ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 14, NUMBER 6, 2005

Cell Transplantation, Vol. 14, pp. 339-351, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Neural Development by Transplanted Human Embryonal Carcinoma Stem Cells Expressing Green Fluorescent Protein

R. Stewart,1 M. Lako,2 G. M. Horrocks,1 and S. A. Przyborski1,3

1School of Biological and Biomedical Science, University of Durham, South Road, Durham DH1 3LE, UK
2Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, NE1 3BZ, UK
3ReInnervate Limited, Old Shire Hall, Old Elvet, Durham, DH1 3HP, UK

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.

Key words: Differentiation; Transplantation; Neural; Stem cell; Human; Cell tracking

Address correspondence to Stefan A. Przyborski, School of Biological and Biomedical Science, University of Durham, South Road, Durham DH1 3LE, UK. Tel: +44 (0)191 3341341; Fax: +44 (0)191 3341201; E-mail: stefan.przyborski@durham.ac.uk

Cell Transplantation, Vol. 14, pp. 353-365, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Intrathecal Grafting of Porcine Chromaffin Cells Reduces Formalin-Evoked c-Fos Expression in the Rat Spinal Cord

J. C. Sol,1 R. Y. Li,1 B. Sallerin,1 S. Jozan,1 H. Zhou,2 V. Lauwers-Cances,3 F. Tortosa,1 P. Chaynes,1 C. Mascott,1 R. Bastide,1 and Y. Lazorthes1

1Laboratory of Pain and Cell Therapy, Rangueil Medical School, 133 route de Narbonne, 31062, Toulouse Cedex, France
2Histology Department, Rangueil Medical School, 133 route de Narbonne, 31062, Toulouse Cedex, France
3Department of Epidemiology, Medical School of Toulouse, 31073 Toulouse Cedex, France

Chromaffin cells from the adrenal gland secrete a combination of neuroactive compounds including catecholamines, opioid peptides, and growth factors that have strong analgesic effects, especially when administered intrathecally. Preclinical studies of intrathecal implantation with xenogeneic bovine chromaffin cells in rats have provided conflicting data with regard to analgesic effects, and recent concern over risk of prion transmission has precluded their use in human clinical trials. We previously developed a new, safer source of adult adrenal chromaffin cells of porcine origin and demonstrated an in vivo antinociceptive effect in the formalin test, a rodent model of tonic pain. The goal of the present study was to confirm porcine chromaffin cell analgesic effects at the molecular level by evaluating neural activity as reflected by spinal cord c-Fos protein expression. To this end, the expression of c-Fos in response to intraplantar formalin injection was evaluated in animals following intrathecal grafting of 106 porcine or bovine chromaffin cells. For the two species, adrenal chromaffin cells significantly reduced the tonic phases of the formalin response. Similarly, c-Fos-like immunoreactive neurons were markedly reduced in the dorsal horns of animals that had received injections of xenogeneic chromaffin cells. This reduction was observed in both the superficial (I-II) and deep (V-VI) lamina of the dorsal horn. The present study demonstrates that both xenogeneic porcine and bovine chromaffin cells transplanted into the spinal subarachnoid space of the rat can suppress formalin-evoked c-Fos expression equally, in parallel with suppression of nociceptive behaviors in the tonic phase of the test. These findings confirm previous reports that adrenal chromaffin cells may produce antinociception by inhibiting activation of nociceptive neurons in the spinal dorsal horn. Taken together these results support the concept that porcine chromaffin cells may offer an alternative xenogeneic cell source for transplants delivering pain-reducing neuroactive substances.

Key words: Cell therapy; Pain; Xenograft; Intrathecal transplantation; c-Fos

Address correspondence to J. C. Sol, M.D., Laboratory of Pain and Cell Therapy, Rangueil Medical School, University Paul Sabatier, 133 route de Narbonne, 31062 Toulouse, France. Tel: (33)5-62-88-90-38; Fax: (33)5-62-88-90-41; E-mail: ylazorth@cict.fr

Cell Transplantation, Vol. 14, pp. 367-374, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effects of Copper and Cross-Linking on the Extracellular Matrix of Tissue-Engineered Arteries

Shannon L. M. Dahl,1 Robert B. Rucker,2 and Laura E. Niklason1,3

1Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
2Department of Nutrition, University of California, Davis, CA 95616, USA
3Department of Anesthesiology, Duke University, Durham, NC 27708, USA

In many cases, the mechanical strengths of tissue-engineered arteries do not match the mechanical strengths of native arteries. Ultimate arterial strength is primarily dictated by collagen in the extracellular matrix, but collagen in engineered arteries is not as dense, as organized, or as mature as collagen in native arteries. One step in the maturation process of collagen is the formation of hydroxylysyl pyridinoline (HP) cross-links between and within collagen molecules. HP cross-link formation, which is triggered by the copper-activated enzyme lysyl oxidase, greatly increases collagen fibril stability and enhances tissue strength. Increased cross-link formation, in addition to increased collagen production, may yield a stronger engineered tissue. In this article, the effect of increasing culture medium copper ion concentration on engineered arterial tissue composition and mechanics was investigated. Engineered vessels grown in low copper ion concentrations for the first 4 weeks of culture, followed by higher copper ion concentrations for the last 3 weeks of culture, had significantly elevated levels of cross-link formation compared to those grown in low copper ion concentrations. In contrast, vessels grown in high copper ion concentrations throughout culture failed to develop higher collagen cross-link densities than those grown in low copper ion concentrations. Although the additional cross-linking of collagen in engineered vessels may provide collagen fibril stability and resistance to proteolysis, it failed to enhance global tissue strength.

Key words: Copper; Tissue engineering; Arteries; Collagen; Cross-links

Address correspondence to Laura E. Niklason, M.D., Ph.D., Department of Biomedical Engineering, Box 90281, 136 Hudson Hall, Duke University, Durham, NC 27708, USA. Tel: (919) 660-5149; Fax: (919) 684-5777; E-ail: nikla001@mc.duke.edu

Cell Transplantation, Vol. 14, pp. 375-389, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Hypothermic Maintenance 0f Hepatocyte Spheroids

Pamela H. Lai,1 Qin Meng,1* Timothy D. Sielaff,2** and Wei-Shou Hu1

1Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455-0132, USA
2Department of Surgery, University of Minnesota, Minneapolis, MN 55455-0132, USA

Primary hepatocytes form spheroids under some culture conditions. These spheroids exhibit many tissue-like ultrastructures and retain many liver-specific functions over a long period of time. They are attractive for many applications employing liver cells. The ability to maintain their viability and functions at a reduced temperature to allow for transportation to the site of their application will facilitate their use. Furthermore, with their structural and functional similarity, they could possibly be used as a model system for studying various liver ischemias. The effect of hypothermic treatment was assessed by oxygen consumption rate, ATP, H2O2, and caspase 8 content, as well as albumin and urea synthesis, during and posttreatment. No single outcome variable gives a superlative quantification of hypothermic damage. Taken together, the hypothermic treatment can be seen as increasingly damaging as the temperature decreases from 21°C to 15°C and 4°C. The addition of the chemical protectants glutathione, N-acetyl-L-cystein (NAC), and tauroursodeoxycholic acid (TUDCA) decreased the damaging effect of hypothermic treatment. This protection effect was even more profound when spheroids were preincubated with the protectant for 24 h, and was most prominent at 4°C. The viability of the hypothermically treated hepatocyte spheroids was confirmed by laser scanning confocal microscopy. The method reported provides a means of maintaining spheroids' viability and may allow for their distribution to application sites at a distance.

Key words: Hepatocyte; Spheroids; Hypothermic; Cold ischemia

Address correspondence to Wei-Shou Hu, 421 Washington Avenue SE, Minneapolis, MN 55455-0132, USA. Tel: (612) 626-7630; Fax: (612) 626-7246; E-mail: acre@cems.umn.edu

*Current address: Department of Chemical Engineering, Zhejiang University, Hanzhou, China.

**Current address: Virginia Piper Cancer Institute, Minneapolis, MN, 55407, USA.

Cell Transplantation, Vol. 14, pp. 391-396, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Effect of Antioxidants and a Caspase Inhibitor on Cryopreserved Rat Hepatocytes

Rie Fujita, Thomas Hui, Marjorie Chelly, and Achilles A. Demetriou

Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA

Hepatocyte transplantation and use of bioartificial liver support systems have been suggested as potential therapies for fulminant hepatic failure. Cryopreservation in liquid nitrogen is presently the major method of long-term storage of isolated hepatocytes. However, cryopreservation can result in low cell recovery and reduction in differentiated function. Several possible mechanisms of cell death during cryopreservation have been proposed. The most important mechanisms appear to be oxidative stress and apoptosis. In this study, we isolated fresh rat hepatocytes and cryopreserved them in three media: University of Wisconsin (UW) solution, an antioxidant-containing medium, and medium containing a caspase inhibitor. Viability and function of hepatocytes cryopreserved in these media were examined. Cryopreservation conditions had no effect on hepatocyte viability after thawing. However, after culture we found significant improvements in viability and function in both antioxidant- and caspase inhibitor-treated hepatocytes at 6 and 24 h.

Key words: Hepatocyte; Cryopreservation; Caspase inhibitor; Antioxidant; Apoptosis

Address correspondence to Achilles A. Demetriou, M.D., Ph.D., Cedars-Sinai Medical Center, 8700 Beverly Blvd., Suite 8215 North Tower, Los Angeles, CA 90048, USA. Tel: 1-310-423-5884; Fax: 1-310-423-0231; E-mail: achilles.demetriou@cshs.org

Cell Transplantation, Vol. 14, pp. 397-401, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Isolated Hepatocytes Versus Hepatocyte Spheroids: In Vitro Culture of Rat Hepatocytes

Giovanni Ambrosino,1 Stefano M. M. Basso,1 Sergio Varotto,1 Enrico Zardi,2 Antonio Picardi,2 and Davide F. D'Amico1

1Department of Surgical and Gastroenterologic Sciences, Liver Transplant Unit, School of Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy
2Interdisciplinary Center for Biomedical Research (CIR), Laboratory of Internal Medicine and Hepatology, University "Campus Bio-Medico," Roma, Italy

The use of hepatocytes that express liver-specific functions to develop an artificial liver is promising. Unfortunately, the loss of specialized liver functions (dedifferentiation) is still a major problem. Different techniques, such as collagen entrapment, spherical multicellular aggregates (spheroids), and coculture of hepatocytes with extracellular matrix, have been used to improve the performance of hepatocytes in culture. The aim of this study was to compare two different models of hepatocyte isolation in culture: isolated hepatocytes (G1) and hepatocyte spheroids (60% hepatocytes, 40% nonparenchymal cells, and extracellular matrix) (G2). To test functional activity of hepatocytes, both synthetic and metabolic, production of albumin and benzodiazepine transformation into metabolites was tested. G2 showed a high albumin secretion, while a decrease after 15 days of culture in G1 was noted. Diazepam metabolites were higher in G2 than in G1 in all samples, but had statistical significance at days 14 and 21 (p < 0.01). The glycogen content, after 30 days of culture, was very low in G1 (14.2 ± 4.4%), while in G2 it was 72.1 ± 2.6% (p < 0.01). Our study confirms the effectiveness of a culture technique with extracellular matrix and nonparenchymal cells. Maintenance of a prolonged functional activity has been related to restoration of cell polarity and close cell-to-cell contact. We showed that isolated hepatocytes maintain their functional activity for a period significantly reduced, when compared to the hepatocyte spheroids. We confirmed the role of extracellular matrix as a crucial component to promote hepatocyte homeostasis, and the close link between cellular architecture and tissue-specific functions.

Key words: Hepatocyte spheroids; Isolation; Culture; Matrix; Benzodiazepine; Albumin

Address correspondence to Giovanni Ambrosino, M.D., at his current address: Department of Surgery-HPB Unit, Divisione Chirurgia I, OSP "San Bortolo," 36100 Vicenza, Italy. E-mail: giovanni.ambrosino@unipd.it

Cell Transplantation, Vol. 14, pp. 403-409, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Peripheral Blood Cytotoxic Lymphocyte Gene Transcript Levels Differ in Patients With Long-Term Type 1 Diabetes Compared to Normal Controls

Dongmei Han,1 Jenifer Leith,1 Rodolfo Alejandro,1,2 Wade Bolton,3 Camillo Ricordi,1,2,4 and Norma S. Kenyon1,2,4

1Diabetes Research Institute, University of Miami School of Medicine, 1450 N.W. 10th Avenue, Miami, FL 33136, USA
2Department of Medicine, University of Miami School of Medicine, 1450 N.W. 10th Avenue, Miami, FL 33136, USA
3Beckman-Coulter Inc., Miami, FL 33136, USA
4Department of Surgery, University of Miami School of Medicine, 1450 N.W. 10th Avenue, Miami, FL 33136, USA

The purpose of this study was to compare mRNA levels of the cytotoxic lymphocyte (CL) gene products: granzyme B (GB), perforin (P), and fas ligand (FasL) in patients with long-term type 1 diabetes and healthy controls. The objective was to utilize this information to follow patients as they undergo islet cell transplantation at our center and to determine if changes in CL gene transcript levels correlate with graft status. We have measured mRNA levels for CL genes in peripheral blood samples from 65 long-term (>5 years) type 1 diabetes patients and 29 healthy controls. Total RNA was extracted from EDTA anticoagulated peripheral blood samples and reverse transcribed into first-strand cDNA using SuperScript II reverse Transcriptase. Quantitative, real-time PCR was utilized to determine CL gene transcript levels. mRNA levels of P and FasL genes were found to be significantly lower for patients with type 1 diabetes compared to normal controls (p < 0.05). However, there was no significant difference for GB mRNA levels between patients and controls (p > 0.05). The decreased expression of P and FasL in patients with long-term type 1 diabetes might contribute to the inability to maintain normal levels of peripheral tolerance, which is essential for protection from autoimmune disease.

Key words: Cytotoxic lymphocyte genes; Type 1 diabetes; Peripheral blood; Granzyme B; Perforin; Fas ligand

Address correspondence to Dongmei Han, Ph.D., Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-2275; Fax: (305) 243-1042; E-mail: dhan@med.miami.edu

Cell Transplantation, Vol. 14, pp. 411-421, 2005
0963-6897/05 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Intrasplenic Transplantation of Encapsulated Genetically Engineered Mouse Insulinoma Cells Reverses Streptozotocin-Induced Diabetes in Rats

Takeshi Aoki,1 Hongxiang Hui,2 Yutaka Umehara,1 Sergio LiCalzi,2 Achilles A. Demetriou,1 Jacek Rozga,1 and Riccardo Perfetti2

1Surgical Research, Department of Surgery, Cedars-Sinai Medical Center, David Geffen UCLA School of Medicine, Los Angeles, CA 90048, USA
2Division of Diabetes, Endocrinology and Metabolism, Cedars-Sinai Medical Center, David Geffen UCLA School of Medicine, Los Angeles, CA 90048, USA

Pancreatic islet transplantation is limited by shortage of donor organs. Although b-cell lines could be used, their secretion of insulin is characteristically glucose independent and immunoisolation is required. Here we show that intrasplenic transplantation of encapsulated glucose-responsive mouse insulinoma cells reversed streptozotocin (STZ)-induced diabetes in rats. MIN-6 cells derived from a transgenic mouse expressing SV 40 large T antigen in pancreatic b-cells were transfected with minigene encoding for human glucagon-like-peptide-1 under the control of rat insulin promoter. The cells were encapsulated in alginate/poly-L-lysine and used for cell transplantation in STZ-diabetic rats. Rats with nonfasting blood glucose (n-FBG) greater than 350 mg/dl were used. In group I rats (n = 6) 20 million encapsulated cells were injected into the spleen. Group II rats (n = 6) received empty capsules. n-FBG was measured biweekly. After 4 and 8 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed in group I; normal rats served as controls. Plasma insulin level was measured every other week (RIA). After 8 weeks, spleens were removed 1 day before sacrifice. In rats transplanted with cells the n-FBG was 100-150 mg/dl until the end of the study. After splenectomy, all cell recipients became diabetic (glucose 400 ± 20 mg/dl). Transplanted rats showed increase in body weight and insulin production (3.3 ± 1.0 ng/ml versus 0.92 ± 0.3 ng/ml; p < 0.01) and had normal IPGTT. Spleens contained capsules with insulin-positive cells. Overall, data from this work indicate that intrasplenic transplantation of xenogeneic encapsulated insulin-producing cells without immunosuppression reversed diabetes in rats. Excellent survival and function of the transplanted cells was due to the fact that the cells were separated from the bloodstream by the immunoisolatory membrane only and insulin was delivered directly to the liver (i.e., in a physiological manner).

Key words: Diabetes; Insulin; Insulinoma cells; Encapsulation; Pancreatic islets; Transplantation technique; Streptozotocin

Address correspondence to Jacek Rozga, M.D., Ph.D., Cedars-Sinai Medical Center, 8700 Beverly Blvd., D-4018, Los Angeles, CA 90048, USA. Tel: 310-423-7702; Fax: 310-423-0224; E-mail: Jacek.Rozga@cshs.org