ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 14, NUMBER 1, 2005

Cell Transplantation, Vol. 14, pp. 1-9, 2005
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Transplantation of Fetal Kidney Cells: Neuroprotection and Neuroregeneration

Yung-Hsiao Chiang,1 Cesario V. Borlongan,2 Feng C. Zhou,3 Barry J. Hoffer,4 and Yun Wang4

1Tri-Service General Hospital, National Defense Medical Center, Taiwan
2Department of Neurology, Medical College of Georgia and Augusta VA Medical Center, Augusta, GA
3Department of Anatomy and Cell Biology, and Stark Neuroscience Research Institute, Indiana University School of Medicine, Indianapolis, IN
4National Institute on Drug Abuse, Intramural Research Program, NIH, Baltimore, MD

Various trophic factors in the transforming growth factor-b (TGF-b) superfamily have been reported to have neuroprotective and neuroregenerative effects. Intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) or bone morphogenetic proteins (BMPs), both members of the TGF-b family, reduce ischemia- or 6-hydroxydopamine (6-OHDA)-induced injury in adult rat brain. Because BMPs and GDNF are highly expressed in fetal kidney cells, transplantation of fetal kidney tissue could serve as a cellular reservoir for such molecules and protect against neuronal injury induced by ischemia, neurotoxins, or reactive oxygen species. In this review, we discuss preclinical evidence for the efficacy of fetal kidney cell transplantation in neuroprotection and regeneration models.

Key words: Fetal kidney; Parkinson's disease; Stroke

Address correspondence to Yun Wang, M.D., Ph.D., National Institute on Drug Abuse, NIH, 5500 Nathan Shock Drive, Baltimore, MD, 21224. Tel: (410)
550-1753; Fax: (410) 550-1425; E-mail: ywang@intra.nida.nih.gov

Cell Transplantation, Vol. 14, pp. 11-19, 2005
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Feasibility and Safety of Autologous Myoblast Transplantation in Patients With Ischemic Cardiomyopathy

Nabil Dib,1 Patrick McCarthy,2 Ann Campbell,1 Michael Yeager,2 Francis D. Pagani,3 Susan Wright,3 W. Robb MacLellan,4 Gregg Fonarow,4 Howard J. Eisen,5 Robert E. Michler,6 Philip Binkley,6 Diane Buchele,6 Ronald Korn,7 Marwan Ghazoul,1 Jonathan Dinsmore,8 Shaun R. Opie,1 and Edward Diethrich1

1Arizona Heart Institute, Phoenix, AZ
2Cleveland Clinic Foundation, Cleveland, OH
3University of Michigan, Ann Arbor, MI
4University of California at Los Angeles, Los Angeles, CA
5Temple University, Philadelphia, PA
6Ohio State University, Columbus, OH
7Scottsdale Medical Imaging Ltd., Scottsdale, AZ
8Diacrin, Inc., Charlestown, MA

Successful autologous skeletal myoblast transplantation into infarcted myocardium in a variety of animal models has demonstrated improvement in cardiac function. We evaluated the safety and feasibility of transplanting autologous myoblasts into infarcted myocardium of patients undergoing concurrent coronary artery bypass grafting (CABG) or left ventricular assist device implantation (LVAD). In addition, we sought to gain preliminary information on graft survival and any potential improvement of cardiac function. Eighteen patients with a history of ischemic cardiomyopathy participated in a phase I, nonrandomized, multicenter pilot study of autologous skeletal myoblast transplantation concurrent with CABG or LVAD implantation. Twelve patients with a history of previous myocardial infarction (MI) and a left ventricular ejection of less than 30% were enrolled in the CABG arm. In a second arm, six patients underwent LVAD implantation as a bridge to heart transplantation and were required to donate their heart for testing at the time of heart transplant. Myoblasts were successfully transplanted in all patients without any acute injection-related complications or significant long-term unexpected adverse events. Follow-up PET scans showed new areas of viability within the infarct scar in CABG patients. Echocardiography measured an average improvement in left ventricular ejection fraction (LVEF) from 25% to 34%. Histological evaluation in four out of five patients who underwent heart transplantation documented survival and engraftment of the skeletal myoblasts within the infarcted myocardium. These interim results demonstrate survival, feasibility, and safety of autologous myoblast transplantation and suggest that this modality may offer a potential therapeutic treatment for end-stage heart disease.

Key words: Myocardial infarction; Myoblast; Cell transplantation; Autologous

Address correspondence to Nabil Dib, M.D., Arizona Heart Institute, 2632 N. 20th Street, Phoenix, AZ 85006. Tel: (602) 266-2200; Fax: (602) 285-0867; E-mail: Ndib@azheart.com

Cell Transplantation, Vol. 14, pp. 21-29, 2005
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Regulation of Trophic Factor Expression by Innervating Target Regions in Intraocular Double Transplants

L. Willis, E. M. Quintero, M. Nelson, and A.-Ch. Granholm

Department of Physiology and Neuroscience and the Center on Aging, Medical University of South Carolina, Charleston, SC 29425

Trophic factors have been found to play a significant role both in long-term survival processes and in more rapid and dynamic processes in the brain and spinal cord. However, little is known regarding the regulation of expression of growth factors, and how these proteins interact on a cell-to-cell basis. We have studied protein levels of one growth factor known to affect the noradrenergic innervation of the hippocampal formation, namely brain-derived neurotrophic factor (BDNF). The purpose of the present study was to determine if appropriate innervation or contact between the LC noradrenergic neurons and their target, the hippocampus, affects expression of this growth factor in either brain region. Fetal brain stem tissue, containing the LC, and hippocampal formation were dissected from embryonic day 17 rat fetuses and transplanted together or alone into the anterior chamber of the eye of adult Fisher 344 rats. The tissue was grown together for 6 weeks, after which the animals were sacrificed and ELISAs for BDNF were undertaken. Transplantation to the anterior chamber of the eye increased the expression of BDNF in the hippocampal but not the brain stem tissue, compared with levels observed in fetal and adult rats in vivo. In addition, double grafting with hippocampal tissue more than tripled BDNF levels in brain stem grafts and doubled BDNF levels in the hippocampal portion of double grafts compared with hippocampal single grafts. Triple grafts containing basal forebrain, hippocampus, and brain stem LC tissue increased brain stem and hippocampal BDNF levels even further. Colchicine treatment of LC-hippocampal double grafts gave rise to a significant decrease in hippocampal BDNF levels to levels seen in single hippocampal grafts, while only a partial reduction of BDNF levels was seen in the brain stem portion of the same double grafts treated with colchicine. The findings suggest that an appropriate hippocampal innervation or contact with its target tissues is essential for regulation of BDNF expression in the brain stem, and that retrograde transport of BDNF can occur between double grafted fetal tissues in oculo.

Key words: Transplantation; Neurotrophins; Locus coeruleus; Hippocampal formation; BDNF

Address correspondence to Ann-Charlotte Granholm, Director, Center on Aging, Department of Physiology and Neuroscience, Medical University of South Carolina, 26 Bee Street, Charleston, SC 29425. Tel: (843) 297-0652; E-mail: granholm@musc.edu

Cell Transplantation, Vol. 14, pp. 31-40, 2005
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Liver Repopulation After Hepatocellular Transplantation: Integration and Interaction of Transplanted Hepatocytes in the Host

Sarah Koenig, Claudia Stoesser, Petra Krause, Heinz Becker, and Peter M. Markus

Department of General Surgery, Georg-August-University Goettingen, 37099 Germany

The mechanisms of donor hepatocyte integration into recipient liver are not fully understood. We investigated mechanisms of both the integration and interaction of transplanted hepatocytes with host liver cells as well as the repopulation of the host organ following intraportal transplantation. Mature hepatocytes were injected into the portal vein of dipeptidylpeptidase IV (DPPIV)-deficient rats pretreated with retrorsine and subjected to 30% partial hepatectomy to ensure selective donor growth. The degree of integration and proliferation was studied by colocalizing transplanted cells (DPPIV positive) with connexin 32, MMP-2, and OX-43 (multilayer immunofluorescence imaging). FACS analysis was established to assess the extent of repopulation quantitatively. Transplanted hepatocytes reached the distal portal spaces and sinusoids within 1 h after injection. A small proportion of cells succeeded in traversing the endothelial barrier through mechanical disruption in both locations. Transplanted hepatocytes lost their membrane-bound gap junctions (connexin 32) during this process. Successful integration of the donor cells required up to 5 days, heralded by gap junction reconstitution and the specific basolateral membrane expression of DPPIV. MMP-2 degraded the extracellular matrix in close proximity to donor cells, providing space for cell division. FACS analysis revealed that more than 37% of the liver was repopulated by cells derived from donors at 2 months after transplantation. Our data demonstrate a high degree of donor cell repopulation of the host organ and provide valuable insight into the specific mechanisms of donor cell integration. Connexin 32 expression in transplanted hepatocytes may serve as an indicator of their effective incorporation and communication within the recipient liver. FACS analysis reveals an accurate method to determine quantitatively the extent of liver repopulation.

Key words: Hepatocyte transplantation; Repopulation; Intercellular contacts; Cell adhesion molecules; Matrix metalloproteinases

Address correspondence to Dr. med. Sarah Koenig, Department of General Surgery, University Hospital, Robert-Koch-Strasse 40, 37099 Goettingen, Germany. Tel: +49-551-398977; Fax: +49-551-396109; E-mail: sarah.koenig@medizin.uni-goettingen.de

Cell Transplantation, Vol. 14, pp. 41-47, 2005
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Monocrotaline, an Alternative to Retrorsine-Based Hepatocyte Transplantation in Rodents

Rafal P. Witek,1 Samantha H. Fisher,1 and Bryon E. Petersen1,2

1Department of Pathology, Immunology and Laboratory Medicine, University of Florida, College of Medicine, Gainesville, FL
2Program for Stem Cell Biology, University of Florida Shands Cancer Center, Gainesville, FL

Retrorsine has been used extensively to inhibit proliferation of resident hepatocytes in various transplantation models. Here we report a successful alternative to currently unavailable retrorsine that can be used in cellular transplantation models. Based on structural and molecular similarities, we investigate the use of monocrotaline (MCT) in cell transplantation studies in rodents. In this study, MCT was given to rats intraperitoneally in two injections 2 weeks apart. Two weeks after the final injection, a partial hepatectomy followed by splenic hepatocyte transplantation was performed. The results indicate that MCT, at two doses of 30 mg/kg, highly enhances liver repopulation by donor hepatocytes following partial hepatectomy and produces 15.3 ± 4.9% liver repopulation within the first 6 weeks following transplantation. Additionally, we tested the effectiveness of MCT in a murine model. Using two injections of 50 mg/kg each, given 2 weeks apart, hepatocyte proliferation in the native liver was inhibited and subsequent oval cell transplants engrafted at 18 ± 21.3% after 16 weeks posttransplantation. In conclusion, MCT can be used as an effective selective pressure for donor hepatocytes in cell transplantation to the liver in rodents.

Key words: Monocrotaline; Hepatocyte proliferation; Retrorsine alternative

Address correspondence to Bryon E. Petersen, Ph.D., Department of Pathology, Immunology and Laboratory Medicine, Room M641 MSB, University of Florida, College of Medicine, PO Box 100275, Gainesville, FL 32610-0275. Tel: (352) 392-6261; Fax: (352) 392-6249; E-mail: Petersen@pathology.ufl.edu

Cell Transplantation, Vol. 14, pp. 49-57, 2005
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hVEGF165 Increases Survival of Transplanted Hepatocytes Within Portal Radicles: Suggested Mechanism for Early Cell Engraftment

Hagit Shani-Peretz,1* Vladislav Tsiperson,1* Gideon Shoshani,2 Ella Veitzman,1 Gera Neufeld,3 and Yaacov Baruch1

1Liver Unit and 2Department of Pediatric Surgery, Rambam Medical Center, and The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096, Haifa, Israel
3Vascular and Tumor Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096, Haifa, Israel

VEGF is a potent angiogenic factor that promotes hepatocyte growth, increases permeability of blood vessels, and induces vasodilatation, and may accelerate engraftment and function of transplanted hepatocytes. The aim was to study the effect of VEGF on early hepatocyte engraftment. Thirty-two Lewis syngeneic female rats underwent 70% partial hepatectomy. Eighteen received 240 ng VEGF165 and 14 received saline for control. Thereafter, intrasplenic transplantation of 107 male hepatocytes was done. Semiquantitative analysis of PCR product of the SRY region of the Y-chromosome was performed. Paraffin-embedded sections were stained for H&E and for PCNA immunostaining. By PCR, male hepatocytes were identified in 8 livers out of 14 VEGF-treated rats at 24-48 h, compared with only 1 liver out of 8 controls. Transplanted cells were seen within portal vessels radicles in 7 out of 14 VEGF-treated rats for as long as 48 h posttransplantation, compared with only one control liver at 24 h. There was no histological sign of cell injury to transplanted or adjacent cells. Two weeks after transplantation male transplanted cells were identified in two out of four rats treated with hVEGF and in one out of six rats treated with saline. No transplanted cells were detected within portal tracts 14 days after transplantation. hVEGF165 enhances the presence of transplanted hepatocytes within portal vessels after transplantation. We suggest an additional mechanism for cell engraftment, whereby transplanted hepatocytes first stick to each other in the portal radicles. Later they become included in the liver parenchyma as groups of organized cells in a process stimulated by VEGF.

Key words: VEGF; Hepatocyte transplantation; Liver regeneration; Partial hepatectomy

Address correspondence to Yaacov Baruch, Liver Unit, Rambam Medical Center, P.O.B. 9602, 31096 Haifa, Israel. Tel: +972-4-854-3049; Fax: +972-4-854-2477; E-mail: ybaruch@rambam.health.gov.il

*These authors contributed equally to this work.

Cell Transplantation, Vol. 14, pp. 59-65, 2005
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Short-Term Culture With the Caspase Inhibitor z-VAD.fmk Reduces Beta Cell Apoptosis in Transplanted Islets and Improves the Metabolic Outcome of the Graft

Marta Montolio, Noèlia Téllez, Montserrat Biarnés, Joan Soler, and Eduard Montanya

Laboratory of Diabetes and Experimental Endocrinology, Endocrine Unit, IDIBELL-Hospital Universitari de Bellvitge, University of Barcelona, Barcelona, Spain

In the initial days after transplantation islets are particularly vulnerable and show increased apoptosis and necrosis. We have studied the effects of caspase inhibition on this early beta cell death in syngeneically transplanted islets. Streptozotocin-diabetic C57BL/6 mice were transplanted with 150 syngeneic islets, an insufficient mass to restore normoglycemia, preincubated with or without the pan-caspase inhibitor z-VAD.fmk 2 h before transplantation. Beta cell apoptosis was increased in control islets on day 3 after transplantation (0.28 ± 0.02%) compared with freshly isolated islets (0.08 ± 0.02%, p < 0.001), and was partially reduced in transplanted islets preincubated with z-VAD.fmk 200 mM (0.14 ± 0.02%, p = 0.003) or with z-VAD.fmk 500 mM (0.17 ± 0.01%, p = 0.012), but not with a lower z-VAD.fmk (100 mM) concentration. Diabetic mice transplanted with islets preincubated with z-VAD.fmk 500 mM showed an improved metabolic evolution compared with control and z-VAD.fmk 200 mM groups. The z-VAD.fmk 500 mM group showed an overall lower blood glucose after transplantation (p = 0.02), and at the end of the study blood glucose values were reduced compared with transplantation day (15.7 ± 3.6 vs. 32.5 ± 0.5 mmol/L, p = 0.001). In contrast, blood glucose was not significantly changed in control and z-VAD.fmk 200 mM groups. Four weeks after transplantation beta cell mass was higher in z-VAD.fmk 500 mM group (0.15 ± 0.02 mg) than in the control group (0.10 ± 0.02 mg) (p = 0.043). In summary, the treatment of freshly isolated islets with the caspase inhibitor z-VAD.fmk reduced the subsequent apoptosis of the islets once they were transplanted and improved the outcome of the graft.

Key words: Islet transplantation; Apoptosis; Caspase; z-VAD.fmk; Beta cell mass

Address correspondence to Eduard Montanya, Endocrine Unit (13-2), IDIBELL-Hospital Universitari Bellvitge, Feixa Llarga, s/n, 08907 L'Hospitalet de Llobregat, Barcelona. Spain. E-mail: montanya@ub.edu

Cell Transplantation, Vol. 14, pp. 67-76, 2005
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Central Necrosis in Isolated Hypoxic Human Pancreatic Islets: Evidence for Postisolation Ischemia

Mauro Giuliani,1* Wolfgang Moritz,1* Elvira Bodmer,1 Daniel Dindo,1 Patrick Kugelmeier,1,2 Roger Lehmann,2 Max Gassmann,3 Peter Groscurth,4 and Markus Weber1

1Department of Visceral and Transplant Surgery, 2Department of Endocrinology and Diabetes, University Hospital Zurich, 8091 Zurich, Switzerland
3Institute of Veterinary Physiology and 4Institute of Anatomy, University of Zurich, 8091 Zurich, Switzerland

A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.

Key words: Islet transplantation; Ischemia; Hypoxia; Apoptosis

Address correspondence to Markus Weber, M.D., Division of Visceral and Transplantation Surgery, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. Tel: +41 1 255 3868; Fax: +41 1 255 4449; E-mail: markus.weber@ usz.ch

*These authors contributed equally to this work.