ognizant Communication Corporation


VOLUME 11, NUMBER 4, 2002

Cell Transplantation, Vol. 11, pp. 313-324, 2002
0963-6897/02 $20.00 + 00
Copyright © 2002 Cognizant Comm. Corp.
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Microencapsulation of Cells Producing Therapeutic Proteins: Optimizing Cell Growth and Secretion

Anne Mari Rokstad,1 Synnøve Holtan,2 Berit Strand,1,2 Bjørg Steinkjer,1 Liv Ryan,1 Bård Kulseng,1 Gudmund Skjåk-Bræk,2 and Terje Espevik1

1Institute of Cancer Research and Molecular Biology and 2Institute of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway

Microencapsulation of genetically engineered cells may have important applications as delivery systems for therapeutic proteins. However, optimization of the microcapsules with regard to mechanical stability, cell growth, and secretion of proteins is necessary in order to evaluate the future use of this delivery technology. We have explored the growth, survival, and secretion of therapeutic proteins from 293-EBNA cells producing endostatin (293 endo cells) and JJN3 myeloma cells producing hepatocyte growth factor (HGF) that have been embedded in various types of alginate capsules. Parameters that affect capsule integrity such as homogenous and inhomogenous gel cores and addition of an outer poly-L-lysine (PLL)-alginate coating were evaluated in relation to cell functions. When cells were encapsulated, the PLL layer was found to be absolutely required for the capsule integrity. The JJN3 and 293 endo cells displayed completely different growth and distribution patterns of live and dead cells within the microcapsules, as shown by 3D pictures reconstructed from images taken with confocal laser scanning microscopy (CLSM). Encapsulated JJN3 cells showed a bell-shaped growth and HGF secretion curve over a time period of 5 months. The 293 endo cells reached a plateau phase in growth after 23 days postencapsulation; however, after around 30 days a fraction of the microcapsules started to disintegrate. Microcapsule disintegration occurred with time irrespective of capsule and cell type, showing that alginate microcapsules possessing relatively high gel strength are not strong enough to keep proliferating cells within the microcapsules for prolonged time periods. Although this study shows that the stability of an alginate-based cell factory can be increased by a PLL-alginate coating, further improvement is necessary with regard to capsule integrity as well as controlling the cell growth before this technology can be used for therapy.

Key words: Alginate microencapsulation; Capsule constructions; Optimizing; Proliferating cells; Endostatin; Confocal laser scanning microscopy

Address correspondence to Anne Mari Rokstad, Institute of Cancer Research and Molecular Biology, Medical Technical Center, Olav Kyrresgt. 3, 7489 Trondheim, Norway. Tel: (47) 73598666; Fax: (47) 73598801; E-mail: anne.m.rokstad@medisin.ntnu.no

Cell Transplantation, Vol. 11, pp. 325-329, 2002
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In Vitro Study of Encapsulation Therapy for Fabry Disease Using Genetically Engineered CHO Cell Line

Y. Naganawa,1 K. Ohsugi,1 R. Kase,2 I. Date,3 H. Sakuraba,2 and N. Sakuragawa1

1Department of Inherited Metabolic Disease, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodiara, Tokyo 187-8502, Japan
2Department of Clinical Genetics, Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo 113-8613, Japan
3Department of Neurological Surgery, Okayama University Medical School, Okayama 700-8558, Japan

Fabry disease is an X-linked recessive disorder caused by a deficiency of the lysosomal hydrolase a-galactosidase A (a-gal). The deficiency of this enzyme leads to the systemic deposition of ceramide trihexoside (CTH) in various tissues and organs. Enzyme replacement using IV doses of recombinant human a-gal produced in CHO cells or in human fibroblasts is currently being evaluated in clinical trials as a potential therapy for this disease. However, it requires lifelong therapy involving a large amount of purified a-gal. As a novel approach for treatment of Fabry disease we used polymer encapsulated Chinese hamster ovary (CHO) cells genetically modified to express a-gal. The secreted high levels of a-gal passed through the semipermeable polymeric membrane. Using coculture system with Fabry fibroblasts, the secreted enzyme was taken up in cells, resulting in reduced accumulation of CTH in Fabry fibroblasts. This in vitro study demonstrated that an encapsulated a-gal-secreting cell line can be used to treat Fabry mice by transplantation in vivo. Judging from the protection against immune rejection by a semipermeable synthetic membrane, this novel approach may be applied to treat patients with Fabry disease and other lysosomal storage diseases.

Key words: Fabry disease; Gene therapy; Encapsulation; CHO cells; a-Galactosidase; Fibroblasts; Ceramide trihexoside (CTH)

Address correspondence to Norio Sakuragawa, M.D., Ph.D., Department of Inherited Metabolic Disease, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodiara, Tokyo 187-8502, Japan. Tel: 81-42-346-1716; Fax: 81-42-346-1746; E-mail: sakuraga@ncnp.go.jp

Cell Transplantation, Vol. 11, pp. 331-349, 2002
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Enhanced Vascularization and Survival of Neural Transplants With Ex Vivo Angiogenic Gene Transfer

Diana Casper,1 Samara J. Engstrom,1 Gautam R. Mirchandani,1 Ann Pidel,1 David Palencia,1 Paul H. Cho,1 Michael Brownlee,2 Diane Edelstein,2 Howard J. Federoff,3 and William J. Sonstein1

Departments of 1Neurological Surgery and 2Medicine, Montefiore Medical Center and the Albert Einstein College of Medicine, Bronx, NY
3Department of Neurology, University of Rochester, School of Medicine and Dentistry, Rochester, NY

Restoration of brain function by neural transplants is largely dependent upon the survival of donor neurons. Unfortunately, in both rodent models and human patients with Parkinson's disease the survival rate of transplanted neurons has been poor. We have employed a strategy to increase the availability of nutrients to the transplant by increasing the rate at which blood vessels are formed. Replication-deficient HSV-1 vectors containing the cDNA for human vascular endothelial growth factor (HSVhvegf) and the bacterial \GK\b-galactosidase gene (HSVlac) have been transduced in parallel into nonadherent neuronal aggregate cultures made of cells from embryonic day 15 rat mesencephalon. Gene expression from HSVlac was confirmed in fixed preparations by staining with X-gal. VEGF expression as determined by sandwich ELISA assay of culture supernatant was up to 322-fold higher in HSVhvegf-infected than HSVlac-infected sister cultures. This peptide was also biologically active, inducing endothelial cell proliferation in vitro. Adult Sprague-Dawley rats received bilateral transplants into the striatum, with HSVlac on one side and HSVhvegf on the other. At defined intervals up to 8 weeks, animals were sacrificed and vibratome sections of the striatum were assessed for various parameters of cell survival and vascularization. Results demonstrate dose-dependent increases in blood vessel density within transplants transduced with HSVhvegf. These transplants were vascularized at a faster rate up to 4 weeks after transplantation. After 8 weeks, the average size of the HSVhvegf-infected transplants was twice that of controls. In particular, the survival of transplanted dopaminergic neurons increased 3.9-fold. Taken together these experiments provide convincing evidence that the rate of vascularization may be a major determinant of neuronal survival that can be manipulated by VEGF gene transduction.

Key words: VEGF; Angiogenesis; Neural transplantation; Parkinson's disease; Dopaminergic neurons; Vascularization; Ex vivo gene therapy

Address correspondence to Dr. Diana Casper, Neurosurgery Lab, Moses Bldg., Room 314, Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467. Tel: (718) 920-4064; Fax: (718) 653-3284; E-mail: casper@aecom.yu.edu

Cell Transplantation, Vol. 11, pp. 351-358, 2002
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Effects of Caspase Inhibitors on Hematopoietic Engraftment After Short-Term Culture

Anne Wiesmann, A. Elena Searles, L. Jeanne Pierce, and Gerald J. Spangrude

Departments of Oncological Sciences, Pathology, and Medicine, Division of Hematology, University of Utah, Salt Lake City, UT 84132

The induction of apoptosis during cytokine-induced proliferation of hematopoietic stem and progenitor cells (HSPC) may result in the loss of hematopoietic function. We tested the ability of several caspase inhibitors to maintain transplantation potential of mouse HSPC during in vitro culture. HSPC were isolated from mouse bone marrow by cell sorting and cultured in the presence of steel factor (STL) with or without various caspase inhibitors. After incubation, cells were harvested and tested for in vitro colony-forming cell (CFC) potential and transplantation activity in both short- and long-term in vivo assays. HSPC required STL to retain CFC activity during a 24-h culture at 37°C, and none of three caspase inhibitors could substitute for STL in this respect. In transplant assays, a twofold higher frequency of animals showed donor-derived blood cells 12 weeks after competitive transplantation of 50 HSPC cultured for 4 h in the presence of STL plus n-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (ac-YVAD) compared with 50 cells cultured in STL alone. To evaluate the effect of ac-YVAD on short-term engraftment, 500 cultured HSPC were transplanted into lethally irradiated mice. Animals transplanted with cells cultured in the presence of ac-YVAD showed a higher survival rate and a faster recovery of platelets and hematocrit compared with animals transplanted with cells cultured in STL alone. We conclude that both the short-term and the long-term engraftment potentials of HSPC cultured in the presence of STL + ac-YVAD were superior to that obtained from cells cultured in STL alone.

Key words: Stem cell transplantation; Cytokine; Ex vivo expansion; Caspase; Apoptosis

Address correspondence to Dr. Gerald Spangrude, University of Utah, Oncological Sciences, 50 North Medical Drive RM 5C334, Salt Lake City, UT 84132. Tel: (801) 585-5544; Fax: (801) 585-3778; E-mail: drblood@path.utah.edu

Cell Transplantation, Vol. 11, pp. 359-368, 2002
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Copyright © 2002 Cognizant Comm. Corp.
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In Vivo Differentiation of Mouse Embryonic Stem Cells Into Hepatocytes

Dongho Choi,1 Hyun-Jeong Oh,1 Uck-Jin Chang,1 Soo Kyung Koo,1 Jean X. Jiang,2 Sue-Yun Hwang,3 Jung-Dal Lee,4 George C. Yeoh,5 Hee-Sup Shin,6 Jin-Sung Lee,6 and Bermseok Oh1

1Division of Genetic Disease, Department of Biomedical Science, National Institute of Health, Seoul 122-701, Korea
2Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229-3900
3Research Institute of Immunology, Catholic Institutes of Medical Science, The Catholic University of Korea, Seoul 137-701, Korea
4Department of Pathology, SongDo Hospital, Seoul 100-453, Korea
5Department of Biochemistry, University of Western Australia, Nedlands, Western Australia, Australia
6Korea Institute of Science and Technology, Seoul 130-650, Korea
7Department of Pediatrics, Yonsei University Medial School, Seoul 120-752, Korea

Embryonic stem (ES) cells have been regarded as a powerful resource for cell replacement therapy. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. Another type of disease that could benefit from the availability of stem cell therapy is liver disease. However, for this potential to be realized, it is necessary to demonstrate the differentiation of ES cells into hepatocytes. To demonstrate the in vivo differentiation potential of mouse ES cells, we injected ES cells into the spleen of immunosuppressed nude mice. Histological analysis of teratomas derived from injected ES cells revealed that some areas contained typical hepatocytes arranged in a sinusoidal structure. The hepatic nature of these cells was further confirmed by showing that transcripts of liver-specific genes were present in the differentiated teratoma using reverse transcriptase-polymerase chain reaction and immunohistochemistry using several liver-specific antibodies including HEP-PAR, phenylalanine hydroxylase, and mouse N-system aminotransferase to identify the respective proteins in the differentiated hepatocytes. This is the first demonstration that mouse ES cells can differentiate in vivo into a mixed population of hepatocytes of varying maturity. This finding extends the potential use of ES cells in the cell replacement therapy by including its possible application for treating liver diseases.

Key words: Embryonic stem cell; Differentiation; Hepatocyte; Stem cell therapy; Transplantation

Address correspondence to Bermseok Oh, Ph.D., Nokbun-Dong 5, Eunpyung-Gu, Seoul, 122-701, Korea. Tel: +82-2-380-1534; Fax: +82-2-388-0924; E-mail: Ohbs@nih.go.kr

Cell Transplantation, Vol. 11, pp. 369-377, 2002
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Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

Makarand V. Risbud,1 Erdal Karamuk,1 René Moser,2 and Joerg Mayer1

1Biocompatible Materials Science and Engineering, Swiss Federal Institute of Technology (ETH)-Zürich, CH-8952, Zürich, Switzerland
2Institute for Biopharmaceutical Research Inc., Matzingen, Switzerland

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan-collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require precoating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

Key words: Textile scaffolds; Hydrogel; HUVECs; Three-dimensional growth; Tissue engineering

Address correspondence to Makarand V. Risbud at his current address: Cell and Tissue Engineering Program, Department of Orthopedic Research, Thomas Jefferson University, 501 Curtis Building, 1015 Walnut Street, Philadelphia, PA 19107. Fax: (215) 995-9159.

Cell Transplantation, Vol. 11, pp. 379-384, 2002
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High Metabolic Function of Primary Human and Porcine Hepatocytes in a Polyurethane Foam/Spheroid Culture System in Plasma From Patients With Fulminant Hepatic Failure

Yo-ichi Yamashita,1 Mitsuo Shimada,1 Eiji Tsujita,1 Ken Shirabe,1 Hiroyuki Ijima,2 Kohji Nakazawa,2 Ryoichi Sakiyama,2 Junji Fukuda,2 Kazumori Funatsu,2 and Keizo Sugimachi1

1Department of Surgery and Science, Graduate School of Medical Sciences, and 2Department of Engineering, Graduate School of Engineering, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan

It has been demonstrated that plasma from patients with fulminant hepatic failure (FHF) interferes extensively with cellular function. We placed primary human and primary porcine hepatocytes in a polyurethane foam (PUF)/spheroid culture system and compared the metabolic functions in the plasma of patients with FHF in a 24-h stationary culture to those in a monolayer culture. The PUF/spheroid culture system using primary human and primary porcine hepatocytes significantly decreased ammonia content during 28-day culture. Fisher's ratio significantly increased at culture days 3 and 7. Tauroursodeoxycholic acid significantly increased and glycochenodeoxycholic acid and taurochenodeoxycholic acid decreased in the FHF patients' plasma at culture day 3. During at least a 24-h culture in the FHF patients' plasma, metabolic functions of primary human and primary porcine hepatocytes were almost identical. The present results indicate that the PUF/spheroid culture system using primary human or primary porcine hepatocytes demonstrated more advantageous metabolic functions in the plasma from patients with FHF than the monolayer culture.

Key words: Fulminant hepatic failure; Human hepatocytes; Porcine hepatocytes; Polyurethane foam/spheroid culture system

Address correspondence to Yo-ichi Yamashita, M.D., Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel: 81-92-642-5469; Fax: 81-92-642-5482; E-mail: yamashi@surg2.med.kyushu-u.ac.jp

Cell Transplantation, Vol. 11, pp. 385-395, 2002
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Transient Hyperproliferation of a Transgenic Human Epidermis Expressing Hepatocyte Growth Factor

Karen E. Hamoen and Jeffrey R. Morgan

Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and Harvard Medical School, Shriners Hospital for Children, Boston, MA

Hepatocyte growth factor (HGF) is a fibroblast-derived protein that affects the growth, motility, and differentiation of epithelial cells including epidermal keratinocytes. To investigate the role of HGF in cutaneous biology and to explore the possibility of using it in a tissue engineering approach, we used retroviral-mediated gene transfer to introduce the gene encoding human HGF into diploid human keratinocytes. Modified cells synthesized and secreted significant levels of HGF in vitro and the proliferation of keratinocytes expressing HGF was enhanced compared with control unmodified cells. To investigate the effects of HGF in vivo, we grafted modified keratinocytes expressing HGF onto athymic mice using acellular dermis as a substrate. When compared with controls, HGF-expressing keratinocytes formed a hyperproliferative epidermis. The epidermis was thicker, had more cells per length of basement membrane, and had increased numbers of Ki-67-positive proliferating cells, many of which were suprabasal in location. Hyperproliferation subsided and the epidermis was equivalent to controls by 2 weeks, a time frame that coincides with healing of the graft. Transient hyperproliferation may be linked to the loss of factors present in the wound that activate HGF. These data suggest that genetically modified skin substitutes secreting HGF may have applications in wound closure and the promotion of wound healing.

Key words: Skin graft; Growth factor; Wound healing; Tissue engineering

Address correspondence to Jeffrey R. Morgan, Ph.D., Shriners Hospital for Children, 51 Blossom Street, Boston, MA, 02114. Tel: (617) 371-4878; Fax: (617) 371-4950; E-mail: jmorgan@sbi.org