ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 17, NUMBER 12, 2008

Cell Transplantation, Vol. 17, pp. 1277-1293, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Administration of Autologous Bone Marrow Stem Cells Into Spinal Cord Injury Patients Via Multiple Routes Is Safe and Improves Their Quality of Life: Comprehensive Case Studies

L. F. Geffner,1 P. Santacruz,1 M. Izurieta,1 L. Flor,1 B. Maldonado,2 A. H. Auad,1 X. Montenegro,1 R. Gonzalez,3 and F. Silva3

1Hospital Luis Vernaza, JBGYE, Guayaquil, Ecuador
2SOLCA, Guayaquil, Ecuador
3DaVinci Biosciences, LLC, Costa Mesa, CA, USA

Presently, there is no cure or effective treatment for spinal cord injury (SCI). Studies in SCI patients have shown that for a treatment to be effective it must primarily improve their quality of life. Numerous studies have shown that stem cells represent an alternative treatment for various disorders and have shown promise in several disease/trauma states. For instance, the use of autologous CD34+ stem cells has been shown to ameliorate symptoms of several disorders such as leukemia, cardiomyopathy, diabetes, and several autoimmune diseases, including multiple sclerosis. For the first time, we report eight case studies of SCI (four acute, four chronic) with approximately 2 years of follow-up that were administered bone marrow stem cells (BMSCs) via multiple routes: directly into the spinal cord, directly into the spinal canal, and intravenous. Magnetic resonance imaging illustrated morphological changes in the spinal cord of some of the patients following BMSCs administration. Comprehensive evaluations demonstrate improvements in ASIA, Barthel (quality of life), Frankel, and Ashworth scoring. Moreover, in order to assess bladder function, we designed a simple numerical clinical scoring system that demonstrates significant changes in bladder function following BMSCs administration. To date, we have administration BMSCs into 52 patients with SCI and have had no tumor formations, no cases of infection or increased pain, and few instances of minor adverse events. These studies demonstrate that BMSCs administration via multiple routes is feasible, safe, and may improve the quality of life for patients living with SCI.

Key words: Spinal cord injury; Bone marrow stem cells (BMSCs); Quality of life

Address correspondence to Francisco Silva, DaVinci Biosciences, 1239 Victoria St., Suite 302, Costa Mesa, CA 92627, USA. Tel (949) 515-2828; Fax: (949) 515-2929; E-mail: fsilva@dvbiosciences.com

Cell Transplantation, Vol. 17, pp. 1295-1304, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Combined Treatment of Intrapancreatic Autologous Bone Marrow Stem Cells and Hyperbaric Oxygen in Type 2 Diabetes Mellitus

Esteban J. Estrada,1 Fabian Valacchi,1 Eduardo Nicora,1 Sergio Brieva,1 Claudio Esteve,1 Laura Echevarria,1 Tatiana Froud,2 Karina Bernetti,2 Shari Messinger Cayetano,2 Omaida Velazquez,5 Rodolfo Alejandro,2 and Camillo Ricordi2,3,4

1Stem Cell Argentina, Buenos Aires, Argentina
2Diabetes Research Institute, University of Miami, Miami, FL, USA
3Wake Forest Institute for Regenerative Medicine, Wake Forest University, Winston-Salem, NC, USA
4Karolinska Institute, Stockholm, Sweden
5Department of Surgery, University of Miami Hospital, Miami, FL, USA

The objective of this study was to determine whether the combination therapy of intrapancreatic autologous stem cell infusion (ASC) and hyperbaric oxygen treatment (HBO) before and after ASC can improve islet function and metabolic control in patients with type 2 diabetes mellitus (T2DM). This prospective phase 1 study enrolled 25 patients with T2DM who received a combination therapy of intrapancreatic ASC and periinfusion HBO between March 2004 and October 2006 at Stem Cells Argentina Medical Center Buenos Aires, Argentina. Clinical variables (body mass index, oral hypoglycemic drugs, insulin requirement) and metabolic variables (fasting plasma glucose, C-peptide, HbA1c, and calculation of C-peptide/glucose ratio) were assessed over quartile periods starting at baseline and up to 1 year follow-up after intervention. Means were calculated in each quartile period and compared to baseline. Seventeen male and eight female patients were enrolled. Baseline variables expressed as means ± SEs were: age 55 ± 2.14 years, diabetes duration 13.2 ± 1.62 years, insulin dose 34.8 ± 2.96 U/day, and BMI 27.11 ± 0.51. All metabolic variables showed significant improvement when comparing baseline to 12 months follow-up, respectively: fasting glucose 205.6 ± 5.9 versus 105.2 ± 14.2 mg/dl, HbA1c 8.8 ± 0.2 versus 6.0 ± 0.4%, fasting C-peptide 1.5 ± 0.2 versus 3.3 ± 0.3 ng/ml, C-peptide/glucose ratio 0.7 ± 0.2 versus 3.5 ± 0.3, and insulin requirements 34.8 ± 2.9 versus 2.5 ± 6.7 U/day. BMI remained constant over the 1-year follow-up. Combined therapy of intrapancreatic ASC infusion and HBO can improve metabolic control and reduce insulin requirements in patients with T2DM. Further randomized controlled clinical trials will be required to confirm these findings.

Key words: Stem cell; Hyperbaric oxygen; Type 2 diabetes; Autologous transplant

Address correspondence to Esteban Estrada, M.D., Stem Cell Argentina, Edison 1789 - (1640) Martínez, Buenos Aires, Argentina. Tel: 01154-11-4-836-2003; Fax: 0054-11-4-717-4996; E-mail: info@stemcellargentina.com or drestradaesteban@yahoo.com.ar and Camillo Ricordi, M.D., Diabetes Research Institute, University of Miami Miller School of Medicine, 1450 N.W. 10 Avenue, Miami, FL 33136, USA. Tel: 305 243-6913; Fax: 305 243-4404; E-mail: ricordi@miami.edu

Cell Transplantation, Vol. 17, pp. 1305-1313, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Human Islet Separation Utilizing a Closed Automated Purification System

A. S. Friberg, M. Ståhle, H. Brandhorst, O. Korsgren, and D. Brandhorst

Department of Oncology, Radiology & Clinical Immunology, Division of Clinical Immunology, Uppsala University Hospital, SE-75185 Uppsala, Sweden

A central step within the human islet isolation process is the separation of islets from contaminating exocrine tissue utilizing linear, continuous density gradients manufactured by means of manually controlled standard gradient makers (SGM). The present study was performed to develop a closed, automated purification system (APS) that customizes density gradient profiles aiming to standardize and optimize human islet purification. Digested human pancreata were pooled, split evenly, and incubated in UW solution according to our standard protocol (n = 11). Continuous density gradient centrifugation was performed in parallel in two refrigerated COBE 2991 cell separators loaded with light (1.076 g/ml) and heavy (1.097 g/ml) Ficoll utilizing either an SGM or two computer-controlled pumps connected to Ficoll-containing bags. Quality control included islet equivalent (IE) yield, purity, in vitro function, and islet cytokine expression. Gradient profiles demonstrated that the APS readily customizes linear and nonlinear gradients. In comparison to the SGM, the APS recovered a higher percentage of the expected volume of continuous gradients (90.0 ± 1.1% vs. 98.2 ± 2.0%, p < 0.05). Islet yield (120,468 ± 15,970 vs. 114,570 ± 15,313 IE, NS) and purity (51.7 ± 4.8% vs. 54.4 ± 4.9%, NS) were nearly identical utilizing the SGM or APS. Decreased MCP-1, IL-6, and IL-8 expression indicated that APS-purified islets were possibly exposed to less proinflammatory stress. Compared to standard procedures, similar success and gentle continuous density gradient separation of human islets is feasible utilizing the APS. The APS facilitates the standardization of this complex procedure according to cGMP standards.

Key words: Human islet isolation; Islet purification; Density gradient purification; Closed system

Address correspondence to Andrew S. Friberg, Department of Oncology, Radiology & Clinical Immunology, University Hospital, Dag Hammarskjölds väg 20 (C11), SE-75185 Uppsala, Sweden. Tel: +46-18-61-13984; Fax: +46-18-61-10222; E-mail: Andrew.Friberg@klinimm.uu.se

Cell Transplantation, Vol. 17, pp. 1315-1322, 2008
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E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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A Meta-Analysis of the Impact of the Two-Layer Method of Preservation on Human Pancreatic Islet Transplantation

Aditya Agrawal,1,2,3 Kurinchi Gurusamy,1 Steve Powis,2 Derek W. Gray,3 Barry Fuller,1 and Brian R. Davidson1

1Department of HPB and Liver Transplant Surgery, Royal Free Hospital, Royal Free and University College School of Medicine, London NW3 2PF, UK
2Department of Nephrology, Royal Free Hospital, Royal Free and University College School of Medicine, London NW3 2PF, UK
3Nuffield Department of Surgery, John Radcliffe Hospital, Oxford OX3 9DU, UK

There are conflicting reports about the effectiveness of perfluorocarbons used in the two-layer method (TLM) of pancreas preservation for human islet transplantation. The mechanism of action is unclear and the optimal role of this method uncertain. The study design was a meta-analysis of the evidence that TLM improves islet isolation outcomes. Pubmed, CENTRAL, EMBASE, Science Citation Index, and BIOSIS were searched electronically in January 2008. After selecting the relevant human trials for meta-analysis data relating to donor variables, study design, primary and secondary islet isolation outcomes were extracted. Electronic searches identified eight unique citations, describing 11 human studies that were eligible for the meta-analysis. When comparing TLM with preservation in University of Wisconsin (UW) solution, there was a statistically significant higher islet yield [WMD 711.55, 95% confidence interval (CI) 140.03-1283.07] in the TLM group. The proportion of transplantable preparations obtained was not significantly different (OR 1.30, 95% CI 0.89-1.88) between the two groups. The rate of successful islet isolations for marginal organs was higher in the TLM group (OR 6.69, 95% CI 1.80-24.87). Improved oxygenation and preservation of cellular bioengertics is thought to be the main underlying mechanism, although no single mechanism has yet been confirmed. There is currently no clear evidence that the TLM is beneficial in human islet transplantation. It may improve the preservation of marginal organs.

Key words: Meta-analysis; Two-layer method; Perfluorocarbon; Organ preservation; Pancreas; Islet transplantation

Address correspondence to Aditya Agrawal, Department of HPB and Liver Transplant Surgery, Royal Free Hospital, Rowland Hill Street, Hampstead, London NW3 2PF, UK. Tel: +44207 830 2757; Fax: +44207 830 2688; E-mail: agrawal_aditya@hotmail.com

Cell Transplantation, Vol. 17, pp. 1323-1336, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Disproportionate Hyperproinsulinemia, b-Cell Restricted Prohormone Convertase 2 Deficiency, and Cell Cycle Inhibitors Expression by Human Islets Transplanted Into Athymic Nude Mice: Insights Into Nonimmune-Mediated Mechanisms of Delayed Islet Graft Failure

Alberto M. Davalli,1 Lucia Perego,1 Federico Bertuzzi,13* Giovanna Finzi,2 Stefano La Rosa,2 Adam Blau,5** Claudia Placidi,2# Rita Nano,3 Luisa Gregorini,4 Carla Perego,6 Carlo Capella,2 and Franco Folli5

1Department of Medicine, Unit of Endocrinology and Metabolic Diseases, San Raffaele Hospital, Milan, Italy
2Department of Pathology, University of Insubria and Ospedale di Circolo, Varese, Italy
3Diabetes Department, Niguarda Hospital, Milan, Italy
4Department of Internal Medicine, University of Milan, Milan, Italy
5Department of Medicine, Division of Diabetes, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
6Institute of General Physiology and Biological Chemistry, University of Milan, Milan, Italy

To learn more about nonimmune-mediated islet graft failure, we transplanted different preparations (preps) of isolated human islets under the kidney capsule of streptozotocin (STZ)-diabetic nude mice. One month after the implantation of 1,000 or 2,000 islets, grafts were harvested for morphological, immunohistochemical, and ultrastructural analysis. Only a single islet prep cured the diabetes out of all the recipients, while the remaining preps showed only partial function after the implantation of 2,000 islets. Transplanted mice showed high circulating proinsulin levels but, with the exclusion of those bearing curative grafts, relatively low mature insulin levels. Engrafted b-cells showed positive carboxypeptidase E (CPE) and prohormone convertase 1 (PC1) staining, while prohormone convertase 2 (PC2) was undetectable. In contrast, PC2 was abundantly expressed by engrafted b-cells. Moreover, engrafted b-cells did not show evidence of replication, and preapoptotic b-cells, with intra- and extracellular amyloid deposition, were detected with electron microscopy. Cell cycle inhibitors p16INK4, p21WAF1, and p27Kip1 were abundantly expressed in the islet grafts and showed a predominant nuclear localization. In conclusion, diabetic nude mice transplanted with human islets showed disproportionate hyperproinsulinemia and graft evidence of b-cell restricted PC2 depletion, amyloid deposition and b-cell death, and lack of b-cell replication with nuclear translocation of p27Kip1 and p21WAF1 that together may contribute to delayed graft failure.

Key words: Islet transplantation; Proinsulin processing; Prohormone convertase 2 (PC2); p16INK4; p21WAF1; p27Kip1; Athymic nude mice

Address correspondence to Alberto M. Davalli, M.D., Department of Medicine, Unit of Endocrinology and Metabolic Diseases, San Raffaele Hospital, Via Olgettina 60, 20132-Milan, Italy. Tel: +39-02-26437168; Fax: +39-02-26433790; E-mail: alberto.davalli@hsr.it or Franco Folli M.D., Ph.D., Department of Medicine, Division of Diabetes, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA. Tel: 210-567-4826; Fax: 210-567-6554; E-mail: folli@uthscsa.edu

*Present affiliation: Diabetes Unit, ISMETT, Palermo, Italy.
**UT San Antonio medical student.
#Student in the Ph.D. Program in Experimental Medicine and Oncology, University of Insubria, Varese.

Cell Transplantation, Vol. 17, pp. 1337-1347, 2008
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E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Flow Cytometric Quantification of Glucose-Stimulated b-Cell Metabolic Flux Can Reveal Impaired Islet Functional Potency

Matthew S. Hanson,1 Anja Steffen,2 Juan S. Danobeitia,1 Barbara Ludwig,2 and Luis A. Fernandez1

1Department of Surgery, Division of Transplantation, University of Wisconsin-Madison, Madison, WI, USA
2Medizinische Klinik und Poliklinik III, University of Carl Gustav Carus, Dresden, Germany

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet b-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+i) (Fura Red), stimulated with glucose, and analyzed on a FACS VantageTM flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet b-cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+i. Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and b-cell glucose responsiveness as indicators of functional potency.

Key words: Islet; b-Cell; Glucose; Mitochondria; Transplantation; Diabetes; Pancreas; Flow cytometry

Address correspondence to Luis A. Fernandez, M.D., Department of Surgery, University of Wisconsin-Madison, H5/301, 600 Highland Avenue, Madison, WI 53792-3236, USA. Tel: 608-262-6664; Fax: 608-262-6280; E-mail: luisf@surgery.wisc.edu

Cell Transplantation, Vol. 17, pp. 1349-1359, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Isolating Human Islets of Langerhans Causes Loss of Decay Accelerating Factor (CD55) on b-Cells

Joo Ho Tai,1* Hongtao Sun,2 Weihua Liu,2 C. W. James Melling,3 Craig Hasilo,3 and David J. G. White1,2,3

1Immunology and Transplantation Research Group, Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
2Department of Pathology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada
3Human Islet Transplantation Program, London Health Sciences Centre, London, Ontario, Canada

It has previously been reported that human decay accelerating factor (DAF; CD55) is not expressed on cells isolated from human islets. We have investigated if this absence is caused by the islet isolation procedure and/or the single cell isolation technique. We focused on loss of DAF expression on b-cells within the intact islet and on isolated individual b-cells. We established that DAF was expressed in islets and on b-cells prior to isolation by in situ analysis in the intact pancreas. In situ immunohistochemistry (IHC) was used to examine DAF expression on human pancreatic islets and isolated islets. A reverse transcriptase-polymerase chain reaction (RT-PCR) specific for human DAF mRNA was developed to measure mRNA levels in situ in islets within the intact pancreas, isolated islets, and purified b-cells. b-Cells were purified by fluorescenceactivated cell sorting. DAF protein expression on these purified cells was measured using flow cytometry. Expression of DAF protein was present on the islets, including b-cells within the human pancreas; however, comparative data from IHC and flow cytometry revealed the absence of DAF protein on b-cells in both isolated islets and single cell preparations. Furthermore, compared to mRNA levels detected by in situ RTPCR in the intact pancreas and in human HEK 293 cells, isolated islets, and purified human b-cells showed downregulation of DAF mRNA. mRNA was detectable in both of these preparations by RT-PCR; levels were lower following both the islet isolation process (53%) and single cell preparation (a further 62%) compared to HEK 293 controls. Human islet allotransplantation might be more successful if either de novo transfer of DAF onto the isolated islets or novel techniques for islet isolation preserving DAF could be developed.

Key words: Decay accelerating factor (DAF; CD55); Islet isolation; Complement; Instant blood-mediated inflammatory reaction (IBMIR); Transplantation; b-Cells

Address correspondence to David J. G. White, F.R.C.Path., Ph.D., Immunology and Transplantation Research Group, Robarts Research Institute, Room 200, SDRI Building, the University of Western Ontario, 1400 Western Road, London, Ontario, Canada N6G 2V4. Tel: 519-663-2946; Fax: 519-663-2938; E-mail: david.white@robarts.ca

*Present address: Imaging Research Laboratories, Robarts Research Institute, the University of Western Ontario and Metabolism & Diabetes and Imaging, Lawson Health Research Institute, St. Joseph's Hospital, London, Ontario, Canada.

Cell Transplantation, Vol. 17, pp. 1361-1370, 2008
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E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Intrahepatic Islet Transplant in the Mouse: Functional and Morphological Characterization

R. Melzi,1 F. Sanvito,2 A. Mercalli,1 K. Andralojc,1,3 E. Bonifacio,4 and L. Piemonti1

1Beta Cell Biology Unit, Diabetes Research Institute, San Raffaele Scientific Institute, Milan, Italy
2Immunohistochemistry of Rodents Unit, San Raffaele Scientific Institute, Milan, Italy
3Animal Physiology and Biochemestry Department, August Cieszkowski University of Agriculture, Poznan, Poland
4Center for Regenerative Therapies Dresden, Dresden University of Technology, Dresden, Germany

Although in a clinical setting islet transplantation is normally performed by percutaneous intrahepatic infusion, the kidney capsule has been the site of choice in nearly all the studies using mice. In the present study, we extensively characterized the mouse model of intraportally transplanted islets with the purpose to propose it as a model to study islet transplantation. C57BL/6 (n = 78) and BALB/C (n = 53) recipients were transplanted with 400 autologous islets alternatively through the portal vein (PV-Tx) or under the kidney capsule (KC-Tx). Glucose concentration during the first hour after syngeneic islet infusion was associated with subsequent long-term function confirming that early events have long-term effects on graft function. In both strains tested the probability to achieve islet function was significantly lower for PV-Tx than KC-Tx. Also in allogeneic models (C57BL/6 to BALB/C, n = 104; BALB/C to C57BL/6, n = 77) the probability to achieve primary function was significantly lower for PV-Tx than KC-Tx and the site of transplantation significantly affected the graft survival. Histological evaluation of livers showed the presence of features (embolism, thrombosis, focal areas of liver necrosis) that are absent in the kidney subcapsular site. Finally, significant differences in the outcome of PV-Tx were observed between the Th type 1 inflammatory-prone C57BL/6 mouse and the type 2 inflammatory-prone BALB/C mouse. Intraportal islet graft model has some features that are more similar to human clinical islet transplantation and should be used as a model to study not only engraftment but also mechanisms of immune suppression and immune tolerance.

Key words: Engraftment; Islet transplantation; Mouse model; Portal vein

Address correspondence to Raffaella Melzi, Beta Cell Biology Unit, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy. Tel: 39 02 26432706; Fax: 39 02 26432871; E-mail: melzi.raffaella@hsr.it

Cell Transplantation, Vol. 17, pp. 1371-1380, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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The Therapeutic Effect of Extracellular Superoxide Dismutase (EC-SOD) Mouse Embryonic Fibroblast (MEF) on Collagen-Induced Arthritis (CIA) Mice

Dong Hoon Yu,1 Myoung Ok Kim,1 Sung Hyun Kim,1 Mi Jung Shin,1 Bong Soo Kim,1 Hei Jung Kim,1 Sang Ryeul Lee,3 Sang Gyu Lee,1 Seung-Ah Yoo,5 Wan Uk Kim,5 Byung Hwa Hyun,4 Young Sik Park,2 Tae Yoon Kim,3 and Zae Young Ryoo1

1School of Life Sciences and Biotechnology, Kyungpook National University, Daegu, 702-701, Korea
2School of Life and Food Sciences, Kyungpook National University, Daegu, 702-701, Korea
3Department of Immunology and Dermatology, College of Medicine, Catholic University, Seoul, 137-040, Korea
4Disease Model Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Korea
5Division of Rheumatology, Department of Internal Medicine, Catholic University of Korea, Seoul, Korea

Rheumatoid arthritis is a chronic inflammatory disease. The generation of reactive oxygen species (ROS) within an inflamed joint has been suggested as playing a significant pathogenic role. Extracellular superoxide dismutase (EC-SOD) is a major scavenger enzyme of ROS, which has received growing attention for its therapeutic potential. To investigate the therapeutic effect of EC-SOD in mice with collagen-induced arthritis (CIA), we used mouse embryonic fibroblast (MEF) of transgenic mice that overexpresses EC-SOD on the skin by using hK14 promoter. DBA/1 mice that had been treated with bovine type II collagen were administrated subcutaneous injections of EC-SOD transgenic MEF (each at 1.4 × 106 cells) on days 28, 35, and 42 after primary immunization. To test EC-SOD activity, blood samples were collected in each group on day 49. The EC-SOD activity was nearly 1.5-fold higher in the transgenic MEF-treated group than in the nontransgenic MEF-treated group (p < 0.05). The severity of arthritis in mice was scored in a double-blind manner, with each paw being assigned a separate clinical score. The severity of arthritis in EC-SOD transgenic MEF-treated mice was significantly suppressed in the arthritic clinical score (p < 0.05). To investigate the alteration of cytokine levels, ELISA was used to measure blood samples. Levels of IL-1b and TNF-a were reduced in the transgenic MEF-treated group (p < 0.05). Abnormalities of the joints were examined by H&E staining. There were no signs of inflammation except for mild hyperplasia of the synovium in the transgenic MEF-treated group. The proliferation of CII-specific T cells was lower in the transgenic MEFtreated mice than in those in the other groups. The transfer of EC-SOD transgenic MEF has shown a therapeutic effect in CIA mice and this approach may be a safer and more effective form of therapy for rheumatoid arthritis.

Key words: Rheumatoid arthritis; Collagen-induced arthritis (CIA); Extracellular superoxide dismutase (EC-SOD); Mouse embryonic fibroblast (MEF)

Address correspondence to Zae Young Ryoo, Ph.D., School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, 1370 Sankyuk-dong, Buk-ku, Daegu 702-701, Korea. Tel: 82-53-950-7361; Fax: 82-53-943-6925; E-mail: jaewoong64@hanmail.net

Cell Transplantation, Vol. 17, pp. 1381-1388, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Porcine Endogenous Retrovirus (PERV) and its Transmission Characteristics: A Study of the New Zealand Designated Pathogen-Free Herd

O. Garkavenko,1 S. Wynyard,1 D. Nathu,1 D. Simond,4 M. Muzina,1 Z. Muzina,1 L. Scobie,2 R. D. Hector,2 M. C. Croxson,3 P. Tan,1 and B. R. Elliott1

1Living Cell Technologies Ltd, Manukau 2025, Auckland, New Zealand
2Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, G4 0BA, UK
3Virology Immunology Department, Auckland Hospital, Auckland, New Zealand
4Centre for Transplantation & Renal Research, Westmead Millennium Institute, Westmead Hospital, Australia

Previously a strategy for monitoring of pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd has previously tested free of infectious agents relevant to xenotransplantation and we present here an in depth study of porcine endogenous retrovirus (PERV) transmission. A panel of assays that describes the constraints for the transmission of PERV has been suggested. It includes a) infectivity test in coculture of DPF pig primary cells with both human and pig target cell lines; b) RT activity in supernatant of stimulated primary cells from DPF pigs; c) viral load in donor's blood plasma; d) PERV proviral copy number in DPF pig genome; e) PERV class C prevalence in the herd and its recombination potential. There was no evidence of PERV transmission from DPF pig tissue to either pig or human cells. Additionally, there was no evidence of PERV RNA present in pig blood plasma. PERV copy number differs in individual pigs from as low as 3 copies to 30 copies and the presence of PERV-C varied between animals and breeds. In all DPF pigs tested, a specific locus for PERV-C potentially associated with the recombination of PERV in miniature swine was absent. Presented data on the PERV transmission allows us to classify the DPF potential donors as "null" or noninfectious pigs.

Key words: Xenotransplantation; Pig viruses; Pig endogenous retrovirus; Infectivity test; "Null" pigs

Address correspondence to O. Garkavenko, Living Cell Technologies Ltd, 19 Laureston Avenue, Otara, Manukau 2025, Auckland, New Zealand. Tel: 64-9-2762690; Fax: 64-9-2762691; E-mail: ogarkavenko@lctglobal.com

Cell Transplantation, Vol. 17, pp. 1389-1401, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Development of a Silk Cable-Reinforced Gelatin/Silk Fibroin Hybrid Scaffold for Ligament Tissue Engineering

Hongbin Fan,1 Haifeng Liu,1 Yue Wang,1 Siew Lok Toh,2,3 and James Cho Hong Goh1,2

1Department of Orthopedic Surgery, National University of Singapore, Singapore
2Division of Bioengineering, National University of Singapore, Singapore
3Department of Mechanical Engineering, National University of Singapore, Singapore

The objective of this study was to develop a silk cable-reinforced gelatin/silk fibroin hybrid scaffold for ligament tissue engineering. The scaffold was fabricated by lyophilizing the cross-linked gelatin and silk fibroin mixture with braided silk cables. Scanning electronic microscopy (SEM) observation showed that microporous gelatin/silk fibroin sponges formed around silk cables mimicked the microstructures of ligament extracellular matrix (ECM). The silk cables significantly increased the tensile strength of the scaffold to meet the mechanical requirements for ligament tissue engineering. The scaffold possessed good cell adhesion property, and when mesenchymal stem cells (MSCs) were seeded on it, cells proliferated profusely. After 2 weeks of culture, seeded MSCs were distributed uniformly throughout the scaffold and were highly viable. Occurrence of cell death during culture was not significant. Deposition of collagen on the scaffold was found to increase with time. Differentiation of MSCs into ligament fibroblasts was verified by expressions of ligament ECM specific genes including collagen type I, collagen type III, and tenascin-C in mRNA and protein level. Immunohistochemistry stains also confirmed the production of key ligament ECM components on the scaffold. The results demonstrate that silk cable-reinforced gelatin/silk fibroin scaffold possesses the appropriate mechanical properties and has enlarged surface area. It is also capable of supporting cell proliferation and differentiation for ligament tissue engineering.

Key words: Silk scaffold; Mesenchymal stem cells; Extracellular matrix; Tissue engineering; Ligament

Address correspondence to James C. H. Goh, Ph.D., Department of Orthopaedic Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore. Tel: 65-65165259; Fax: 65-67765322; E-mail: dosgohj@nus.edu.sg

Cell Transplantation, Vol. 17, pp. 1403-1414, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
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Liver After Hepatocyte Transplantation for Liver-Based Metabolic Disorders in Children

Alberto Quaglia,1 Sharon C. Lehec,1 Robin D. Hughes,1 Ragai R. Mitry,1 A. S. Knisely,1 Stephen Devereaux,2 Julie Richards,2 Mohamed Rela,1 Nigel D. Heaton,1 Bernard C. Portmann,1 and Anil Dhawan1

1Institute of Liver Studies, King's College Hospital and King's College London School of Medicine, London, UK
2Department of Haematology, King's College Hospital, London, UK

There are limited data regarding donor hepatocyte engraftment into recipient liver after human hepatocyte transplantation (HHTx). We reviewed the explant livers of seven children with metabolic disorders [ornithine-transcarbamylase deficiency (one), coagulation factor VII deficiency (three), Crigler-Najjar syndrome (one), progressive familial intrahepatic cholestasis type 2 (PFIC-2) deficiency (two)] who received allograft hepatocytes by intraportal infusion with improvement in phenotype, although all later underwent liver transplantation (LT). Immunohistochemistry for bile salt export protein (BSEP) in the PFIC-2 patients and genetic typing following laser capture microdissection (LCM) of liver cells in the others were used to identify donor hepatocytes in recipient explant livers. Explant livers usually showed a preserved lobular architecture. In one patient, hepatocytes were identified inside portal vein thrombi. No donor hepatocytes in liver cell plates were identified immunohistochemically or by genetic typing. HHTx was generally followed by partial recovery of metabolic function; the procedure was well tolerated; any increase in portal vein pressure was transient. Hepatocytes were identified in portal vein thrombi, even months after portal vein infusion. Further studies are needed to monitor donor hepatocytes in vivo, to quantify better the efficacy of the procedure and to find ways of improving engraftment and function.

Key words: Engraftment; Factor VII deficiency; Crigler-Najjar syndrome; Ornithine transcarbamylase deficiency; Progressive familial intrahepatic cholestasis type 2 (PFIC-2)

Address correspondence to Professor Anil Dhawan, Institute of Liver Studies, King's College Hospital, Denmark Hill, London SE5 9RS, UK. Tel: 0044 20 3299 4408; Fax: 0044 20 3299 4228; E-mail: anil.dhawan@kcl.ac.uk

Cell Transplantation, Vol. 17, pp. 1415-1421, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Brief Communication
Repopulation by Endogenous Hepatocytes Does Not Reconstitute Liver Mass in Rats Treated With Retrorsine

Sergio Laconi, Silvia Doratiotto, Stefania Montisci, Paolo Pani, and Ezio Laconi

Department of Biomedical Sciences and Biotechnology, Section of Experimental Pathology, University of Cagliari, Cagliari, Italy

The retrorsine (RS)-based model for massive liver repopulation was laid on the hypothesis that transplanted cells can proliferate in the recipient liver if the growth capacity of endogenous hepatocytes is persistently impaired. In order to directly test this hypothesis, we examined the long-term response to 2/3 partial hepatectomy (PH) in rats pretreated with RS, according to the protocol for liver repopulation. Rats were given RS or saline and 4 weeks later they underwent PH; they were killed up to 16 weeks thereafter. Liver weights, liver DNA, and protein content were significantly lower in the RS group throughout the experimental time considered (e.g., at 16 weeks post-PH relative liver weight was 1.99 ± 0.30% in RS group vs. 3.06 ± 0.5% in controls). Regenerative nodules were present in RS-treated livers; they occupied about 3% of the liver at 2 weeks post-PH and this value increased to nearly 50% at 8 weeks and to >95% at 16 weeks. In conclusion, RS-treated rat liver is unable to recover its original mass for several months following PH, despite the development of regenerative nodules. This long-lasting effect is likely to contribute to the growth of transplanted hepatocytes, leading to massive liver repopulation.

Key words: Retrorsine; Cell cycle block; Regenerative nodules; Cyclin D1

Address correspondence to Ezio Laconi, Department of Biomedical Sciences and Technologies, Section of Experimental Pathology, University of Cagliari, Via Porcell, 4, 09125-Cagliari, Italy. Tel: +39-070-675-8682; Fax: +39-070-662574; E-mail: elaconi@unica.it

Cell Transplantation, Vol. 17, pp. 1423-1428, 2008
0963-6897/08 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Brief Communication
The Effect of Cryopreservation on Umbilical Cord Blood Endothelial Progenitor Cell Differentiation

Xiaomei Lu, Steve J. Proctor, and Anne M. Dickinson

Haematological Sciences, School of Clinical & Laboratory Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK

Endothelial progenitor cells (EPCs) has been shown to be present in umbilical cord blood (UCB) in addition to hematopoietic stem cells. Cryopreservation is the accepted method for long-term storage of UCB. However, whether EPCs can be derived from cryopreserved UCB samples is unclear. The aim of this study was to investigate the differentiation potential of EPCs from cryopreserved CB samples. CD34??cells were isolated from fresh or frozen and thawed UCB using magnetic beads. Cells were then cultured on fibronectincoated plates containing endothelial differentiation medium. After 4-5 weeks in culture, endothelial-like cells were generated from fresh UCB samples, but not cryopreserved UCB samples. Examining this further, both fresh and frozen/thawed UCB MNCs were stained with Annexin V-PE and 7-actinomycin D (7-AAD) using flow cytometry. We found that there were a significant number of apoptotic cells in cryopreserved UCB samples compared to fresh UCB samples. In conclusion, cryopreservation induced UCB cell apoptosis and impaired EPC differentiation.

Key words: Cord blood; Endothelial progenitor cells; Cryopreservation; Differentiation

Address correspondence to Dr. Xiaomei Lu at her present address: Stem Cell and Gene Therapy Group, Molecular Gastroenterology, Leeds Institute of Molecular Medicine, St. James University Hospital, University of Leeds, Leeds LS9 7TF, UK. Tel: 0044-1133438422; Fax: 0044-1133438702; E-mail: x.lu@leeds.ac.uk