ognizant Communication Corporation

CELL TRANSPLANTATION
The Regenerative Medicine Journal

ABSTRACTS
VOLUME 14, NUMBER 8, 2005

Cell Transplantation, Vol. 14, pp. 527-536, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

b1 Integrin as a Xenoantigen in Fetal Porcine Mesencephalic Cells Transplanted Into the Rat Brain

Caroline Martin, Benoît Melchior, Véronique Nerrière-Daguin, Philippe Naveilhan, Jean-Paul Soulillou, and Philippe Brachet

Institut National de la Santé et de la Recherche Médicale, Unité 643 and Institut de Transplantation et de Recherche en Transplantation, F-44093 Nantes, France

Xenografts of fetal porcine mesencephalic cells implanted into the rat striatum are generally rejected within several weeks. The fetal donor mesencephalon predominantly consists of neurons, but also contains microglial and endothelial cells, which are more immunogenic. In the present work, we investigated the occurrence of donor endothelial cells in grafts of porcine mesencephalic cells implanted into the rat striatum. Pig endothelial cells were monitored by immunochemical methods, using a monoclonal antibody (mAb) that recognizes a peptidic epitope of the porcine b1 integrin, and isolectin IB4, for the staining of the Gala1,3Gal epitope. The analysis also involved the detection of the pig hyaluronate receptor CD44, and the cell adhesion molecule CD31. The anti-b1 integrin mAb revealed endothelial-like cells in grafts of porcine mesencephalic cells as soon as 1 week after implantation. A similar staining pattern was obtained with the IB4 lectin. Unlike aortic endothelial cells, these pig brain-derived endothelial-like cells were not recognized by the anti-CD44 antibody. They also failed to express the CD31 adhesion molecule, a fact which suggests that they remained poorly mature, even in grafts maintained during 45 days in immunosuppressed rats. Interestingly, a strong expression of β1 integrin immunoreactivity was noticed in a large proportion (80%) of the cells freshly dissociated from the fetal pig mesencephalic tissue. The immunoreactivity decreased progressively after transplantation of the cells into the rat brain. This observation suggests that dissociated neuroblasts are capable of a temporary expression of b1 integrin. This molecule is known to participate in the process of cell sorting and migration in the developing brain. Hence, its expression could be the hallmark of a rescue mechanism triggered by the disruption of the cell/matrix interactions during the dissociation of the fetal mesencephalon. This disruption might account for part of the dramatic cell death process that occurs during the manipulation of the donor tissue.

Key words: Xenotransplantation; Brain; Pig neuron; Endothelial cell; b1 integrin; Graft rejection

Address correspondence to Philippe Brachet, INSERM U643, CHU Hotel Dieu, 30 bd Jean Monnet, F-44093 Nantes, France. E-mail: pbrachet@nantes.inserm.fr




Cell Transplantation, Vol. 14, pp. 537-550, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Creatine Supplementation Improves Dopaminergic Cell Survival and Protects Against MPP+ Toxicity in an Organotypic Tissue Culture System

Robert H. Andres,1 Angelique D. Ducray,1 Alberto Pérez-Bouza,1 Uwe Schlattner,2 Alexander W. Huber,1 Sandra H. Krebs,1 Rolf W. Seiler,1 Theo Wallimann,2 and Hans R. Widmer1

1Department of Neurosurgery, University Hospital, 3010 Berne, Switzerland
2Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), 8093 Zurich, Switzerland

Cell replacement therapy using mesencephalic precursor cells is an experimental approach for the treatment of Parkinson's disease (PD). A significant problem associated with this procedure is the poor survival of grafted neurons. Impaired energy metabolism is considered to contribute to neuronal cell death after transplantation. Creatine is a substrate for mitochondrial and cytosolic creatine kinases (CK) and buffers cellular ATP resources. Furthermore, elevated cellular creatine levels facilitate metabolic channeling and show antiapoptotic properties. Exogenous creatine supplementation therefore might offer a tool for improvement of dopaminergic neuron survival. The present study aimed at investigating the effects of creatine on cell survival of rat embryonic day 14 (E14) ventral mesencephalic neurons grown as organotypic free-floating roller tube (FFRT) cultures. We found that the brain-specific isoform of CK (BB-CK) and the ubiquitous mitochondrial isoform (uMt-CK) are expressed at high levels in FFRT cultures and colocalize with tyrosine hydroxylase immunoreactive (TH-ir) cells. Exposure of these cultures to creatine induced an increase in the content of the BB-CK isotype. Creatine (5 mM) administration starting at day in vitro (DIV) 7 resulted in a significant increase (+35%) in TH-ir cell density at DIV21. In addition, we observed that creatine treatment provided neuroprotection against 1-methyl-4-phenyl pyridinium ion (MPP+)-induced TH-ir cell loss in the FFRT culture system, resulting in a significantly higher density (+19%) of TH-ir neurons in creatine-treated cultures compared to corresponding controls. The decrease of TH-ir neurons in the MPP+-treated group corresponded with an increase in immunoreactivity for active caspase-3, an effect that was not seen in the group receiving creatine supplementation. In conclusion, our data imply that creatine administration is beneficial for the survival of TH-ir neurons encountering harmful conditions.

Key words: Ventral mesencephalic cell cultures; Creatine; Tyrosine hydroxylase; Free-floating roller tubes; MPP+; Parkinson's disease

Address correspondence to Hans R. Widmer, Ph.D., Department of Neurosurgery, University Hospital, CH-3010 Berne, Switzerland. Tel: +41 31 632 27 70; Fax: +41 31 382 24 14; E-mail: hanswi@insel.ch




Cell Transplantation, Vol. 14, pp. 551-564, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Survival of Rat or Mouse Ventral Mesencephalon Neurons After Cotransplantation With Rat Sertoli Cells in the Mouse Striatum

R. Shamekh,1,2 J. Newcomb,1,2 J. Mallery,1,2 C. J. Cassady,1,2 S. Saporta,1,2,3 D. F. Cameron,1,2,3 P. R. Sanberg,1,2,4,5 and A. E. Willing1,2,3,4,5

1Center of Excellence for Aging & Brain Repair, University of South Florida, College of Medicine, Tampa, FL 33612, USA
2Department Neurosurgery, University of South Florida, College of Medicine, Tampa, FL 33612, USA
3Department of Anatomy, University of South Florida, College of Medicine, Tampa, FL 33612, USA
4Department of Pharmacology and Therapeutics, University of South Florida, College of Medicine, Tampa, FL 33612, USA
5Department of Pathology, University of South Florida, College of Medicine, Tampa, FL 33612, USA

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

Key words: Parkinson's disease; Xenograft; Microglia; Astrocytes; Dopaminergic neurons

Address correspondence to Dr. A. E. Willing, University of South Florida, Department of Neurosurgery, MDC 78, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA. E-mail: awilling@hsc.usf.edu




Cell Transplantation, Vol. 14, pp. 565-571, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Seeding of Corneal Wounds by Epithelial Cell Transfer From Micropatterned PDMS Contact Lenses

Christopher J. Pino,1 Frederick R. Haselton,1,2 and Min S. Chang2

1Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USA
2Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, TN 37235, USA

Persistent corneal wounds result from numerous eye disorders, and to date, available treatments often fail to accelerate reepithelialization, the key initial step in wound healing. To speed reepithelialization, we explored a cell-transfer transplant method utilizing polydimethylsiloxane (PDMS) contact lenses to deliver epithelial cells derived from limbal explants directly within a corneal wound. Human primary epithelial cells and an immortalized corneal epithelial cell line (HCE-SV40) grew well on PDMS contact lenses and their morphology and growth rates where similar to cells grown on tissue culture polystyrene. To initially study cell transfer from PDMS, HCE-SV40 cells were seeded onto PDMS with or without micropatterned posts. After a day in culture, HCE-SV40 cells attached to the unpatterned PDMS uniformly, whereas on micropatterned PDMS they appeared to attach primarily between posts. The cell-covered PDMS contacts were then placed cell-side down onto tissue culture plastic and, after 1, 2, or 3 days, the PDMS contact was removed and the transferred cells were trypsinized and counted. Micropatterned PDMS contact lenses with 100-μm-diameter posts and a post height of 40 μm transferred three times as many cells as unpatterned PDMS. Cell transfer to a wounded cornea was tested in a pig cornea organ culture model de-epithelialized by alkali treatment. Post micropatterned PDMS contact lenses were seeded with labeled HCE-SV40 cells at a density 50,000 cells/cm2 and applied to the wounded pig corneas. After 24, 48, or 96 h of application, PDMS contact lenses were removed, corneas fixed with formaldehyde, and sectioned. After 48 h, epithelial cells transferred from post micropatterned contact lenses to provide 35% epithelial coverage of denuded pig corneas; after 96 h coverage was 65%. We conclude that cell transfer from epithelial-coated PDMS contact lenses micropatterned with posts provides a promising approach to reepithelialize corneal surfaces.

Key words: Cornea; Wound healing; Primary epithelial cells; Transfer; Contact lens

Address correspondence to Rick Haselton, Box 1510 Station B, Vanderbilt University, Nashville TN, 37235, USA. Tel: (615) 322-6622; Fax: (615) 343-7919; E-mail: rick.haselton@vanderbilt.edu




Cell Transplantation, Vol. 14, pp. 573-583, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Selection of Cell Source for Ligament Tissue Engineering

Zigang Ge,1 James Cho Hong Goh,1,2 and Eng Hin Lee1

1Department of Orthopaedic Surgery, National University of Singapore, Singapore, 119260
2Division of Bioengineering, National University of Singapore, Singapore, 119260

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as a-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and a-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.

Key words: Mesenchymal stem cell; Fibroblast; Anterior cruciate ligament; Tissue engineering

Address correspondence to James C. H. Goh, Ph.D., Department of Orthopedic Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119260. Tel: (65)-67724423; Fax: (65)-67744082; E-mail: dosgohj@nus.edu.sg




Cell Transplantation, Vol. 14, pp. 585-594, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Effects of Cryopreservation on Human Hepatocytes Obtained From Different Sources of Liver Tissue

Claire Terry, Ragai R. Mitry, Sharon C. Lehec, Paolo Muiesan, Mohamed Rela, Nigel D. Heaton, Robin D. Hughes, and Anil Dhawan

Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, Denmark Hill, London, SE5 9RS, UK

Successful cryopreservation of human hepatocytes is important to establish hepatocyte banks for clinical use or in vitro research. The availability of donor tissue from unused liver segments/lobes and non-heart-beating donors (NHBD) has provided newer sources of hepatocytes. The quality of hepatocytes at the time of cryopreservation is important as cells isolated from liver tissue of borderline quality may not withstand the stresses associated with cryopreservation and subsequent thawing. Human hepatocytes were cryopreserved after isolation from mainly donor tissues (n = 40). In vitro assessment of the viability and function of the fresh and thawed cryopreserved hepatocytes was performed. Viability, attachment efficiency, enzyme activity, and albumin production of hepatocytes were all significantly decreased, and LDH leakage significantly increased, on thawing after cryopreservation. The viability of cryopreserved hepatocytes isolated from tissue rejected for orthotopic liver transplantation (36 ± 15%) was significantly lower than those isolated from tissue where part was used for liver transplantation (47 ± 14%, p = 0.002), but there were no significant differences in functional parameters. The viability of cryopreserved hepatocytes isolated from NHBD tissue (29 ± 9%, p = 0.001) and from steatotic donor tissue (35 ± 11%, p = 0.019) was significantly lower than those isolated from normal donor tissue (49 ± 14%). There was no difference in functional parameters, except for albumin production of hepatocytes from NHBD tissue (2.9 ± 1.0 mg/h/mg protein) being significantly lower than those from normal donor tissue (4.8 ± 2.8 mg/h/mg protein, p = 0.03). The viability and attachment efficiency of cryopreserved hepatocytes isolated from liver tissue from resections for tumors was significantly higher, and the LDH leakage significantly lower, than those isolated from all donor tissue. Hepatocytes isolated from NHBD and steatotic tissue were more vulnerable to the effects of cryopreservation. Further research is required to improve hepatocyte isolation and cryopreservation protocols for different types of liver tissue.

Key words: Hepatocytes; Cryopreservation; Donor tissue; Transplantation

Address correspondence to Dr. Anil Dhawan, Institute of Liver Studies, King's College Hospital, Denmark Hill, London, SE5 9RS, UK. Tel: +44 20 7346 3578; Fax: +44 20 7346 3760; E-mail: anil.dhawan@kcl.ac.uk




Cell Transplantation, Vol. 14, pp. 595-606, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Subcutaneous Transplantation of Islets Into Streptozocin-Induced Diabetic Rats

Craig R. Halberstadt,1 Deana Williams,1 Dwaine Emerich,2 Moses Goddard,2 Alfred V. Vasconcellos,2 William Curry,1 Aashim Bhatia,1 and Paul F. Gores 1

1Department of General Surgery, Carolinas Medical Center, Charlotte, NC 28232, USA
2LCT BioPharma, Inc., Providence, RI, USA

Pancreatic islet transplantation into type 1 diabetic patients is currently being performed by intraportal infusion. This method, albeit reproducible, has some disadvantages including potential development of portal hypertension, hemorrhage, and an inability to retrieve or detect the transplanted tissue. Other transplant sites have been examined in animal models including the omentum, peritoneal cavity, and the spleen. A transplant site that has not been successful in supporting functional islet tissue transplantation in humans is the subcutaneous space due primarily to the lack of a well-defined vascular bed. This site has many favorable characteristics such as ease of access for transplantation and potential for removal of the transplanted tissue with a minimally invasive surgical procedure. This report addresses the evaluation of a subcutaneously placed device for the support of rat syngeneic islet transplantation in a streptozocin-induced diabetic model. The data generated support the use of this device for islet engraftment. In addition, beta cell function in this device compared favorably with the function of islets transplanted to the renal subcapsular space as well as islets within the native pancreas.

Key words: Diabetes; Subcutaneous transplant; Islets; Syngeneic; Rat; IVGTT

Address correspondence to Craig Halberstadt, Carolinas Medical Center, Department of General Surgery, Cannon Research Building, P.O. Box 32861, Charlotte, NC 28232-2861, USA. Tel: (704) 355-2846; Fax: (704) 355-7203; E-mail: chalberstadt@carolinas.org