Canada

Recent advances in clinical islet transplantation have clearly demonstrated that this procedure can provide excellent glycemic control and often insulin independence in a population of patients with type 1 diabetes. A key limitation in the widespread application of clinical islet transplantation is the requirement of 10,000 islet equivalents/kg in most recipients, generally derived from two or more cadaveric donors. It has been determined that a majority of the transplanted islets fail to engraft and become fully functional. In this review article, the factors that contribute to this early loss of islets following transplantation are discussed in depth.

Key words: Islet transplantation; Apoptosis; Hypoxia; Revascularization; IBMIR

Address correspondence to Juliet Emamaullee, Ph.D., Surgical Medical Research Institute, 1074 Dentistry-Pharmacy Centre, University of Alberta, Edmonton, AB T6G 2N8, Canada. Tel: 780-492-4656; Fax: 780-492-1627; E-mail: juliete@ualberta.ca

Clinical Islet Transplant Program, University of Alberta and Capital Health Authority, Edmonton, Alberta, Canada

Current good manufacturing practice (cGMP) islet processing facilities provide an ultraclean environment for the safe production of clinical grade islets for transplantation into immunosuppressed diabetic recipients. The objective of this study was to monitor the rate of microbial contamination in islet products after implementation of good manufacturing practice conditions. Fluid samples for microbial contamination were collected at the following steps: from the pancreas transport solution upon arrival of the organ (n = 157), after surface decontamination of the pancreas with antiseptic agents (n = 89), from islet supernatant at the end of the isolation (n = 104), and from islet supernatant as a final transplantable product after culture (n = 53). Bacterial, fungal, and mycoplasma cultures were conducted for 2, 2, and 3 weeks, respectively. Microbial contamination was detected in 31% of transport solution. The contamination was not associated with the presence of the duodenum during the preservation, cold ischemia time, or procurement team (local vs. distant). Surface decontamination of the pancreas resulted in clearance of 92% of the microbial contamination. Six preparations at the end of the isolation revealed microbial growth. All were de novo contamination during the processing. Fifty-three preparations that met our release criteria in terms of product sterility were transplanted into type 1 diabetic patients. In two instances, positive culture of the islet preparation was reported after transplantation had occurred. No patient showed any clinical findings suggestive of infection or any radiological abnormalities suggestive of abscess; a single dose of antibiotic coverage was given routinely to recipients prior to islet infusion. Although transport solution carries a high risk of microbial contamination, most contaminants become undetectable during islet processing. Microbial contamination in final products is rare, but de novo contamination still occurs during processing even under cGMP conditions.

Key words: Microbial contamination; Quality control; Islet isolation; Antibiotics

Address correspondence to Jonathan R. T. Lakey, Ph.D., at his current address: CEO, Clinical Islet Transplantation Group Inc., Edmonton, Alberta, Canada T6G 2C8. Tel: (780) 407-8749; Fax: (780) 407-8760; E-mail: Jonlakey@rogers.blackberry.net

¹Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
²Division of Reproduction and Endocrinology, King's College, London, UK

Transplantation of microencapsulated islets may reduce hyperglycemia in the absence of immunosuppression. However, the efficiency of microencapsulated islet transplantation is low, requiring more islets to achieve normoglycemia than in vascularized islet transplantation. Exendin-4 (a glucagon-like receptor agonist) has been previously shown to improve islet transplantation outcome in rodents. We investigated whether this treatment would enhance the function of microencapsulated islets in vitro and in vivo. Encapsulated or naked islets were cultured with or without exendin-4 for 72 h. To test in vitro function, insulin release and glucose oxidation rates were measured in the absence or presence of exendin-4. In addition, in vivo function of a minimal mass of 350 microencapsulated islets was assessed by syngeneic transplantation into the peritoneal cavity of alloxan-diabetic mice. Glucose oxidation rates of microencapsulated islets were improved by 72-h pretreatment with exendin-4. Insulin release was increased both acutely after glucose stimulation and over a 40-h culture period by the presence of exendin-4. Transplantation outcome of microencapsulated islets cultured with exendin-4 was initially improved, but by day 7 there were no differences compared with control cultured microencapsulated islets. Culture of microencapsulated islets with exendin-4 increases glucose oxidation and insulin release rates, but the increased function seen in vitro was not enough to improve the long term outcome in a transplantation model.

Key words: Diabetes; Islet transplantation; Microencapsulation; GLP-1; Exendin-4; Alginate

Address correspondence to Dr. Aileen King, Hodgkin Building, Guy's Campus, King's College, London, London, SE1 1UL, UK. Tel: +44 207 848 6279; Fax: +44 207 848 6280; E-mail: aileen.king@kcl.ac.uk

¹Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL 33136, USA
²Department of Dermatology and Cutaneous Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, 33136, USA
³Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL 33136, USA
⁴DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL 33136, USA

Tacrolimus is an immunosuppressive agent used in solid organ and islet transplantation. Its topical form has shown benefit in the treatment of inflammatory skin conditions. Although tacrolimus has a wide spectrum of side effects, dermatological complications related to systemic tacrolimus therapy are limited in the literature. Atopic dermatitis (AD) is a chronic pruritic cutaneous condition that usually begins in infancy and is characterized by an increased Th2 response. We report the case of a patient with type 1 diabetes mellitus (T1DM) and history of AD latent for 10 years who developed severe dermatitis and alopecia 5 months after undergoing allogeneic islet transplantation and initiating a steroid-free immunosuppressive regimen with sirolimus and tacrolimus maintenance. After exclusion of other possible causes for the progression and exacerbation of the clinical presentation of AD, discontinuation of tacrolimus and introduction of mycophenolate mofetil resulted in full remission of the symptoms. The beneficial effects of tacrolimus withdrawal suggest a cause-effect relationship between this adverse event and the utilization of the drug. Islet graft function remained stable after modification of the therapeutic regimen (stable glycemic control and unchanged C-peptide).

Key words: Atopic dermatitis; Alopecia areata; Diabetes; Immunosuppression; Islet transplantation; Tacrolimus

Address correspondence to Rodolfo Alejandro, M.D., Diabetes Research Institute (R-134), 1450 NW 10th Avenue, Miami, FL 33136, USA. Tel: (305) 243-5324; Fax: (305) 243-1058; E-mail: ralejand@med.miami.edu

*These two authors contributed equally to this report.

Department of Ophthalmology, Lund University Hospital, Lund, Sweden

In this study we wanted to examine how an adult neuroretina from an animal with an eye similar to the human one survives in vitro. We also wanted to investigate how the culture process affects the adult retina when used in a transplantation paradigm. Full-thickness neuroretinal sheets from adult porcine eyes were dissected into pieces measuring 3 mm in diameter. These were kept in culture for 1-3 days. After this time, the explants were fixed or transplanted subretinally to adult pigs, which were killed after 72-74 days. Transplanted eyes, as well as tissue kept in culture only, were processed for hematoxylin and eosin staining and immunohistochemistry. Explants kept 1 day in vitro (DIV) displayed the normal morphology. In these specimens, single pyknotic cells were evident in the outer nuclear layer (ONL) and ganglion cell layer, but were more frequent in the inner nuclear layer (INL). After longer times in vitro, severe degenerative changes appeared. Transplanted explants kept 1 DIV prior to transplantation exhibited normal retinal lamination in two out of four specimens. Transducin and recoverin labeling revealed photoreceptors with inner segments in these grafts. Rod bipolar cells displayed a normal morphology. Vertically arranged Müller cells were also seen in the laminated grafts. Two of the three transplants kept 2 DIV displayed minimal lamination. Eyes with transplants kept 3 DIV prior to transplantation displayed degenerated grafts in all eyes. This study shows that adult porcine neuroretinal explants kept in culture for 1 day display a normal morphology in their major part. Additionally, 1-day explants can survive transplantation with retained morphology even after several months. This indicates the possibility of storing adult donor tissue between harvest and transplantation. The culture system may also be used in the future as a tool for manipulating retinal donor tissue prior to transplantation.

Key words: Histology; Photoreceptors; In vitro; Pig; Retinal explants; Cell biology

Address correspondence to Karl Engelsberg, Department of Ophthalmology, Lund University Hospital, SE-221 85 Lund, Sweden. Tel: +46 46 2220771; Fax: +46 46 2220774; E-mail: karl.engelsberg@med.lu.se

¹Fondazione IRCCS Ospedale Maggiore, Department of Neurological Sciences, Stem Cell Laboratory, Dino Ferrari Center, University of Milan, Milan, Italy
²Fondazione Matarelli, Ospedale Fatebenefratelli e Oftalmico, Laboratory of Matarelli Foundation for Blood Diseases, Milan, Italy
³Department of Neurology and Laboratory of Neuroscience, Dino Ferrari Center, University of Milan-Medical School, IRCCS Istituto Auxologico Italiano, Milan, Italy
⁴Fondazione IRCCS Ospedale Maggiore, Bone Marrow Transplantation Center, University of Milan-Medical School, Milan, Italy

In animal models of neurological disorders for cerebral ischemia, Parkinson's disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFR^p75) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophin's expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.

Key words: Mesenchymal stem cells; Transplantation; Neurotrophin; Astroglial cells

Address correspondence to Yvan Torrente, Stem Cell Laboratory, Department of Neurological Sciences, University of Milan, Padiglione Ponti, Fondazione IRCCS Ospedale Maggiore of Milan, via Francesco Sforza 35, 20122 Milan, Italy. Tel: +39 02-55033874; Fax: +39-02-50320430; E-mail: torrenteyvan@hotmail.com

¹Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8512, Japan
²Department of Immunology and Medicine, St. Marianna University School of Medicine, Kawasaki 216-8512, Japan
³Department of Orthopaedic Surgery, Keio University School of Medicine, Tokyo 160-8582, Japan
⁴Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan

The present study investigated whether plasma could be useful as a scaffold for cell transplantation in rats with spinal cord injury (SCI). Transplantation of cells with plasma promoted the recovery of SCI-induced motor dysfunction. Immunohistochemical analysis revealed that the grafted cells had differentiated into the neural lineage. When dissociated neural precursor cells were cultured with plasma, extensive neurite outgrowth was observed along with increased expression of p35 and NF68. Neural markers were also expressed by the cultured cells. Culture with plasma reduced thymidine incorporation, but promoted cell growth and increased the RNA contents. These findings suggest that the cells underwent differentiation into neurons in the presence of plasma. In conclusion, plasma could be a promising scaffold for cell transplantation therapy.

Key words: Neural precursor cell; Plasma; Serum; BBB score; Spinal cord injury

Address correspondence to Mitsuko Takenaga, Ph.D., Institute of Medical Science, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki 216-8512, Japan. Tel: +81-44-977-8111; Fax: +81-44-976-3747; E-mail: m2take@marianna-u.ac.jp

¹Second Department of Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
²Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6b-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E₂ (PGE₂), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.

Key words: Cryopreservation; Hepatocyte transplantation; Organic anion transporter; Cytochrome P450; Transporter

Address correspondence to Tomotake Koizumi, M.D., Second Department of Surgery, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Tel: +81-3-3784-8541; Fax: +81-3-3784-5835; E-mail: t-koiz@med.showa-u.ac.jp

¹Department of Simulation Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
²Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
³Department of Transplantation Immunology, Kyoto University Hospital, Kyoto 606-8507, Japan

Green tea polyphenols are known to protect allogenic donor tissues from acute rejection by their recipients. This immunosuppressive effect may be generated by a unique chemical property of the major component, epigallocatechin-o-gallate (EGCG), which can block specific cell surface molecules of the donor tissues. To test this hypothesis, we examined the effects of EGCG on the murine mixed lymphocyte reactions. EGCG treatment of stimulator cells significantly attenuated the proliferation of responder T cells. The proliferation did not recover upon the secondary stimulations by fresh untreated cells or exogenous IL-2. Flow cytometric analyses showed that EGCG treatment decreased the staining intensities of various cell surface molecules including MHC II, which plays a major role in antigen presentation, and B7.1, B7.2, and their ligand, CD28, which are required for costimulatory signals in T-cell activation. These results suggest that an anergic state of alloreactive T cells may be induced by either weakening of antigen signaling or blockage of costimulatory signals with EGCG. Other possible mechanisms behind the immunosuppressive effect and a potential use of EGCG treatment of donor tissues in transplantation medicine are discussed.

Key words: Costimulatory signals; Allorecognition; Polyphenol; EGCG

Address correspondence to Suong-hyu Hyon, Ph.D., Department of Simulation Medical Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Tel/Fax: 81-75-751-4109; E-mail: biogen@frontier.kyoto-u.ac.jp

Department of Cell Biology, Beijing Institute of Basic Medical Sciences, Beijing 100850, China

Previous data have demonstrated that mesenchymal stem cells (MSCs) can exert immunomodulatory activity in vitro, in which of the process nearly all kinds of immune cell subsets are involved. However, there is still a paucity of information about whether and why MSCs inhibit the ongoing immune responses in vivo. Working in a murine splenocyte transfusion model across the major histocompatibility barrier (C57BL/6 --> BALB/c, H2^b --> H2^d), we have found that MSC coinfusion prolongs the mean survival time (MST) of the recipient mice in a dose-dependent manner and reduces graft-versus-host-associated histopathology in comparison to the allosplenocyte transfusion controls. In vivo eGFP tracing with polymerase chain reaction analysis revealed that grafted MSCs could migrate and settle into the lungs, spleen, liver, intestine, and skin shortly after administration. Further investigations into the functional characteristics of intrasplenic lymphocytes showed that their proliferation and cytotoxic activity against P815 cells (H2^d) were significantly restrained by MSC cotransfer. FACS analysis demonstrated that MSC infusion not only increased the proportion of CD4⁺ subset but also decreased that of CD8⁺ cells at the belated observation points, resulting in the increase of the ratio of CD4⁺/CD8⁺ cells. Also, in contrast to the slight increase of the proportion of CD4⁺CD25⁺ T regulatory cells (Tregs) in MSC cotransfer mice, the ratio of Tregs/CD8⁺ cells was dramatically elevated. Furthermore, RT-PCR analysis on the cytokine array of IL-2, IL-4, IL-12, TNF-a, and TGF-b in recipient splenocytes implied the Th1 to Th2 polarization. Therefore, it is deducible that alteration in the proportions of different T-lymphocyte subsets may be one of the main mechanisms by which grafted MSCs suppress the ongoing immune responses in vivo. The study here might provide some new clues for the design of therapeutic approaches for MSC transplantation.

Key words: Mesenchymal stem cells; In vivo; Immunomodulation; Lymphocyte subsets; Murine model

Address correspondence to Dr. Ning Mao, Department of Cell Biology, Beijing Institute of Basic Medical Sciences, 27 Tai-Ping Road, Beijing 100850, China. Tel: 8610-66931318; Fax: 8610-68213039; E-mail: maoning@nic.bmi.ac.cn or maoning931318yahoo.com

*These authors contributed equally to this work.