ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 16, NUMBER 7, 2007

Cell Transplantation, Vol. 16, pp. 675-684, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Consistency and Safety of Cell Banks for Research and Clinical Use: Preliminary Analysis of Fetal Skin Banks

Aurelie Quintin,11,2 Nathalie Hirt-Burri,3 Corinne Scaletta,4 Constantin Schizas,4 Dominique P. Pioletti,2 and Lee Ann Applegate1,4

1Orthopedic Cell Therapy Unit, University Hospital, Lausanne, Switzerland
2Laboratoire de Biomécanique en Orthopédie (EPFL-HORS), Institut de Biomécanique Translationnelle, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
3Department of Pediatric Surgery, University Hospital, Lausanne, Switzerland
4Hôpital Orthopédique de la Suisse Romande (HORS), Lausanne, Switzerland

Current restrictions for human cell-based therapies have been related to technological limitations with regards to cellular proliferation capacity, maintenance of differentiated phenotype for primary human cell culture, and transmission of communicable diseases. We have seen that cultured primary fetal cells from one organ donation could possibly meet the exigent and stringent technical aspects for development of therapeutic products. We could develop a master cell bank (MCB) of 50 homogenous ampoules of 4-5 million cells each from one fetal organ donation (skin) in short periods of time compared to other primary cell types. Safety tests were performed at all stages of the cell banking. MCB ampoules could create a working cell bank to be used for clinical or research use. Monolayer culture of fetal skin cells had a life span of 12-17 passages, and independent cultures obtained from the same organ donation were consistent for protein concentration (with 1.4-fold maximal difference between cultures) as well as gene expression of MMP-14, MMP-3, TIMP-3, and VEGF (1.4-, 1.9-, 2.1-, and 1.4-fold maximal difference between cultures, respectively). Cell cultures derived from four independent fetal skin donations were consistent for cell growth, protein concentration, and gene expression of MDK, PTN, TGF-xb1, and OPG. As it is the intention that banked primary fetal cells can profit from the potential treatment of hundreds of thousands of patients with only one organ donation, it is imperative to show consistency, tracability, and safety of the process, including donor tissue selection, cell banking, cell testing, and growth of cells in upscaling for the preparation of cell transplantation.

Key words: Fetal cells; Fibroblasts; Cell banking; Safety; Tissue engineering

Address correspondence to Dr. Lee Ann Laurent-Applegate, Orthopedic Cell Therapy Unit, University Hospital, CHUV, PAV 03, Rm 121, 1011 Lausanne, Switzerland. Tel: +41 21 314 3150; Fax: +41 21 887 8414; E-mail: Lee.Laurent-Applegate@chuv.ch

Cell Transplantation, Vol. 16, pp. 685-696, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Good Manufacturing Practice-Compliant Expansion of Marrow-Derived Stem and Progenitor Cells for Cell Therapy

Martin H. Gastens,1 Kristin Goltry,2 Wolfgang Prohaska,1 Diethelm Tschöpe,3 Bernd Stratmann,3 Dirk Lammers,3 Stanley Kirana,3 Christian Götting,1 and Knut Kleesiek1

1Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, 32545 Bad Oeynhausen, Germany
2Aastrom Bioscience, Ann Arbor, MI 48105, USA
3Diabeteszentrum, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, 32545 Bad Oeynhausen, Germany

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 106 total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

Key words: GMP-compliant; Cell therapy; Bone marrow; Closed process

Address correspondence to Dr. M. Gastens, Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstr. 11, D-32545 Bad Oeynhausen, Germany. Tel: +49-5731-97-1390; Fax: +49-5731-97-2307; E-mail: gastens@gmx.de

Cell Transplantation, Vol. 16, pp. 697-705, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Aging Reduces the Neuroprotective Capacity, VEGF Secretion, and Metabolic Activity of Rat Choroid Plexus Epithelial Cells

Dwaine F. Emerich, Patricia Schneider, Briannan Bintz, Jebecka Hudak, and Christopher G. Thanos

LCT BioPharma, Inc., Providence, RI, USA

Delivery of neurotrophic molecules to the brain has potential for preventing neuronal loss in neurodegenerative disorders. Choroid plexus (CP) epithelial cells secrete numerous neurotrophic factors, and encapsulated CP transplants are neuroprotective in models of stroke and Huntington's disease (HD). To date, all studies examining the neuroprotective potential of CP transplants have used cells isolated from young donor animals. Because the aging process significantly impacts the cytoarchitecture and function of the CP the following studies determined whether age-related impairments occur in its neuroprotective capacity. CP was isolated from either young (3-4 months) or aged (24 months) rats. In vitro, young CP epithelial cells secreted more VEGF and were metabolically more active than aged CP epithelial cells. Additionally, conditioned medium from cultured aged CP was less potent than young CP at enhancing the survival of serum-deprived neurons. Finally, encapsulated CP was tested in an animal model of HD. Cell-loaded or empty alginate capsules (control group) were transplanted unilaterally into the rat striatum. Seven days later, the animals received an injection of quinolinic acid (QA; 225 nmol) adjacent to the implant site. Animals were tested for motor function 28 days later. In the control group, QA lesions severely impaired function of the contralateral forelimb. Implants of young CP were potently neuroprotective as rats receiving CP transplants were not significantly impaired when tested for motor function. In contrast, implants of CP from aged rats were only modestly effective and were much less potent than young CP transplants. These data are the first to directly link aging with diminished neuroprotective capacity of CP epithelial cells.

Key words: Choroid plexus; Xenotransplant; VEGF; Encapsulation; Aging; Alginate

Address correspondence to Dwaine F. Emerich, 241 Anan Wade Rd., Glocester, RI 02857, USA. Tel: 401-499-6662; Fax: 401-823-0466; E-mail: ED3FJM@aol.com

Cell Transplantation, Vol. 16, pp. 707-715, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effect of Pretransplant Preconditioning by Whole Body Hyperthermia on Islet Graft Survival

H. Brandhorst,1 M. Olbrich,2 A. Neumann,3 H. Jahr,3 and D. Brandhorst1

1Department of Oncology, Radiology & Clinical Immunology, University Hospital, 75185 Uppsala, Sweden
2Department of Internal Medicine, University Hospital Eilbeck, 22081 Hamburg, Germany
3Third Medical Department, University Hospital, 35385 Giessen, Germany

Previous observations in heat-shocked pig islets revealed the ambivalent character of the stress response simultaneously inducing processes of protection and apoptosis. To clarify whether the proapoptotic character of the stress response is reduced in heat-exposed islets still embedded in their native environment, hyperthermia was performed in the present study either as whole body hyperthermia (WBH) prior to pancreas resection or as in vitro heat shock (HS) after isolation. HS (42°C/45 min) was induced in donors 12 h before isolation (WBH, n = 32) or in freshly isolated islets prior to 12 h of culture at 37°C (in vitro HS, n = 25). Islets continuously incubated at 37°C served as controls (n = 34). Proinflammatory treatment was performed with H2O2, DETA-NO, or a combination of IL-1b, TNF-a, and IFN-g. Quality assessment included islet yield, viability staining, static glucose incubation, and nude mouse transplantation. WBH was significantly less effective than in vitro HS to induce HSP70 overexpression and to increase islet resistance against inflammatory mediators. Although characterized by an unaltered Bax to Bcl-2 ratio, islets subjected to WBH partially failed to restore sustained normoglycemia in diabetic nude mice. The inflammatory response observed in the pancreas of WBH-treated rats was associated with significantly reduced viability that seems to have a higher predictive value for posttransplant outcome compared to islet in vitro function or mitochondrial activity. In contrast, in vitro HS significantly decreased transcript levels of Bcl-2, but did not affect posttransplant function compared to sham-treated islets. These findings suggest that WBH is primarily associated with increased necrosis as a secondary tissue type-specific effect of pancreas damage while in vitro HS mainly induces apoptosis.

Key words: Rats; Islet isolation; Heat shock; Inflammation; Apoptosis; Whole body hyperthermia

Address correspondence to Daniel Brandhorst, Department of Oncology, Radiology & Clinical Immunology, University Hospital Uppsala, Dag Hammarskjölds väg 20, 75185 Uppsala, Sweden. Tel: +46-70-4250636; Fax: +46-18-611-0222; E-mail: Daniel.Brandhorst@klinimm.uu.se

Cell Transplantation, Vol. 16, pp. 717-728, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Adult-Derived Human Liver Mesenchymal-Like Cells as a Potential Progenitor Reservoir of Hepatocytes?

Mustapha Najimi,1 Dung Ngoc Khuu,1 Philippe Antoine Lysy,1 Nawal Jazouli,1 Jorge Abarca,2 Christine Sempoux,3 and Etienne Marc Sokal1

1Université catholique de Louvain, Laboratory of Pediatric Hepatology & Cell Therapy, B-1200 Brussels, Belgium
2Université catholique de Louvain, Radiodiagnostic, Cliniques universitaires St-Luc, B-1200, Brussels, Belgium
3Université catholique de Louvain, Anatomo-pathology, Cliniques universitaires St-Luc, B-1200 Brussels, Belgium

It is currently accepted that adult tissues may develop and maintain their own stem cell pools. Because of their higher safety profile, adult stem cells may represent an ideal candidate cell source to be used for liver cell therapies. We therefore evaluated the differentiation potential of mesenchymal-like cells isolated from adult human livers. Mesenchymal-like cells were isolated from enzymatically digested adult human liver and expanded in vitro. Cell characterization was performed using flow cytometry, RT-PCR, and immunofluorescence, whereas the differentiation potential was evaluated both in vitro after incubation with specific media and in vivo after intrasplenic transplantation of uPA+/+-SCID and SCID mice. Adult-derived human liver mesenchymal-like cells expressed both hepatic and mesenchymal markers among which albumin, CYP3A4, vimentin, and a-smooth muscle actin. In vitro differentiation studies demonstrated that these mesenchymal-like cells are preferentially determined to differentiate into hepatocyte-like cells. Ten weeks following intrasplenic transplantation into uPA+/+-SCID mice, recipient livers showed the presence of human hepatocytic cell nodules positive for human albumin, prealbumin, and a-fetoprotein. In SCID transplanted liver mice, human hepatocyte-like cells were mostly found near vascular structures 56 days posttransplantation. In conclusion, the ability of isolated adult-derived liver mesenchymal stem-like cells to proliferate and differentiate into hepatocyte-like cells both in vitro and in vivo leads to propose them as an attractive expandable cell source for stem cell therapy in human liver diseases.

Key words: Stem cells; Transplantation; SCID mice; Differentiation; Cell therapy

Address correspondence to Mustapha Najimi, Ph.D., Laboratory of Pediatric Hepatology and Cell Therapy, Avenue Hippocrate 10/1301, 1200 Bruxelles, Belgium. Tel: +32 2 764 5283; Fax: +32 2 764 8909; E-mail: mustapha.najimi@pedi.ucl.ac.be

Cell Transplantation, Vol. 16, pp. 729-739, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Novel Cell Seeding System Into a Porous Scaffold Using a Modified Low-Pressure Method to Enhance Cell Seeding Efficiency and Bone Formation

Ichiro Torigoe,1 Shinichi Sotome,1,2 Akio Tsuchiya,1,3 Toshitaka Yoshii,1 Makoto Takahashi,1 Shigenori Kawabata,1 and Kenichi Shinomiya1,3,4,5

1Section of Orthopaedic and Spinal Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan
2Section of Regenerative Therapeutics for Spine and Spinal Cord, Graduate School, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan
3The 21st Century Center of Excellence (COE) Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, 101-0062, Japan
4Hard Tissue Genome Research Center, Tokyo Medical and Dental University, Tokyo, 101-0062, Japan
5Core to Core Program for Advanced Bone and Joint Science, Tokyo Medical and Dental University, Tokyo, 101-0062, Japan

The efficient seeding of cells into porous scaffolds is important in bone tissue engineering techniques. To enhance efficiency, we modified the previously reported cell seeding techniques using low-pressure conditions. In this study, the effects of low pressure on bone marrow-derived stromal cells (BMSCs) of rats and the usefulness of the modified technique were assessed. There was no significant difference found in the proliferative and osteogenic capabilities among various low-pressure (50-760 mmHg, 1-10 min) conditions. To analyze the efficacies of the cell seeding techniques, BMSCs suspended in the plasma of rats were seeded into porous b-tricalcium phosphate (b-TCP) blocks by the following three procedures: 1) spontaneous penetration of cell suspension under atmospheric pressure (SP); 2) spontaneous penetration and subsequent low pressure treatment (SPSL), the conventional technique; and 3) spontaneous penetration under low pressure conditions (SPUL), the modified technique. Subsequently, these BMSCs/b-TCP composites were used for the analysis of cell seeding efficiency or in vivo bone formation capability. Both the number of BMSCs seeded into b-TCP blocks and the amount of bone formation of the SPUL group were significantly higher than those of the other groups. The SPUL method with a simple technique permits high cell seeding efficiency and is useful for bone tissue engineering using BMSCs and porous scaffolds.

Key words: Tissue engineering; Bone formation; Porous scaffolds; Bone marrow-derived stromal cells (BMSCs); Cell seeding method; Low pressure

Address correspondence to Shinichi Sotome, M.D., Ph.D., Section of Regenerative Therapeutics for Spine and Spinal Cord, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. Tel: +81-3-5803-5272; Fax: +81-3-5803-5272; E-mail: sotome.orth@tmd.ac.jp

Cell Transplantation, Vol. 16, pp. 741-749, 2007
0963-6897/07 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Improvement of Urethral Sphincter Deficiency in Female Rats Following Autologous Skeletal Muscle Myoblasts Grafting

Christophe Praud,1 Philippe Sebe,2 Anne-Sophie Biérinx,3 and Alain Sebille3

1INSERM U582, Institut de Myologie, Université Pierre et Marie Curie-Paris 6, Groupe Hospitalier Pitié Salpêtrière, Paris F-75013, France
2AP-HP, Service d'Urologie, Hôpital Tenon, 75020 Paris, France
3Atelier de régénération neuromusculaire, Université Pierre et Marie Curie-Paris 6, 75012 Paris, France

Sphincteric deficiency is the most common cause of urinary incontinence in humans. Various treatments have lead to disappointing results due to a temporary benefit. Recent studies raised the possibility that sphincteric deficiency could be treated by implanting skeletal myoblasts. In the present study, we developed in the female rat a model of chronic sphincteric defect to assess the benefit of myoblast injection. Sphincter deficiency was induced by freezing, longitudinal sphincterotomy, and notexin injection, respectively, to obtain a reproducible and irreversible incontinence. Autologous tibialis anteriors were cultured to be injected in the best model. Functional results were evaluated by measuring the urethral pressure with an open catheter. Histology was performed in the excised urethras. Of the three techniques, only longitudinal sphincterotomy caused definitive incontinence by irreversibly destroying the striated sphincter muscle fibers: a 45% decrease of the closure pressure was observed 21 days after the sphincterotomy. At this time, we injected myoblasts at the sphincterotomy site. In the sham-injected group (n = 18), the closure pressure decrease was not significantly modified 21 days after injection. By comparison, a return to near normal value was observed after cell grafting (n = 21). These results and those obtained by others strongly suggest that the use of myoblasts could be a potential innovative therapy for urethral deficiencies leading to incontinence.

Key words: Urinary incontinence; Urethral pressure; Notexin; Sphincterotomy

Address correspondence to Christophe Praud, INSERM U582, Institut de Myologie, Batiment Babinski, Hôpital de la Pitiè-Salpétrière, 47 boulevard de l'Hôpital, 75651 Paris cedex 13, France. Tel: 33 1 42 16 57 12; Fax; 33 1 42 16 57 00; E-mail: c.praud@institut-myologie.org