ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 15, NUMBER 7, 2006

Cell Transplantation, Vol. 15, pp. 563-577, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bone Marrow Stromal Cells for Repair of the Spinal Cord: Towards Clinical Application

Rishi D. S. Nandoe,1,3,4 Andres Hurtado,1,4 Allan D. O. Levi,1,2 Andre Grotenhuis,3 and Martin Oudega1,2,4

1The Miami Project to Cure Paralysis, University of Miami, School of Medicine, Miami, FL 33136, USA
2Department of Neurological Surgery, University of Miami, School of Medicine, Miami, FL 33136, USA
3 Department of Neurological Surgery, Radboud University Medical Center Nijmegen, Nijmegen, 6525 GC, The Netherlands
4International Center for Spinal Cord Injury, Kennedy Krieger Institute and the Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Stem cells have been recognized and intensively studied for their potential use in restorative approaches for degenerative diseases and traumatic injuries. In the central nervous system (CNS), stem cell-based strategies have been proposed to replace lost neurons in degenerative diseases such as Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (Lou Gehrig's disease), or to replace lost oligodendrocytes in demyelinating diseases such as multiple sclerosis. Stem cells have also been implicated in repair of the adult spinal cord. An impact to the spinal cord results in immediate damage to tissue including blood vessels, causing loss of neurons, astrocytes, and oligodendrocytes. In time, more tissue nearby or away from the injury site is lost due to secondary injury. In case of relatively minor damage to the cord some return of function can be observed, but in most cases the neurological loss is permanent. This review will focus on in vitro and in vivo studies on the use of bone marrow stromal cells (BMSCs), a heterogeneous cell population that includes mesenchymal stem cells, for repair of the spinal cord in experimental injury models and their potential for human application. To optimally benefit from BMSCs for repair of the spinal cord it is imperative to develop in vitro techniques that will generate the desired cell type and/or a large enough number for in vivo transplantation approaches. We will also assess the potential and possible pitfalls for use of BMSCs in humans and ongoing clinical trials.

Key words: Bone marrow stromal cell (BMSC); Spinal cord injury; Implantation; Regeneration; Clinical trial

Address correspondence to Martin Oudega, Ph.D., The International Center for Spinal Cord Injury, Kennedy Krieger Institute and the Department of Neurology, Johns Hopkins University School of Medicine, 707 N Broadway, Baltimore, MD 21205, USA. Tel: 443-923-9248; Fax: 443-923-9215; E-mail: oudega@kennedykrieger.org

Cell Transplantation, Vol. 15, pp. 579-593, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transplanted Human Neural Precursor Cells Migrate Widely But Show no Lesion-Specific Tropism in the 6-Hydroxydopamine Rat Model of Parkinson's Disease

M. Jain,1 R. J. E. Armstrong,1 S. Elneil,2 and R. A. Barker1,3

1Cambridge University Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK
2Dpartment of Obstetrics and Gynaecology, Level 2, The Rosie Hospital, Robinson Way, Cambridge, CB2 2SW, UK
3Department of Neurology, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ, UK

Parkinson's disease (PD), while primarily associated with degeneration of nigrostriatal dopamine neurons, is now increasingly recognized to have more widespread cell loss and so the most effective cell replacement therapy should target all these neuronal losses. Neural precursor cells might be ideal in this regard as in certain circumstances they have been shown to migrate widely following transplantation into the CNS. The aim of this study was to investigate whether transplanted human expanded neural precursor cells (hENPs) could migrate to sites of established or evolving pathology in the adult brain using the 6-hydroxydopamine (6-OHDA) rat model of PD. hENPs were grafted into the striatum prior to, at the same time as, or after the animals received a 6-OHDA lesion to the medial forebrain bundle. The presence of donor cells was then assessed in a distant site of cell loss (substantia nigra) or sites where cell death would not be expected (frontal cortex and globus pallidus). Donor cells were found distant from the site of implantation but the migration of these hENPs was not significantly greater in the 6-OHDA-lesioned brain and the cells did not specifically target the site of cell loss in the substantia nigra. The temporal relationship of grafting relative to the lesion, and therefore dopaminergic cell death, did not affect the migration of hENPs nor their differentiation. We conclude that while transplanted hENPs are capable of migration away from the site of implantation, they show no specific tropism for sites of ongoing or established nigral dopaminergic cell loss in this lesion model. Therefore, the use of such cells to replace the range of neurons lost in PD is likely to require a deeper understanding of the migratory cues in the damaged adult brain and some manipulation of these cells prior to transplantation.

Key words: Neural stem cells; Progenitor cells; Neural transplantation; Migration; Substantia nigra; Cortex

Address correspondence to Roger A. Barker, Cambridge University Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge, CB2 2PY, UK. Tel: +44 1223 331160; Fax: +44 1223 331174; E-mail: rab46@cam.ac.uk

Cell Transplantation, Vol. 15, pp. 595-602, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

In Vitro Culture Duration Does Not Impact the Ability of Encapsulated Choroid Plexus Transplants to Prevent Neurological Deficits in an Excitotoxin-Lesioned Rat Model of Huntington's Disease

Dwaine F. Emerich and Christopher G. Thanos

LCT BioPharma, Inc., Providence, RI, USA

Delivery of neurotrophic molecules to the CNS is a potential treatment strategy for preventing the neuronal loss accompanying many neurological disorders. Choroid plexus (CP) epithelial cells secrete a cocktail of neurotrophic factors, and encapsulated CP transplants are neuroprotective in animal models of stroke and Huntington's disease (HD). Prior to clinical use, it is essential to identify and optimize parameters such as the length of time that transplant products such as encapsulated CP can be maintained. In the present study, neonatal porcine CP was encapsulated within alginate microcapsules and maintained in vitro for 1, 2, or 7 months. The encapsulated cells remained viable (>80%) at all time points and were transplanted unilaterally into the rat striatum. Seven days later, the same animals received unilateral injections of quinolinic acid (QA; 225 nmol) adjacent to the implant site. Separate groups of animals served as controls and received QA alone. After surgery, animals were periodically evaluated for weight loss and were tested for motor function 14 days post-QA. In controls, QA lesions produced a significant loss of body weight and impaired function of the contralateral forelimb. In contrast, implants of CP were potently neuroprotective as rats receiving CP transplants did not lose body weight and were not significantly impaired when tested for motor function. These benefits were independent of the length of time that the cells were held in vitro and demonstrate that the potential potency of alginate encapsulated CP cells can be retained for extremely long periods of time in vitro.

Key words: Choroid plexus; Xenotransplant; Huntington's disease; Encapsulation

Address correspondence to Dwaine F. Emerich, LCT BioPharma, 4 Richmond Square, Providence, RI 02906, USA. E-mail: ED3FJM@aol.com

Cell Transplantation, Vol. 15, pp. 603-612, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Retinal Progenitor Cell Xenografts to the Pig Retina: Immunological Reactions

Karin Warfvinge,1 Jens F. Kiilgaard,2 Henry Klassen,3* Parisa Zamiri,4 Erik Scherfig,2 Wayne Streilein,4 Jan U. Prause,3* and Michael J. Young4

1Department of Ophthalmology, Lund University Hospital, Lund, Sweden
2Eye Department, Rigshospitalet and Eye Pathology Institute, Copenhagen University, Copenhagen, Denmark
3Department of Ophthalmology, UC Irvine, Irvine, CA, USA
4Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA

We evaluated the host response to murine retinal progenitor cells (RPCs) following transplantation to the subretinal space (SRS) of the pig. RPCs from GFP mice were transplanted subretinally in 18 nonimmunosuppressed normal or laser-treated pigs. Evaluation of the SRS was performed on hematoxylin-eosin (H&E)-stained sections. Serum samples were taken from naive and RPC-grafted pigs and mouse-reactive antibody responses were assessed. At 1 week, histology showed a few perivascular lymphocytes consistent with a mild retinal vasculitis, and depigmentation of the RPE with large numbers of mononuclear inflammatory cells in the choroid near the transplantation site. Large choroidal infiltrates were evident at 2-5 weeks. Serum from naive and RPC-xenografted pigs contained significant levels of preformed IgG and IgM antibodies against murine antigens. Xenogeneic RPCs transplanted to the porcine SRS induced mononuclear infiltration in the choroid with graft rejection occurring over 2-5 weeks. Serum analysis confirmed that mice and pigs are discordant species; however, a cell-mediated acute mechanism appears to be responsible, rather than an antibody-mediated rejection.

Key words: Xenografts; Stem cell; Transplant; Retina

Address correspondence to Michael J. Young, Ph.D., Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA. Tel: 617-912-7419; Fax: 617-912-0101; E-mail: mikey@vision.eri.harvard.edu

*Current address: Singapore Eye Research Institute, Singapore.

Cell Transplantation, Vol. 15, pp. 613-620, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Resolution of Neurotoxicity and b-Cell Toxicity in an Islet Transplant Recipient Following Substitution of Tacrolimus With MMF

Tatiana Froud,1,3 David A. Baidal,1 Gaston Ponte,1 Jacqueline V. Ferreira,1,2 Camillo Ricordi,1,3 and Rodolfo Alejandro1,2

1Diabetes Research Institute, University of Miami School of Medicine, Miami, FL 33136, USA
2Department of Medicine, University of Miami School of Medicine, Miami, FL 33136, USA
3Department of Surgery, University of Miami School of Medicine, Miami, FL 33136, USA

Calcineurin inhibitors such as tacrolimus have well-recognized efficacy in organ transplantation but side effects of nephrotoxicity, neurotoxicity, and b-cell toxicity that can be particularly detrimental in islet transplantation. Neuro- and nephrotoxicity have been demonstrated in multiple islet transplant recipients despite the relatively low serum maintenance levels typically used (3-5 ng/ml). We describe a single patient in whom symptoms and signs of neurotoxicity necessitated substitution of tacrolimus with mycophenolate mofetil (MMF), which resulted in complete symptom resolution over the subsequent 9 months. Concomitantly noted were an almost immediate improvement in glycemic control and an improved response to stimulation testing, suggesting remission of tacrolimus-induced b-cell toxicity and insulin resistance. At 18 months post-"switch," 30 months posttransplant, the patient remains insulin independent with good glycemic control. The goal to remove calcineurin inhibitors from regimens of islet transplantation is a worthy one.

Key words: Islet; Transplantation; Tacrolimus; Neurotoxicity; b-Cell toxicity; Function

Address correspondence to Alejandro, Rodolfo, M.D., Diabetes Research Institute (R-134), University of Miami School of Medicine, 1450 NW 10 Avenue, Miami, FL 33136, USA. Tel: (305) 243-5374; Fax: (305) 243-1058; E-mail: ralejand@med.miami.edu

Cell Transplantation, Vol. 15, pp. 621-636, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Tetracycline-Regulated Expression of VEGF-A in Beta Cells Induces Angiogenesis: Improvement of Engraftment Following Transplantation

Zoltan Mathe,1,2* Philippe Dupraz,3* Chris Rinsch,3 Bernard Thorens,4 Domenico Bosco,2 Marie Zbinden,5 Philippe Morel,2 Thierry Berney,2 and Michael S. Pepper5,6,7

1Transplantation and Surgical Department, Semmelweis University, Budapest, H-1089, Hungary
2Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals, Geneva; CH-1211, Switzerland
3Isotis SA Isotis SA, Lausanne, CH-1005, Switzerland
4Institut of Pharmacology and Toxicology, University of Lausanne, Lausanne, CH-1005, Switzerland
5Department of Cell Physiology and Metabolism, University Medical Center, CH-1211, Geneva, Switzerland
6NetCare Molecular Medicine Institute, Unitas Hospital, Lyttleton, Pretoria, South Africa
7Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, SA-0140, South Africa

Early revascularization of pancreatic islet cells after transplantation is crucial for engraftment, and it has been suggested that vascular endothelial growth factor-A (VEGF-A) plays a significant role in this process. Although VEGF gene therapy can improve angiogenesis, uncontrolled VEGF secretion can lead to vascular tumor formation. Here we have explored the role of temporal VEGF expression, controlled by a tetracycline (TC)-regulated promoter, on revascularization and engraftment of genetically modified beta cells following transplantation. To this end, we modified the CDM3D beta cell line using a lentiviral vector to promote secretion of VEGF-A either in a TC-regulated (TET cells) or a constitutive (PGK cells) manner. VEGF secretion, angiogenesis, cell proliferation, and stimulated insulin secretion were assessed in vitro. VEGF secretion was increased in TET and PGK cells, and VEGF delivery resulted in angiogenesis, whereas addition of TC inhibited these processes. Insulin secretion by the three cell types was similar. We used a syngeneic mouse model of transplantation to assess the effects of this controlled VEGF expression in vivo. Time to normoglycemia, intraperitoneal glucose tolerance test, graft vascular density, and cellular mass were evaluated. Increased expression of VEGF resulted in significantly better revascularization and engraftment after transplantation when compared to control cells. In vivo, there was a significant increase in vascular density in grafted TET and PGK cells versus control cells. Moreover, the time for diabetic mice to return to normoglycemia and the stimulated plasma glucose clearance were also significantly accelerated in mice transplanted with TET and PGK cells when compared to control cells. VEGF was only needed during the first 2-3 weeks after transplantation; when removed, normoglycemia and graft vascularization were maintained. TC-treated mice grafted with TC-treated cells failed to restore normoglycemia. This approach allowed us to switch off VEGF secretion when the desired effects had been achieved. TC-regulated temporal expression of VEGF using a gene therapy approach presents a novel way to improve early revascularization and engraftment after islet cell transplantation.

Key words: Diabetes; VEGF; Regulated gene expression; Beta cell; Islet transplantation

Address correspondence to Prof. Michael S. Pepper, NetCare Molecular Medicine Institute, Unitas Hospital, Clifton Avenue, 0140 Lyttleton, Pretoria, South Africa. Tel: +27(0)12-677-8504; Fax: +27(0)12-677-8505; E-mail: mpepper@doctors.netcare.co.za

*These authors contributed equally to this work.

Cell Transplantation, Vol. 15, pp. 637-645, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Transplantation of Murine Bone Marrow Stromal Cells Under the Kidney Capsule to Secrete Coagulation Factor VIII

Taekeun Oh,1* Alexandra Peister,2 Kazuo Ohashi,3 and Frank Park1

1Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA, USA
2Department of Biochemistry, Tulane University, New Orleans, LA, USA
3First Department of Surgery, Nara Medical University, Nara, Japan

Ectopic cell transplantation has been studied as an alternative to whole organ transplantation or as a method to produce secretable proteins for genetic disorders. In this study, bone marrow stromal cells isolated from C57Bl/6 mice were genetically modified to express either lacZ- or B-domain-deleted human factor VIII. In vitro modification of the isolated bone marrow stromal cells was initially performed by transducing increased doses of VSV-G pseudotyped lentiviral vectors expressing lacZ. At a MOI of 25, all of the bone marrow stromal cells were X-gal positive, which maintained their ability to expand and differentiate prior to transplantation into mice. Extremely poor engraftment was observed in the liver, but transplantation of the bone marrow stromal cells expressing lacZ under the kidney capsule resulted in long-term viable X-gal-positive cells for at least 8 weeks (length of study). In vitro expression of human factor VIII was detected in a dose-dependent manner following bone marrow stromal cell with a factor VIII-expressing lentiviral vector. Transplantation of the factor VIII-expressing bone marrow stromal cells under the kidney capsule led to long-term therapeutic expression in the mouse plasma (1-3 ng/ml; n = 4-5 mice/group) for 8 weeks. This study demonstrated that ectopic transplantation of bone marrow stromal cells under the kidney capsule can be effective as a method to express secretable proteins in vivo.

Key words: Lentiviral vectors; Bone marrow stromal cells; Human factor VIII; Ectopic transplantation; Kidney capsule

Address correspondence to Frank Park, Ph.D., Medical College of Wisconsin, Department of Medicine, 8701 Watertown Plank Road, H4100, Milwaukee, WI 53226, USA. Tel: 414-456-8103; Fax: 414-456-6312; E-mail: fpark@mcw.edu

*Current address: Department of Internal Medicine, Chungbuk National University Hospital, Cheongju, South Korea.

Cell Transplantation, Vol. 15, pp. 647-658, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Human Umbilical Cord Blood Progenitor Cells Are Attracted to Infarcted Myocardium and Significantly Reduce Myocardial Infarction Size

Robert J. Henning,1 Jose D. Burgos,1 Leo Ondrovic,2 Paul Sanberg,3 John Balis,4 and Michael B. Morgan4

1Department of Medicine, University of South Florida College of Medicine and the James A. Haley VA Hospital, Tampa, FL, USA
2Department of Surgery, University of South Florida College of Medicine, Tampa, FL, USA
3Department of Neuroscience, University of South Florida College of Medicine, Tampa, FL, USA
4Department of Pathology, University of South Florida College of Medicine and the James A. Haley VA Hospital, Tampa, FL, USA

We are investigating the effects of human umbilical cord blood mononuclear progenitor cells (HUCBC) for the treatment of acute myocardial infarction because human cord blood is a readily available and an abundant source of primitive cells that may be beneficial in myocardial repair. However, there is currently no scientific consensus on precisely when to inject stem/progenitor cells for the optimal treatment of acute myocardial infarction. We used an in vitro assay to determine the attraction of infarcted rat myocardium at 1, 2, 2.5, 3, 6, 12, 24, 48, and 96 h after left anterior descending coronary artery (LAD) occlusion from 45 rats for HUCBC in order to determine the optimal time to transplant HUCBC after myocardial infarction. Our assay is based on the migration of fluorescent DAPI-labeled HUCBC from wells in an upper chamber of a modified Boyden apparatus through a semiporous polycarbonate membrane into wells in a lower chamber that contain either normal or infarcted myocardium. DAPI-labeled HUCBC (100,000) were placed in each of the separate wells above the membrane that corresponded to normal or infarct homogenate in the lower wells. The greatest HUCBC migration to infarcted myocardium occurred at 2 h and 24 h after LAD occlusion in comparison with normal controls. A total of 76,331 ± 3384 HUCBC migrated to infarcted myocardium at 2 h and 69,911 ± 2732 at 24 h after LAD occlusion (both p < 0.001) and significantly exceeded HUCBC migration to normal heart homogenate. The HUCBC migration remained greatest at 2 and 24 h after LAD occlusion when the number of migrated cells was adjusted for the size of each myocardial infarction. Injection of 106 HUCBC in saline into infarcted myocardium of non immunosuppressed rats within 2 h (n = 10) or at 24 h (n = 5) after LAD occlusion resulted in infarction sizes 1 month later of 6.4 ± 0.01% and 8.4 ± 0.02% of the total left ventricular muscle area, respectively, in comparison with infarction sizes of 24.5 ± 0.02% (n = 10) in infarcted rat hearts treated with only saline (p < 0.005). Acute myocardial infarction in rats treated with only saline increased the myocardial concentration of tumor necrosis factor-a (TNF-a) from 6.9 ± 0.8% to 51.3 ± 4.6%, monocyte/macrophage chemoattractant protein (MCP-1) from 10.5 ± 1.1% to 39.2 ± 2.0%, monocyte inflammatory protein (MIP) from 10.6 ± 1.6% to 23.1 ± 1.5%, and interferon-g (INF-g) from 8.9 ± 0.3% to 25.0 ± 1.7% between 2 and 12 h after coronary occlusion in comparison with known controls (all p < 0.001). In contrast, the myocardial concentrations of these cytokines in rat hearts treated with HUCBC did not significantly change from the controls at 2, 6, 12, and 24 h after coronary occlusion. The present investigations suggest that infarcted myocardium significantly attracts HUCBC, that HUCBC can substantially reduce myocardial infarction size, and that HUCBC can limit the expression of TNF-a, MCP-1, MIP, and INF-g in acutely infarcted myocardium.

Key words: Stem cells; Cell therapy; Myocardial infarction; Coronary artery disease

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, Tampa, FL 33612, USA. E-mail: rhenning@hsc.usf.edu

Cell Transplantation, Vol. 15, pp. 659-663, 2006
0963-6897/06 $90.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Use of Repeating Dispensers to Increase the Efficiency of the Intramuscular Myogenic Cell Injection Procedure

Marlyne Goulet, Daniel Skuk, and Jacques P. Tremblay

Unité de recherche en Génétique humaine, Centre Hospitalier de l'Université Laval, Québec, QC, Canada G1V 4G2

Intramuscular myoblast transplantation in humans and nonhuman primates requires precise repetitive cell injections very close to each other. Performed with syringes operated manually throughout large regions, this procedure takes a lot of time, becoming tiring and thus imprecise. We tested two repetitive dispensers with Hamilton syringes as cell injection devices to facilitate this procedure. Monkeys received intramuscular allotransplantations of &beta;-galactosidase-labeled myoblasts, using either a monosyringe or a multisyringe repeating dispenser. The monosyringe repeating dispenser allowed performing cell injections faster and easier than with a manually operated syringe. The multisyringe dispenser accelerated the procedure still more, but it was not ergonomic. Biopsies of the myoblast-injected sites 1 month later showed abundant &beta;-galactosidase-positive myofibers, with the same density and morphological pattern observed following myoblast transplantation with a syringe operated manually. We recommend the monosyringe repeating dispenser for myoblast transplantation in skeletal muscles and maybe in the heart.

Key words: Cell delivery; Muscle precursor cell; Repeating dispensers; Skeletal muscle

Address correspondence to Daniel Skuk, M.D., Unité de recherche en Génétique humaine (RC 9300), Centre Hospitalier de l'Université Laval, 2705 boulevard Laurier, Québec, QC, Canada G1V 4G2. Tel: (418) 654-2186; Fax: (418) 654-2207; E-mail: Daniel.Skuk@anm.ulaval.ca