ognizant Communication Corporation


VOLUME 13, NUMBERS 7/8, 2004

Cell Transplantation, Vol. 13, pp. 725-727, 2004
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Healing a Broken Heart With Stem Cells

David M. Stern,1 David C. Hess,1,2,3 and Cesar V. Borlongan1,2,3

1School of Medicine and 2Department of Neurology and Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA
3Research and Affiliations Service Line, Augusta VAMC, Augusta, GA

We discuss here the rapid progress of stem cell therapy in myocardial infarction. In particular, we focus on the issue of transdifferentiation as a "hallmark" of the stem cell's potential to replace damaged cells of the heart. A study by Henning and colleagues in this issue of Cell Transplantation supports the alternative notion of a nontransdifferentiation-mediated protection of the heart as an equally robust mechanism underlying the therapeutic potential of stem cells.

Key words: Stem cell therapy; Myocardial infarction; Transdifferentiation; Therapeutic potential

Address correspondence to Dr. Cesar V. Borlongan, Department of Neurology, BI-3080, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-3200. Tel: (706) 733-0188, Ext. 2485; Fax: (706) 721-7619; E-mail: cborlongan@mail.mcg.edu

Cell Transplantation, Vol. 13, pp. 729-739, 2004
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Human Umbilical Cord Blood Mononuclear Cells for the Treatment of Acute Myocardial Infarction

Robert J. Henning,1 Hamdi Abu-Ali,1 John U. Balis,2 Michael B. Morgan,2 Alison E. Willing,3 and Paul R. Sanberg3

Departments of 1Medicine, 2Pathology, and 3Neurosurgery and the Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, and the James A. Haley Veterans' Hospital, Tampa, FL

Cell transplantation is a new treatment to improve cardiac function in hearts that have been damaged by myocardial infarction. We have investigated the use of human umbilical cord blood mononuclear progenitor cells (HUCBC) for the treatment of acute myocardial infarction. The control group consisted of 24 normal rats with no interventions. The infarct + vehicle group consisted of 33 rats that underwent left anterior descending coronary artery (LAD) ligation and after 1 h were given Isolyte in the border of the infarction. The infarct + HUCBC group consisted of 38 rats that underwent LAD ligation and after 1 h were given 106 HUCBC in Isolyte directly into the infarct border.  Immunosuppression was not given to any rat. Measurements of left ventricular (LV) ejection fraction, LV pressure, dP/dt, and infarct size were determined at baseline and 1, 2, 3, and 4 months. The ejection fraction in the controls decreased from 88 ± 3% to 78 ± 4% at 4 months (p = 0.03) as a result of normal aging. Following infarction in the infarct + vehicle group, the ejection fraction decreased from 87 ± 4% to 51 ± 3% between 1 and 4 months (p < 0.01). In contrast, the ejection fraction of the infarcted + HUCBC-treated rat hearts decreased from 87 ± 4% to 63 ± 3% at 1 month, but progressively increased to 69 ± 6% at 3 and 4 months, which was different from infarct + vehicle group rats (p < 0.02) but similar to the controls. At 4 months, anteroseptal wall thickening in infarct + HUCBC group was 57.9 ± 11.6%, which was nearly identical to the control anteroseptal thickening of 59.2 ± 8.9%, but was significantly greater than the infarct + vehicle group, which was 27.8 ± 7% (p < 0.02). dP/dtmax increased by 130% in controls with 5.0 mg of phenylephrine (PE)/min (p < 0.001). In the infarct + vehicle group, dP/dtmax increased by 91% with PE (p = 0.01). In contrast, in the infarct + HUCBC group, dP/dtmax increased with PE by 182% (p < 0.001), which was significantly greater than the increase in dP/dtmax in the infarct + vehicle group (p = 0.03) and similar to the increase in the controls. Infarct sizes in the infarct + HUCBC group were smaller than the infarct + vehicle group and averaged 3.0 ± 2.8% for the infarct + HUCBC group versus 22.1 ± 5.6% for infarct + vehicle group at 3 months (p < 0.01); at 4 months they averaged 9.2 ± 2.0% for infarct + HUCBC group versus 40.0 ± 9.2% for the infarct + vehicle group (p < 0.001). The present experiments demonstrate that HUCBC substantially reduce infarction size in rats without requirements for immunosuppression. As a consequence, LV function measurements, determined by LV ejection fraction, wall thickening, and dP/dt, are significantly greater than the same measurements in rats with untreated infarctions.

Key words: Umbilical cord blood cells; Stem cells; Acute myocardial infarction; Infarct size; Left ventricular (LV) function; Ejection fraction; dP/dt

Address correspondence to Robert J. Henning, M.D., Professor of Medicine, University of South Florida College of Medicine/James A. Haley Hospital, 13000 Bruce B. Downs Blvd., Tampa, FL 33612. Tel: (813) 978-5873; Fax: (813)-978-5874; E-mail: rhenning@hsc.usf.edu

Cell Transplantation, Vol. 13, pp. 741-748, 2004
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G-CSF Promotes Bone Marrow Cells to Migrate Into Infarcted Mice Heart, and Differentiate Into Cardiomyocytes

Shinya Fukuhara,1,2 Shinji Tomita,1,3* Takeshi Nakatani,4 Yoshinori Ohtsu,1 Michiko Ishida,1 Chikao Yutani,2 and Soichiro Kitamura3

1Department of Regenerative Medicine & Tissue Engineering, 2Department of Pathology, 3Department of Cardiovascular Surgery, and 4Department of Organ Transplantation, National Cardiovascular Center, Osaka, Japan

A recent study showed that granulocyte-colony stimulating factor (G-CSF) treatment improved the infarcted cardiac function. Although mobilized stem cells may affect it, the mechanism is unclear. In this study, we investigated the origins of stem cells and phenotypic changes of the migrated cells, and evaluated the efficacy of G-CSF. Eighteen C57BL/6 mice were irradiated (900 cGy) and GFP mouse-derived bone marrow cells (GFP-BMC: 106 cells) were injected via a tail vein followed by splenectomy 4 weeks later. Ligation of the left descending coronary artery was performed 2 weeks later. Recombinant human G-CSF (200 mg/kg/day) was injected for 3 days before and 5 days after ligation (group 1, n = 10). Saline was injected in group 2 (n = 8). Four weeks after infarction, hearts and other organs were fixed for histology. The survival rate after postoperative day 3 in group 1 was 100%, while that in group 2 was 50% (p = 0.03). Bone marrow-derived GFP cells (BMD-GFP) in group 1 (103.3 ± 71.9/mm2) were located at the infarcted border area significantly more than those in group 2 (43.6 ± 23.7/mm2) (p < 0.0001). BMD-GFP cells were positive for troponin I (16.6%), myosin heavy chain-slow (16.7%), and nestin (8.8%) in group 1. Ki-67-positive BMD-GFP in group 1 (10.0 ± 7.0/mm2) were significantly more than those in group 2 (4.8 ± 6.1/mm2) (p = 0.01). G-CSF increased the survival rate after infarction. G-CSF promoted BMC to migrate into the infarcted border area. Bone marrow was one of the origins of regenerated cardiomyocytes.

Key words: Bone marrow; Stem cells; Cardiomyocytes; Regeneration; G-CSFAddress correspondence to Shinya Fukuhara, Department of Cardiology,

Address correspondence toShinya Fukuhara, Department of Cardiology, Saiseikai-Suita Hospital 1-2, Kawazonotoyo, Suita, Osaga, Japan 564-0013. Tel: +81-6-6382-1521, ext. 4123; Fax: +81-6-6382-2498; E-mail: shinyafukuhara@hotmail.com

*Current address: Cardiothoracic Surgery Unit, Auckland City Hospital, Auckland, New Zealand.

Cell Transplantation, Vol. 13, pp. 749-754, 2004
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Evaluation of Surgical Techniques for Neuronal Cell Transplantation Used in Patients With Stroke

Douglas Kondziolka,1 Gary K. Steinberg,2 Sean B. Cullen,3* and Michael McGrogan3*

1Department of Neurological Surgery, University of Pittsburgh and the McGowan Institute for Regenerative Medicine, Pittsburgh, PA
2Department of Neurosurgery, Stanford University, Stanford, CA
3Layton BioScience, Inc., Sunnyvale, CA

Transplantation of cultured neuronal cells was performed in two human clinical trials after safety and efficacy was demonstrated in animal models of stroke. The studies tested the utility of human neuronal cellular transplantation into and around the small stroke volume. We developed a stereotactic surgical technique for cell delivery and evaluated that method in 26 patients with basal ganglia region motor stroke. Human neuronal cells (hNT cells; LBS neurons) were delivered frozen then thawed and formulated on the morning of surgery. Patients in a first trial received 2 or 6 million cells in three or nine implants, and in a second trial, 5 or 10 million in 25 implants. A novel cell delivery cannula was designed, manufactured, tested, and used in surgery. Immediate postoperative CT scans and later serial MR scans were used to evaluate the surgical site. Tests on the cell implantation cannula showed that the cells were not damaged and remained viable after injection. All patients underwent uncomplicated surgeries. Cells could be implanted within a 2-h period, maintaining viability of the preparation. Serial evaluations (maximum 5 years) showed no cell-related adverse serologic or imaging-defined effects. One patient had burr hole drainage of an asymptomatic chronic subdural hematoma. Human neuronal cells can be produced in culture and implanted stereotactically into the brains of patients with stroke. Surgical cell delivery did not lead to new neurological deficits, and imaging studies showed no adverse effects. The cannula used allowed precise injection of the clinical cell dose within a time period that maintained cell viability.

Key words: Stroke; Transplantation; Paralysis; Neurons; Infarction; Stereotactic surgery

Address correspondence to Douglas Kondziolka, M.D., Suite B-400, UPMC, 200 Lothrop Street, Pittsburgh, PA 15213. Tel: (412) 647-6782; Fax: (412) 647-0989; E-mail: kondziolkads@upmc.edu

*The authors no longer work for Layton BioScience, Inc.

Cell Transplantation, Vol. 13, pp. 755-763, 2004
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Formation and Structure of Transplantable Tissue Constructs Generated in Simulated Microgravity From Sertoli Cells and Neuron Precursors

Don F. Cameron,1,2 J. J. Hushen,1 L. Colina,1 J. Mallery,1 A. Willing,1,2,3 P. R. Sanberg,1,3 and Samuel Saporta1,2,3

Departments of 1Anatomy, 2Neurosurgery, and the 3Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL 33612

Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT--specific for NT2 cells) and cholera toxin subunit B (CTb--specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.

Key words: Sertoli cells; NT2 cells; Simulated microgravity coculture; Morphology; Parkinson's disease

Address correspondence to Don F. Cameron, Ph.D., Department of Anatomy, MDC-6, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel: (813) 974-9434; Fax: (813) 974-2058; E-mail: dcameron@hsc.usf.edu

Cell Transplantation, Vol. 13, pp. 765-773, 2004
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The Potential of Bone Marrow Stem Cells to Correct Liver Dysfunction in a Mouse Model of Wilson's Disease

Katrina J. Allen,1 Daphne M. Y. Cheah,1 Xiao Ling Lee,1 Nicole E. Pettigrew-Buck,1 Jim Vadolas,1 Julian F. B. Mercer,2 Panayiotis A. Ioannou,1 and Robert Williamson1

1Cell and Gene Therapy Group, Murdoch Childrens Research Institute, Department of Paediatrics University of Melbourne, Royal Children's Hospital, Parkville, Victoria, 3052, Australia
2Centre for Cell and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood, Victoria, 3125, Australia

Metabolic liver diseases are excellent targets for correction using novel stem cell, hepatocyte, and gene therapies. In this study, the use of bone marrow stem cell transplantation to correct liver disease in the toxic milk (tx) mouse, a murine model for Wilson's disease, was evaluated. Preconditioning with sublethal irradiation, dietary copper loading, and the influence of cell transplantation sites were assessed. Recipient tx mice were sublethally irradiated (4 Gy) prior to transplantation with bone marrow stem cells harvested from normal congenic (DL) littermates. Of 46 transplanted tx mice, 11 demonstrated genotypic repopulation in the liver. Sublethal irradiation was found to be essential for donor cell engraftment and liver repopulation. Dietary copper loading did not improve cell engraftment and repopulation results. Both intravenously and intrasplenically transplanted cells produced similar repopulation successes. Direct evidence of functionality and disease correction following liver repopulation was observed in the 11 mice where liver copper levels were significantly reduced when compared with mice with no liver repopulation. The reversal of copper loading with bone marrow cells is similar to the level of correction seen when normal congenic liver cells are used. Transplantation of bone marrow cells partially corrects the metabolic phenotype in a mouse model for Wilson's disease.

Key words: Bone marrow; Irradiation; Liver; Transplantation; tx mouse; Wilson's disease

Address correspondence to Dr. Katrina Allen, Cell and Gene Therapy Group, Murdoch Childrens Research Institute, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria, 3052, Australia. Tel: (+613) 8341-6236; Fax: (+613) 9348-1391; E-mail: katie.allen@rch.org.au

Cell Transplantation, Vol. 13, pp. 775-781, 2004
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A Simple and Effective Method to Improve Intrasplenic Rat Hepatocyte Transplantation

Valeria Sigot,1 María G. Mediavilla,1 Graciela Furno,3 Joaquín V. Rodríguez,2 and Edgardo E. Guibert1

1Biología Molecular, Dto. Cs. Biológicas, 2Farmacología, Dto. Cs. Fisiológicas, 3Estadística, Dto. Matemáticas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Santa Fe, Argentina

Transplanted hepatocytes integrate, survive, and express their specific functions in the liver parenchyma. The aim of this study was to determine whether a large number of hepatocytes could move from the spleen to the liver when the cells are injected together with sodium nitroprusside, and if the improved hepatocyte migration may be related with portal vein dilatation. Wistar rats were transplanted in the spleen with fluorescent-labeled hepatocytes alone or together with sodium nitroprusside. At 1, 3, 6, and 24 h after the transplant, the liver from recipient animals was removed and morphometric analyses were performed. Portal and arterial pressures were also measured immediately after intrasplenic injection of a solution of sodium nitroprusside, hepatocytes alone, or hepatocytes plus sodium nitroprusside. Intrasplenically injected sodium nitroprusside produced a transient drop in arterial pressure and a sustained reduction in portal pressure. During hepatocyte transplantation it increased the number of transplanted cells migrating to the liver after 3 h. Sodium nitroprusside simultaneously injected with hepatocytes in the spleen allowed more cells to migrate into the liver of the host animal without risk in animal survival.

Key words: Hepatocyte transplantation; Sodium nitroprusside; Vasodilator; Portal pressure; Intrasplenic injection; Fluorescence microscopy

Address correspondence to Edgardo E. Guibert, Biología Molecular, Dto. Cs. Biológicas, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Santa Fe, Argentina. Tel/Fax: 0054-341-4393400; E-mail: eguibert@fbioyf.unr.edu.ar

Cell Transplantation, Vol. 13, pp. 783-791, 2004
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Cytotoxic Immune Response to a Xenogeneic Bioartificial Liver

Scott L. Nyberg, Bruce Amiot, Joseph Hardin, Edwina Baskin-Bey, and Jeffrey L. Platt

Division of Solid Organ Transplantation, Division of Biochemistry and Molecular Biology, Transplantation Biology Program, Mayo Clinic, Rochester, MN 55905

Prior studies have suggested the possibility of immune-mediated death of xenogeneic hepatocytes in a bioartificial liver (BAL) during hemoperfusion. This study was designed to elucidate how immunity may cause death of xenogeneic hepatocytes in the BAL. Healthy dogs were treated with a BAL containing hollow fiber membranes with large pores (200 nm) or small pores (400 kDa). The immune response of recipient dogs to BAL therapy was monitored over 3 h of treatment. We observed significantly greater loss of viability of hepatocytes in the 200 nm group compared with the 400 kDa group (p < 0.001). Low viability after treatment with the large pore membrane was associated with positive staining for dog IgG, dog IgM, and dog complement on dead hepatocytes. Significant levels of dog antibody were detected in samples of BAL medium from the 200 nm group. These canine antibodies were cytotoxic to porcine hepatocytes. In contrast, medium from the 400 kDa group contained only trace levels of dog IgG and were noncytotoxic. We conclude that antibody-mediated cytotoxicity contributed to the death of hepatocytes during treatment with a xenogeneic BAL. Immune-mediated death of hepatocytes was reduced by increasing selectivity of the BAL membrane.

Key words: Porcine hepatocyte; Bioartificial liver; Xenoreactive antibody

Address correspondence to Scott L. Nyberg M.D., Ph.D., 200 First Street S.W., Rochester, MN 55905. Tel: (507) 266-6772; Fax: (507) 266-2810; E-mail: nyberg.scott@mayo.edu

Cell Transplantation, Vol. 13, pp. 793-799, 2004
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cDNA Microarray Analysis in Hepatocyte Differentiation in Huh 7 Cells

Yo-ichi Yamashita,1 Mitsuo Shimada,1 Norifumi Harimoto,1 Shinji Tanaka,1 Ken Shirabe,1 Hiroyuki Ijima,2 Kohji Nakazawa,2 Junji Fukuda,2 Kazumori Funatsu,2 and Yoshihiko Maehara1

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

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of a hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we previously demonstrated the upregulation of hepatocyte-specific function by spheroid formation in polyurethane foam and by culturing with the histone deacetylase inhibitor, trichostatin A (TSA), in a human hepatoma cell line (Huh 7). In this article we analyze the gene expression profile using cDNA microarray (1281 genes) in spheroid formation or culturing with TSA in Huh 7 to determine the target genes in hepatocyte differentiation. In both the spheroid formation and in the culture with TSA, the Oct-3/4 transcription factor was upregulated more than twofold, while the early growth response-1 (EGR-1) transactivator was downregulated less than 0.5-fold. These results indicate that expressions of Oct-3/4 and EGR-1 may be key factors in the induction of hepatocyte differentiation in Huh 7.

Key words: cDNA microarray; Hepatocyte differentiation; Hybrid artificial liver support system (HALSS)

Address correspondence to Yo-ichi Yamashita, M.D. Ph.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: harimoto@surg2.med.kyushu-u.ac.jp

Cell Transplantation, Vol. 13, pp. 801-808, 2004
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Effect of Inspired Oxygen on Portal and Hepatic Oxygenation: Effective Arterialization of Portal Blood by Hyperoxia

Stephen J. Hughes,1,3 Wenxuan Yang,2 Maciej Juszczak,1,3 Gareth L. Jones,1,3 Stephen H. Powis,3 Alexander M. Seifalian,2 and Martin Press1

Departments of 1Endocrinology, 2Surgery, and 3Centre for Nephrology, Royal Free Campus, Royal Free & University College Medical School, London, UK

Because hypoxia may compromise the survival of intraportally transplanted pancreatic islets, we have measured portal blood flow and both portal and hepatic oxygenation in normal and diabetic rats breathing graded inspired oxygen concentrations. Portal blood flow and hepatic tissue oxygenation were measured using a transonic flowmeter and near infrared spectroscopy while gas analysis was carried out on portal venous blood samples. The effects of breathing 13%, 21%, 50%, or 100% oxygen were compared in animals with steptozotocin-induced diabetes and in controls. In diabetic rats breathing 21% oxygen, portal blood flow was significantly lower than in controls (7.2 ± 0.7 vs. 9.1 ± 0.8 ml/min, p < 0.05). In both groups, breathing 100% oxygen significantly increased portal flow (to 8.4 ± 1.0 and 12.2 ± 0.7 ml/min, respectively). This effect was not secondary to hepatic arterial vasoconstriction because it was not prevented by hepatic artery ligation. In controls, breathing 100% oxygen increased portal pO2 from 5.0 ± 0.9 to 14.4 ± 1.4 kPa (p < 0.05) and portal venous oxygen saturation (PSaO2) from 53.9 ± 12.1% to 92.9 ± 1.4% (p < 0.05), a value not significantly different from peripheral (arterial) saturation. Similarly, in diabetic animals pO2 rose from 5.6 ± 0.3 to 11.7 ± 0.4 kPa (p < 0.01) and SO2 from 55.5 ± 5.2% to 88.5 ± 0.6% (p < 0.05). Hepatic oxyhemoglobin rose and deoxyhemoglobin fell reciprocally as a function of the inspired oxygen concentration. Improved hepatic oxygenation observed in animals breathing oxygen-enriched gas mixtures results from an increase in splanchnic blood flow coupled with a marked increase in portal oxygen saturation. This effective arterialization of portal blood may have important consequences for the success of intraportal transplantation of pancreatic islets.

Key words: Experimental transplantation; Islets; Hyperoxia; Portal oxygenation

Address correspondence to Martin Press, Department of Endocrinology, Royal Free Hospital, Pond St., London NW3 2QG UK. Tel: 020 7830 2171; Fax: 020 7830 2171; E-mail martin.press@royalfree.nhs.uk

Cell Transplantation, Vol. 13, pp. 809-816, 2004
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Bioreactor Microcarrier Cell Culture System (Bio-MCCS) for Large-Scale Production of Autologous Melanocytes

Jin Yu Liu, Jürg Hafner, Galya Dragieva, and Günter Burg

Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland

Restoration of cutaneous pigmentation can be achieved in stable vitiligo by autologous cultured melanocyte transplantation. It was the goal of this study to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous melanocytes in large scale. In this Bio-MCCS, porcine gelatin microbeads were used as microcarriers, spinning bottle as fermented tank. Autologous melanocytes were able to attach to and proliferate on the gelatin microbeads in serum-free melanocyte medium in the Bio-MCCS, reaching up to 24-fold the cells seeded on day 15 (MTT assay). These autologous melanocytes cultured on gelatin microbeads could leave the microbeads and proliferate on the bottom of tissue culture flasks. Although Pluronic F68 has been widely used to protect animal cells from hydrodynamic stress in animal cell bioreactors, Pluronic F68 at a concentration of 0.25-1.0% showed no significant protective effects on the autologous melanocytes cultured on the microbeads and subjected to mechanical stress in the Bio-MCCS. This Bio-MCCS using porcine gelatin microbeads as microcarriers enabled large-scale production of autologous melanocytes, offering a potential treatment for large-area stable vitiligo by direct administration of the melanocytes cultured on the gelatin microbeads to the vitiliginous site.

Key words: Melanocyte; Cell transplantation; Bioreactor; Vitiligo; Cell culture

Address correspondence to Prof. Dr. med. Günter Burg, Chairman, Department of Dermatology, University Hospital of Zurich, GlorriaStr. 31, CH-8091, Zürich, Switzerland. Tel: 41-1-255-2550; Fax: 41-1-2553999; E-mail: burg@derm.unizh.ch

Cell Transplantation, Vol. 13, pp. 817-821, 2004
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A Comparison Between Real-Time Quantitative PCR and DNA Hybridization for Quantitation of Male DNA Following Myoblast Transplantation

Erika Bosio,1 Tracey F. Lee-Pullen,1,2 Clayton T. Fragall,1 Manfred W. Beilharz,1 Alayne L. Bennett,1 Miranda D. Grounds,2 Stuart I. Hodgetts,2 and Leanne M. Sammels1

1Discipline of Microbiology, School of Biomedical and Chemical Sciences, and 2School of Anatomy and Human Biology, The University of Western Australia, Perth, W.A., 6009, Australia

The transplantation of muscle precursor cells (myoblasts) is a potential therapy for Duchenne muscular dystrophy. A commonly used method to detect cell survival is quantitation of the Y chromosome following transplantation of male donor cells into female hosts. This article presents a direct comparison between real-time quantitative PCR (Q-PCR) and the DNA hybridization (slot-blot) technique for quantitation of Y chromosome DNA. Q-PCR has a significantly greater linear quantitation range and is up to 40-fold more sensitive at low concentrations of male DNA, detecting as little as 1 ng of male DNA in each female tibialis anterior (TA) muscle. At high male DNA concentrations, accurate quantitation by Q-PCR is 2.5 times higher than the maximum possible with slot-blot. In conclusion, Q-PCR has a higher dynamic range and is more efficient than slot-blot analysis for the detection of donor cell engraftment in a transsexual transplantation model.

Key words: Myoblast transplantation; Real-time quantitative PCR; DNA hybridization; Slot-blot; Male donor cell quantitation

Address correspondence to Dr. Manfred W. Beilharz, Discipline of Microbiology (M502), School of Biomedical and Chemical Sciences, University of Western Australia, QEII Medical Centre, Nedlands, Perth, W.A. Australia 6009. Tel: 61-8-9346 2217; Fax: 61-8-9346 2912; E-mail: beilharz@cyllene.uwa.edu.au

Cell Transplantation, Vol. 13, pp. 823-831, 2004
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Maintenance of Functional Human Cancellous Bone and Human Hematopoiesis in NOD/SCID Mice

Frédérique Hubin,1 Chantal Humblet,1 Zakia Belaid,1 Roland Greimers,2 Jacques Boniver,2 Albert Thiry,2 and Marie-Paule Defresne1

1Department of Cytology and Histology and 2Department of Pathological Anatomy, University of Liège, Liège, 4000, Belgium

Attempts were made to establish models to study interactions between marrow stromal cells and hematopoietic cells in vivo. The approach was to create a NOD-SCID-hu murine model of long-term human hematopoiesis by implantation of a human adult bone fragment. Nine to 12 weeks posttransplantation, human CD45+ cells were detected in the blood and the spleen of some mice. The histology of the human transplant showed that human bone fragment was viable at 9 weeks. Moreover, vessels of human origin, as assessed by immunohistochemical detection of human b2-microglobulin, were observed in the mouse tissue surrounding the transplanted human fragment.

Key words: Hematopoiesis; Human bone fragment; NOD/SCID mice; In vivo model

Address correspondence to Frédérique Hubin, Department of Cytology and Histology, University of Liège, BAT.B23, avenue de l'Hopital, 3, 4000 Liège 1, Belgium. Tel: 003243662403; Fax: 003243662919; E-mail: F.Hubin@ulg.ac.be

Cell Transplantation, Vol. 13, pp. 833-838, 2004
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Lack of Cytomegalovirus Transmission After Pancreatic Islet Transplantation

Neal R. Barshes,1 Timothy C. Lee,1 F. Charles Brunicardi,1 Amy Mote,1 A. Paige Schock,1 Rodolfo Alejandro,2 Camillo Ricordi,2 and John A. Goss1

1Baylor College of Medicine, Michael E. DeBakey Department of Surgery, Houston, TX
2Diabetes Research Institute, University of Miami, Miami, FL

In spite of antiviral prophylaxis, the transmission rate of cytomegalovirus (CMV) after solid organ transplantation remains high. In contrast, CMV transmission has never been reported following pancreatic islet transplantation (PIT). Eleven (seven CMV seronegative, four CMV seropositive) recipients underwent a total of 26 PITs. Following PIT recipients were monitored clinically and tested monthly for CMV antigenemia. Valganciclovir was given to all patients for 100 days after each PIT. Follow-up ranged from 6 to 24 months (median 14.5 months). Pancreatic islet grafts were procured from 18 CMV seropositive and 8 seronegative donors (69% and 31% of donors, respectively). In total there were 6 R+D+, 3 R+D-, 12 R-D+, and 5 R-D- PITs. No patient developed CMV antigenemia or symptoms consistent with CMV infection at any time following PIT. Routine posttransplant testing of PIT recipients demonstrated that neither CMV transmission nor CMV infection occurred after PIT.

Key words: Pancreatic islet transplantation (PIT); Cytomegalovirus (CMV); b-Cell replacement; Antiviral prophylaxis

Address correspondence to John A. Goss, M.D., Michael E. DeBakey Department of Surgery, Baylor College of Medicine, 6550 Fannin, Suite 1628, Houston, TX 77030. Tel: (713) 798-8355; E-mail: jgoss@bcm.tmc.edu

Cell Transplantation, Vol. 13, pp. 839-844, 2004
0963-6897/04 $20.00 + 00
Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Dose-Dependent Neurochemical and Functional Protection Afforded by Encapsulated CNTF-Producing Cells*

Dwaine F. Emerich

LCT BioPharma, Inc., 241 Anan Wade Road, Glocester, RI 02857

Intracerebral delivery of hCNTF has shown considerable neuroprotective potential in animal models of Huntington's disease (HD). The present study describes the relationship between a range of hCNTF doses and the resulting behavioral and neurochemical (striatal ChAT and GAD activity) protection in a rodent model of HD. Encapsulated BHK delivering a range of hCNTF doses were implanted into the lateral ventricle ipsilateral to an intrastriatal quinolinic acid (QA) injection. Results demonstrated a dose-dependent effect of hCNTF with complete, partial, and no observable neuroprotection occurring with preimplant doses of hCNTF of 30.8, 8.6, and 0.8-2.1 ng hCNTF/24 h, respectively. These data continue to support the use of cellular delivery of hCNTF for HD and will facilitate the optimization of this approach in the clinical situation.

Key words: CNTF; Gene therapy; Huntington's disease; Encapsulation; Xenotransplantion; Striatum; Neurodegeneration

Address correspondence to Dwaine F. Emerich, Ph.D., VP Research, LCT BioPharma, Inc., 241 Anan Wade Road, Glocester, RI 02857. Fax: (401) 823-0466; E-mail: ed3fjm@aol.com

*These data were collected while the author was an employee at CytoTherapeutics, Inc., Providence, RI.