ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 16, NUMBER 6, 2007

Cell Transplantation, Vol. 16, pp. 563-577, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Autologous Transplantation of Muscle-Derived CD133+ Stem Cells in Duchenne Muscle Patients

Y. Torrente,1 M. Belicchi,1 C. Marchesi,1 G. D'Antona,2 F. Cogiamanian,1 F. Pisati,1 M. Gavina,1 R. Giordano,3 R. Tonlorenzi,4 G. Fagiolari,1 C. Lamperti,1 L. Porretti,3 R. Lopa,3 M. Sampaolesi,4 L. Vicentini,5 N. Grimoldi,1 F. Tiberio,1 V. Songa,6 P. Baratta,6 A. Prelle,1 L. Forzenigo,7 M. Guglieri,8 O. Pansarasa,2 C. Rinaldi,2 V. Mouly,9 G. S. Butler-Browne,9 G. P. Comi,1 P. Biondetti,7 M. Moggio,1 S. M. Gaini,5 N. Stocchetti,6 A. Priori,1 M. G. D'Angelo,8 A. Turconi,8 R. Bottinelli,2 G. Cossu4 P. Rebulla,3 and N. Bresolin1,8

1Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Department of Neurological Sciences, Dino Ferrari Center, University of Milan, Italy
2Department of Experimental Medicine, Human Physiology Unit, University of Pavia, Pavia, Italy
3Centro Trasfusionale e di Immunologia dei Trapianti, Ospedale Maggiore Policlinico, University of Milan, Milan, Italy
4Stem Cell Research Institute, San Raffaele Hospital, Italy
5Department of Surgery, Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, University of Milan, Italy
6Department of Anesthesia and Critical Care Medicine, Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, University of Milan, Italy
7Radiology Unit, Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, University of Milan, Italy
8IRCCS Eugenio Medea, Bosisio Parini, Italy
9CNRS, Cytoskeleton and Development, Paris, France

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of musclederived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

Key words: Autologous stem cell transplantation; Muscular dystrophy; CD133

Address correspondence to Dr. Yvan Torrente, Department of Neurological Science, University of Milan, Stem Cell Laboratory, Policlinico Hospital, via Francesco Sforza 35, 20122 Milan, Italy. Tel: +39-02-55033874, Fax: +39-02-50320430; E-mail: torrenteyvan@hotmail.com

Cell Transplantation, Vol. 16, pp. 579-585, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Ex Vivo Expansion and Transplantation of Hematopoietic Stem/Progenitor Cells Supported by Mesenchymal Stem Cells From Human Umbilical Cord Blood

Guo-Ping Huang,1* Zhi-Jun Pan,2* Bing-Bing Jia,1 Qiang Zheng,2 Chun-Gang Xie,1 Jiang-Hong Gu,3 Ian K. McNiece,4 and Jin-Fu Wang1

1College of Life Sciences, Zi Jin Gang Campus, Zhejiang University, Hangzhou 310058, P. R. China
2The Second Affiliated Hospital, Zhejiang University, Hangzhou 310006, P. R. China
3The Obstetrics Department, Hangzhou Traditional Chinese Medical Hospital, Hangzhou 310027, P. R. China
4Johns Hopkins Oncology Center, Division of Hematologic Malignancies, Baltimore, MD 21231, USA

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTCICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colonyforming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45-55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

Key words: UCB-derived mesenchymal stem cells; Transplantation; NOD/SCID mice; Ex vivo expansion of hematopoietic stem/progenitor cells

Address correspondence to Jin-Fu Wang, Institute of Cell Biology, College of Life Sciences, Zi Jin Gang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China. Tel: +86-571-88206592; Fax: +86-571-85128776; E-mail: wjfu@zju.edu.cn

*These authors contributed equally to this study

Cell Transplantation, Vol. 16, pp. 587-594, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

A Closed System for Islet Isolation and Purification Using the COBE2991 Cell Processor May Reduce the Need of Clean Room Facilities

R. A. Klaffschenkel,1 A. Biesemeier,1 M. Waidmann,2 H. Northoff,2 W. Steurer,1 A. Königsrainer,1 and N. Lembert1

1Department of General, Visceral, and Transplantation Surgery, University of Tübingen, 72076 Tübingen, Germany
2Department of Transfusion Medicine, University of Tübingen, 72076 Tübingen, Germany

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 ± 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were than purified by adding a gradient (bottom loading). Using this closed system 1098 ± 489 IEQ per gram pancreas were purified with a total cell viability of 67 ± 10% and a b-cell viability of 41 ± 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of b-cells was still 56 ± 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-a to 40 ± 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.

Key words: Porcine islets; COBE2991; GMP; Islet transplantation

Address correspondence to PD Dr. Nicolas Lembert, Department of General, Visceral, and Transplantation Surgery, University of Tübingen, Hoppe Seyler Straße 3, 72076 Tübingen, Germany. Tel: +49 7071 29 87326; E-mail: Lembert9@gmx.de

Cell Transplantation, Vol. 16, pp. 595-607, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Toward Maximizing the Success Rates of Human Islet Isolation: Influence of Donor and Isolation Factors

Gaston M. Ponte,1* Antonello Pileggi,1,2* Shari Messinger,1,3 Angel Alejandro,1,4 Hirohito Ichii,1,2 David A. Baidal,1 Aisha Khan,1 Camillo Ricordi,1,2 John A. Goss,5 and Rodolfo Alejandro1,4

1Cell Transplant Center and Clinical Islet Transplant Center, Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
2DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
3Department of Epidemiology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
4Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
5Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA

In order to make islet transplantation a therapeutic option for patients with diabetes there is an urgent need for more efficient islet cell processing to maximize islet recovery. Improved donor management, organ recovery techniques, implementation of more stringent donor criteria, and improved islet cell processing techniques may contribute to enhance organ utilization for transplantation. We have analyzed the effects of donor and islet processing factors on the success rate of human islet cell processing for transplantation performed at a single islet cell processing center. Islet isolation outcomes improved when vasopressors, and in particular pitressin, and steroids were used for the management of multiorgan donors. Higher islet yields were obtained from adult male donors, BMI >25 kg/m2, adequate glycemic control during hospital stay, and when the pancreas was retrieved by a local surgical team. Successful isolations were obtained in 58% of the cases when >=4 donor criteria were met, and even higher success rates (69%) were observed when considering >=5 criteria. Our data suggest that a sequential, integrated approach is highly desirable to improve the success rate of islet cell processing.

Key words: Pancreatic islets; Islet transplantation; Organ selection; Islet isolation; Donor management; Pancreas preservation; Pancreas recovery; Islet purification

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

Cell Transplantation, Vol. 16, pp. 609-620, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Encapsulation of Porcine Islets Permits Extended Culture Time and Insulin Independence in Spontaneously Diabetic BB Rats

Lawrence S. Gazda,1,2 Horatiu V. Vinerean,1 Melissa A. Laramore,1 Carolyn H. Diehl,2,3 Richard D. Hall,1,4 Albert L. Rubin,2,3 and Barry H. Smith1,2,3

1The Rogosin Institute-Xenia Division, Xenia, OH 45385, USA
2The Rogosin Institute, New York, NY 10021, USA
3New York-Presbyterian Hospital and Weill Medical College of Cornell University, New York, NY 10021, USA
4Bob Evans Farms, Inc., Columbus, OH 43207, USA

The ability to culture porcine islets for extended times allows for both their functional assessment and the assurance of their microbiological safety prior to transplantation. We have previously shown that agaroseencapsulated porcine islets can be cultured for at least 24 weeks. In the current study, porcine islet agarose macrobeads cultured for up to 67 weeks were assessed for their ability to restore normoglycemia, respond to an intraperitoneal glucose challenge, maintain spontaneously diabetic BB rats free of insulin therapy for more than 6 months, and for their biocompatibility. Porcine islets were encapsulated in agarose macrobeads and subjected to weekly static perifusion assays for the assessment of insulin production. After in vitro culture for either 9, 40, or 67 weeks, 56-60 macrobeads were transplanted to each spontaneously diabetic BB rat. Transplanted rats were monitored daily for blood glucose levels. Glucose tolerance tests and assessments for porcine C-peptide were conducted at various intervals throughout the study. Normoglycemia (100-200 mg/dl) was initially restored in all islet transplanted rats. Moderate hyperglycemia (200-400 mg/dl) developed at around 30 days posttransplantation and continued throughout the study period of 201-202 days. Importantly, all rats that received encapsulated porcine islets continued to gain weight and were free of exogenous insulin therapy for the entire study. Porcine C-peptide (0.2-0.9 ng/ml) was detected in the serum of islet recipients throughout the study period. No differences were detected between recipient animals receiving islet macrobeads of various ages. These results demonstrate that the encapsulation of porcine islets in agarose macrobeads allows for extended culture periods and is an appropriate strategy for functional and microbiological assessment prior to clinical use.

Key words: Porcine islets; Islet culture; Xenotransplantation; Encapsulation

Address correspondence to Lawrence S. Gazda, The Rogosin Institute-Xenia Division, 740 Birch Road, Xenia, OH 45385, USA. Tel: (937) 374-3116; Fax: (937) 374-3261; E-mail: lgazda@rixd.org

Cell Transplantation, Vol. 16, pp. 621-627, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
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Distribution of Intraportally Implanted Microspheres and Fluorescent Islets in Mice

Erik von Seth,1 Daniel Nyqvist,2 Arne Andersson,1 Per-Ola Carlsson,1,3 Martin Köhler,2 Göran Mattsson,1 Astrid Nordin,1 Per-Olof Berggren,2 and Leif Jansson1

1Department of Medical Cell Biology, Uppsala University, SE-75123 Uppsala, Sweden
2The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital Solna, SE-17176 Stockholm, Sweden
3Department of Medical Sciences, Uppsala University, Uppsala University Hospital, SE-75185 Uppsala, Sweden

The aim of the study was to evaluate the distribution of intraportally transplanted islets in mice. We initially administered 2000 polystyrene microspheres with a diameter of 50 mm intraportally into normoglycemic C57BL/6 mice. In separate experiments other mice were injected similarly with 300 microspheres each with a diameter of 100 or 200 mm. One week later the animals were killed, and the lungs and livers were removed and divided into lobes. The number of microspheres in each individual liver lobe and in the lungs was counted using a stereomicroscope. In other experiments, athymic C57BL/6 mice were similarly implanted with 250 islets isolated from transgenic mice expressing the enhanced yellow fluorescent protein in the islet cells. The distribution of microspheres and islets was independent of size, and fairly homogenous within the liver, with the exception of the caudate lobe, which contained fewer microspheres and islets, respectively. Approximately one third of all microspheres and islets were present as aggregates. Eighty-five to 90% of the implanted microspheres were identified in the liver sections, whereas 60-65% of the implanted isletswere recovered. Aggregates or single fluorescent cells were observed in the liver of islet-implanted mice. We conclude that islets and microspheres implanted into the liver distribute fairly homogenously and quite a few of them exist as aggregates or, with respect to islets, as fragments.

Key words: Pancreatic islets; Microspheres; Engraftment; Liver; Distribution

Address correspondence to Leif Jansson, Department of Medical Cell Biology, Biomedical Centre, Husargatan 3, Box 571; SE-751 23 Uppsala, Sweden. Tel: ?46 18 4714396; Fax: ?46 18 4714059; E-mail: Leif.Jansson@medcellbiol.uu.se

Cell Transplantation, Vol. 16, pp. 629-637, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
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Hepatocyte Transplantation for Glycogen Storage Disease Type Ib

Kwang-Woong Lee,1 Ji-Hyun Lee,2 Sung Wook Shin,3 Sung Joo Kim,1 Jae Won Joh,1 Doo-Hoon Lee,4 Jong-Won Kim,5 Hwa-Young Park,6 Soo-Youn Lee,5 Hwan Hyo Lee,1 Jin Wan Park,1 Shi-Yeon Kim,2 Hee-Hoon Yoon,4 Doo-Hee Jung,4 Yon Ho Choe,6 and Suk-Koo Lee1

1Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
2Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
3Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
4Department of Chemical and Biochemical Engineering, Dongguk University, Seoul, Korea
5Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
6Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders with an incidence of 1 in 100,000. The two major subtypes are GSD-Ia, caused by a deficiency of glucose-6-phosphatase (G6Pase), and GSD-Ib, caused by a deficiency of glucose-6-phosphate transporter (G6PT). We report that a substantial improvement was achieved following several infusions of hepatocytes in a patient with GSD-Ib. Hepatocytes were isolated from the unused cadaveric whole livers of two donors. At the first transplantation, approximately 2 × 109 cells (2% of the estimated recipient's total hepatocytes) were infused. Seven days later 1 × 109 (1% of liver mass) cryopreserved hepatocytes from the same donor were infused, and an additional 3 × 109 (3% of liver mass) cells from the second donor were infused 1 month after the second transplantation. After the hepatocyte transplantation, the patient showed no hypoglycemic symptoms despite the discontinuation of cornstarch meals. Liver biopsies on posttransplantation days 20 and 250 showed a normal level of glucose-6-phosphatase activity in presolubilization assay that was very low before transplantation. This was the first and successful clinical hepatocyte transplantation in Korea. In this study, hepatocyte transplantation allowed a normal diet in a patient with GSD-Ib, with substantial improvement in their quality of life. Hepatocyte transplantation might be an alternative to liver transplantation and dietary therapy in GSD-Ib.

Key words: Hepatocyte; Transplantation; Glycogen storage disease

Address correspondence to Suk-Koo Lee, M.D., Ph.D., Department of Surgery, Samsung Medical Center, Sungkyunkwan University, 50 Ilwondong, Gangnam-Gu, Seoul, 135-710 Korea. Tel: 82-2-3410-3464; Fax: 82-2-3410-0040; E-mail: sklee@smc.samsung.co.kr

Cell Transplantation, Vol. 16, pp. 639-647, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
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Cryopreservation-Induced Nonattachment of Human Hepatocytes: Role of Adhesion Molecules

Claire Terry, Robin D. Hughes, Ragai R. Mitry, Sharon C. Lehec, and Anil Dhawan

Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK

Good quality cryopreserved human hepatocytes are becoming an important source for clinical hepatocyte transplantation. However, the process of cryopreservation leads to both structural and functional impairment of hepatocytes. The aim of this study was to investigate the mechanisms of cryopreservation-induced nonattachment in human hepatocytes. Hepatocytes were cryopreserved after isolation from unused donor liver tissue. Cell attachment to collagen-coated plates was measured. A cDNA gene array system for 96 cell adhesion-related molecules was used to determine mRNA expression in fresh and cryopreserved hepatocytes. Two cell adhesion molecule proteins were investigated further: b1-integrin, a cell-matrix adhesion molecule, and E-cadherin, a cell-cell adhesion molecule. Attachment efficiency was significantly decreased after cryopreservation of human hepatocytes. Twenty-two genes were downregulated after cryopreservation including integrins, cadherins, catenins, and matrix metalloproteinases (MMPs). b1-Integrin gene and protein expression were significantly decreased in cultured cryopreserved hepatocytes compared to fresh hepatocytes. There was a significant correlation between loss of b1-integrin and attachment in cryopreserved cells. Degradation of E-cadherin was increased in cryopreserved hepatocytes. The process of cryopreservation leads to downregulation of cell adhesion molecules at the gene and the cellular level. New cryopreservation protocols are needed to prevent these effects on cell attachment.

Key words: Hepatocyte; Cryopreservation; Attachment; b1-Integrin; E-cadherin

Address correspondence to Professor Anil Dhawan, Paediatric Liver Service, King's College Hospital, Denmark Hill, London SE5 9RS, UK. Tel: ?44 (0)20-3299-3578; Fax: ?44 (0)20-3299-4228; E-mail: anil.dhawan@kcl.ac.uk

Cell Transplantation, Vol. 16, pp. 649-661, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Culture of Keratinocytes for Transplantation Without the Need of Feeder Layer Cells

Neeltje A. Coolen,1 Michelle Verkerk,1 Linda Reijnen,1 Marcel Vlig,1 Antoon J. van den Bogaerdt,1 Melanie Breetveld,2 Susan Gibbs,2 Esther Middelkoop,1,3 and Magda M. W. Ulrich1,2

1Association of Dutch Burns Centres, 1940 EA Beverwijk, The Netherlands
2Department of Dermatology, VU University Medical Centre, 1007 MB Amsterdam, The Netherlands
3Department of Plastic, Reconstructive and Hand Surgery, VU University Medical Centre, 1007 MB Amsterdam, The Netherlands

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

Key words: Epidermis; Culture; Burn wounds; Collagen type IV; Skin equivalents

Address correspondence to Magda M. W. Ulrich, Ph.D., Association of Dutch Burn Centres, P.O. Box 1015, 1940 EA Beverwijk, The Netherlands. Tel: ?31 251 275506; Fax: ?31 251 216059; E-mail: mulrich@burns.nl

Cell Transplantation, Vol. 16, pp. 663-674, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Inhibition of Apoptosis by Expression of Antiapoptotic Proteins in Recombinant Human Keratinocytes

Claudia Y. U. Choi,1 Kerstin Reimers,1 Christina Allmeling,1 Susanne Kall,1 Yeong-Hoon Choi,2 and Peter M. Vogt1

1Department of Plastic, Hand and Reconstructive Surgery, Medical School Hannover, D-30625 Hannover, Germany
2Department of Cardiac Surgery, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA

The Fas ligand/Fas interaction plays an important role in the regulation of immune responses. Allografted cells undergo Fas-mediated apoptosis induced by CD8+ T cells. Our objective was to prevent human keratinocytes from immunologically induced apoptosis. We focused on three proteins with inhibitory function on Fas-mediated apoptosis. Human keratinocytes were transfected with either Flip, Faim, or Lifeguard (LFG). The treatment proved to be practicable and efficient. The recombinant keratinocytes with expression of our target proteins were cocultured with CD8+ T cells and the apoptotic activity was then evaluated. Activation of caspase-8 was detectable in control but not in the recombinant cells. Quantitative analysis revealed significant induction of T-cell-induced apoptosis in nontransfected keratinocytes (p = 0.04, n = 12) but not in Flip (p = 0.66), Faim (p = 0.42), or LFG (p = 0.44) expressing cells. Our results suggest that heterotopic expression of antiapoptotic proteins can induce the resistance of keratinocytes to a major mechanism of rejection.

Key words: Apoptosis; Keratinocytes; Allotransplantation; Inhibition of rejection

Address correspondence to Dr. Claudia Choi, Department of Plastic, Hand and Reconstructive Surgery, Medical School Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Tel: (+49) 511/906-3917; Fax: (+49) 511/906-3606; E-mail: Choi.Claudia@MH-Hannover.de