ognizant Communication Corporation

The Regenerative Medicine Journal

VOLUME 15, NUMBER 1, 2006

Cell Transplantation, Vol. 15, pp. 1-12, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Genetic Modification of Hepatocytes Towards Hepatocyte Transplantation and Liver Tissue Engineering

Hiroyuki Kuge,1 Kazuo Ohashi,1 Takashi Yokoyama,1 Hiromichi Kanehiro,1 Michiyoshi Hisanaga,1 Fumikazu Koyama,1 Ginny L. Bumgardner,2 Ken-Ichiro Kosai,3 and Yoshiyuki Nakajima1

1Department of Surgery, Nara Medical University, Nara, Japan
2Department of Surgery, Ohio State University Medical Center, Columbus, OH, USA
3Division of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute for Brain Diseases, Kurume University, Kurume, Japan

Cell-based therapies, including liver tissue engineering following hepatocyte transplantation, have therapeutic potential for several types of liver diseases. Modifications in the methodology to manipulate the donor hepatocytes in a more simple and timely manner prior to transplantation would enhance the therapeutic efficacy of this procedure. Conventional approach for vector-mediated gene transduction to the isolated hepatocytes has been performed under primary culture conditions that routinely require several days to complete. In our study, we have established a clinically feasible approach that requires only 1 h of infection time with an adenoviral vector system that results in an extremely efficient transduction efficiency (>80%). To optimize transduction efficiency and sustain normal cellular function, we determined that the isolated hepatocytes should be maintained in UW solution as a suspension medium and infected with adenoviral vectors (Ad) for no more than 1 h at a MOI of 1. To establish if the isolated hepatocytes could be used as a source for cell-based therapies, we transplanted the Ad-transduced hepatocytes into the liver or under the kidney capsule. When the cells were transplanted into the liver, Ad-transduced hepatocytes cultured in suspension conditions were found to have a significantly higher survival rate (p < 0.01) than Ad-transduced hepatocytes cultured under standard conditions. We also confirmed that these Ad-transduced hepatocytes have ability to survive long term and were able to engineer a biologically active hepatic tissue under the kidney capsule. Finally, we obtained high level of transduction into canine, porcine, and human isolated hepatocytes in a suspension solution mixed with Ad. In conclusion, the present studies demonstrate that isolated hepatocytes could be genetically modified using Ad when kept in a suspension solution. For this reason, this cell-modified technique could be used for the treatment of liver-targeted diseases and/or disorders.

Key words: Hepatocyte transplantation; Gene modification; Ex vivo gene therapy; Adenoviral vector; Liver tissue engineering

Address correspondence to Kazuo Ohashi, M.D., Ph.D., Department of Surgery, Nara Medical University 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. Tel: +81(744) 22-3051; Fax: +81(744) 24-6866; E-mail: kohashi@naramed-u.ac.jp

Cell Transplantation, Vol. 15, pp. 13-22, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Hepatocyte Transplantation in the Long Evans Cinnamon Rat Model of Wilson's Disease

Seon Mee Park,1 Kim Vo,2 Michel Lallier,2 Alexis-Simon Cloutier,2 Pierre Brochu,3 Fernando Alvarez,1 and Steven R. Martin1

1Department of Pediatrics, Hôpital Sainte-Justine, Université de Montréal, Montréal, Qc, Canada
2Department of Surgery, Hôpital Sainte-Justine, Université de Montréal, Montréal, Qc, Canada
3Department of Pathology, Hôpital Sainte-Justine, Université de Montréal, Montréal, Qc, Canada

Wilson's disease (WD), caused by a mutation in the P-type copper transporting ATPase (Atp7b) gene, results in excessive accumulation of copper in the liver. Long Evans Cinnamon rats (LEC) bear a mutation in the atp7b gene and share clinical characteristics of human WD. To explore hepatocyte transplantation (HT) as therapy for metabolic liver diseases, 8-week-old LEC rats (n = 12) were transplanted by intrasplenic injection of hepatocytes from donor Long Evans (LE) rats. Immunosuppression was maintained with intraperitoneal tacrolimus. The success of HT was monitored at 24 weeks of life. Serum aminotransferases and bilirubin peaked at 14-21 weeks in both HT rats and nontransplanted controls, but at 24 weeks, survival was 97% in LEC-HT versus 63% in controls. All transplanted rats showed restored biliary copper excretion and reduced liver iron concentration associated with increased ceruloplasmin oxidase activity. Liver tissue expressed atp7b mRNA (11.9 ± 13.6%) indicative of engraftment of normal cells in 7 of 12 HT rats, associated with a reduced liver copper concentration compared to untreated LEC rats. Periportal islets of normal appearing hepatocytes, recognized by atp7b antibody, were observed in transplanted livers while lobular host cells showed persistent pleomorphic changes and inflammatory infiltrates. In conclusion, transplantation of normal hepatocytes prevented fulminant hepatitis, reduces chronic inflammation, and improved 6-month survival in LEC rats. Engraftment of transplanted cells, which express atp7b mRNA, repopulated the recipient liver with normal functional capacity.

Key words: Hepatocyte transplantation; LEC rat; Wilson's disease

Address correspondence to Dr. Michel Lallier, Department of Surgery, Hôpital Sainte-Justine, 3175, Côte Sainte-Catherine, Montréal, Québec, H3T 1C5, Canada. Tel: 514-345-4931; E-mail: michel_lallier@ssss.gouv.qc.ca

Cell Transplantation, Vol. 15, pp. 23-33, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cryopreserved Fetal Liver Cell Transplants Support the Chronic Failing Liver in Rats With CCl4-Induced Cirrhosis

Olga V. Ochenashko,1 Nataliya A. Volkova,1 Svetlana P. Mazur,1 Alexander Yu. Somov,1 Barry J. Fuller,2 and Alexander Yu. Petrenko1

1Department of Biochemistry, Institute for Problems of Cryobiology and Cryomedicine, Kharkov, 61015, Ukraine
2University Department of Surgery, Hampstead Campus, Royal Free & University College Medical School, London, UK

Hepatocyte transplantation is a promising method for supporting hepatic function in a broad spectrum of liver diseases. The aim of this work was to test the efficacy of human fetal liver cells to support the chronic failing liver in an experimental model of carbon tetrachloride (CCl4)-induced cirrhosis in rats. Liver cirrhosis was induced by intraperitoneal administration of CCl4 at a dose of 0.2 ml (50% v/v solution)/100 g body weight, twice a week for 3 months in rats. Ten days after stopping CCl4 administration (experimental day 0), rats received intrasplenic injection of cryopreserved fetal liver cells (FLC, 1 x 107 cells in 0.3 ml medium). As a cirrhotic control group, CCl4-induced cirrhotic rats were used with intrasplenic injection of an equal volume of medium alone. Animals were sacrificed on experimental day 15. Human fetal liver cell transplantation almost completely prevented the death of cirrhotic animals during the 2 weeks after treatment, while high ongoing mortality was seen in the cirrhotic control group. Cell transplantation into the spleen normalized total bilirubin and TBARSs levels and increased albumin levels in blood serum, as well as restoring mitochondrial function and liver detoxification function (assessed by cytochrome P450 contents and activity) compared with the activities seen in the cirrhosis control group. In parallel with this restoration of biochemical and functional liver indices, morphological patterns of liver recovery or regeneration after liver cell transplantation were demonstrated in day 15 samples by light microscopy. These were absent in the group that had received only medium alone.

Key words: Fetal liver cells; Human; Cryopreservation; Liver cell transplantation; CCl4-induced cirrhosis; Rat liver function

Address correspondence to Barry J. Fuller, University Department of Surgery, Hampstead Campus, Royal Free & University College Medical School, London, UK. Tel: (00 44) (0)20 7 472 6111; E-mail: b.fuller@rfc.ucl.ac.uk

Cell Transplantation, Vol. 15, pp. 35-43, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Analysis of the Effects of Cryopreservation on Rat Hepatocytes Using SELDI-TOF Mass Spectrometry

Claire Terry,1 Matthew Bailey,2 Ragai R. Mitry,1 Sharon C. Lehec,1 Anil Dhawan,1 and Robin D. Hughes1

1Institute of Liver Studies, King's College London School of Medicine, London, UK
2Department of Nutrition and Dietetics, King's College London, London, UK

Successful cryopreservation of hepatocytes is essential to the future of hepatocyte transplantation as a treatment for liver disease, and also for the improved in vitro use of hepatocytes for research. However, hepatocyte function is adversely affected by even the best cryopreservation protocols. To investigate possible mechanisms for these changes, total cell lysates were prepared from fresh and cryopreserved rat hepatocytes and the proteome profiles compared using SELDI-TOF-MS ProteinChip® technology. In addition, in vitro functional assays (viability, attachment efficiency, and lactate dehydrogenase leakage) were performed on the corresponding fresh and cryopreserved hepatocytes. Sixty-one peptides were identified as being significantly changed after cryopreservation. Thirty-seven peaks were significantly increased and 24 were significantly decreased after cryopreservation. The peak intensity of a number of these peptides was found to correlate with the in vitro function of the hepatocytes. Seven peptides correlated with in vitro function after cryopreservation and 10 peptides correlated with both fresh and cryopreserved function. The peptides significantly decreased after cryopreservation could include cytosolic enzymes or cofactors, which leaked out of the cells due to cryopreservation-induced membrane damage. The peptides significantly increased after cryopreservation could be retained products of cleavage of larger intracellular polypeptides and proteins or the result of aggregation of peptides caused by physical changes in the cell due to the cryopreservation process. Proteome profiling using SELDI-TOF-MS could be a useful tool to assess the effects of isolation and cryopreservation of hepatocytes, particularly if the findings are extended to human hepatocytes.

Key words: Cryopreservation; Proteome; Hepatocytes; Cell damage

Address correspondence to Dr. Robin Hughes, Institute of Liver Studies, King's College London School of Medicine, Bessemer Road, London SE5 9PJ, UK. Tel: +44-(0)20-7346-3137; Fax: +44-(0)20-7346-3760; E-mail: robin.hughes@kcl.ac.uk

Cell Transplantation, Vol. 15, pp. 45-53, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Sertoli Cells Induce Systemic Donor-Specific Tolerance in Xenogenic Transplantation Model

R. Shamekh,1* N. S. El-Badri,2,3,4* S. Saporta,1,2,3 C. Pascual,4 P. R. Sanberg,2,3 and D. F. Cameron1,2

1Department of Anatomy, University of South Florida, College of Medicine, Tampa, FL 33612, USA
2Department of Neurosurgery, University of South Florida, College of Medicine, Tampa, FL 33612, USA
3Center for Aging and Brain Repair, University of South Florida, College of Medicine, Tampa, FL 33612, USA
4All Children Hospital, University of South Florida, College of Medicine, Tampa, FL 33612, USA

Cell therapy is a potentially powerful tool in the treatment of many grave disorders including leukemia, immune deficiencies, autoimmune diseases, and diabetes. However, finding matched donors is challenging and recipients may suffer from the severe complications of systemic immune suppression. Sertoli cells, when cotransplanted with both allo- and xenograft tissues, promote graft acceptance in the absence of systemic immunosuppression. How Sertoli cells do this is not, as yet, clearly defined. We have examined the ability of Sertoli cells to produce systemic immune tolerance. For this purpose, Sertoli cells were injected into an otherwise normal C57/BL6 mouse host via the lateral tail vein. No other immunosuppressive protocols were applied. Six to 8 weeks posttransplantation, blood was collected for analysis of cytokine levels. Tolerance to donor cells was determined by mixed lymphocytic culture, and production of T-cell-dependent antibody was determined by an in vitro anti-sheep red blood cell plaque-forming assay. Results showed a marked modulation of immune cytokines in the transplanted mouse host and donor-specific transplantation tolerance was achieved. Tolerant mouse lymphocytes maintained a competent humoral antibody response. Additionally, C57/BL6 mice transplanted with rat Sertoli cells tolerated rat skin grafts significantly longer than control non-Sertoli cell transplanted mice. We conclude that systemic administration of rat Sertoli cells across xenogenic barrier induces transplantation tolerance without altering systemic immune competence. These data suggest that Sertoli cells may be used as a novel and potentially powerful tool in cell transplantation therapy.

Key words: Sertoli cells; Systemic tolerance; Xenogenic transplantation model

Address correspondence to Dr. Don F. Cameron, Department of Anatomy, MDC 6, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA. E-mail: dcameron@hsc.usf.edu

*These authors contributed equally to this work.

Cell Transplantation, Vol. 15, pp. 55-65, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effects of Simulated Microgravity on the Morphology and Function of Neonatal Porcine Cell Clusters Cultured With and Without Sertoli Cells

G. Luca,1* M. Calvitti,2* C. Nastruzzi,3 G. Macchiarulo,1 E. Becchetti,2 L. M. Neri,4 S. Capitani,4 G. Basta,1 P. Brunetti,1 R. Calafiore,1 and D. F. Cameron5

1Department of Internal Medicine and Endocrine and Metabolic Sciences, University of Perugia, Perugia, Italy
2Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy
3Department of Chemistry and Technology of the Drugs, School of Pharmacy, University of Perugia, Perugia, Italy
4Department of Morphology and Embriology, Section of Human Anatomy, University of Ferrara, Ferrara, Italy
5partment of Anatomy, University of South Florida College of Medicine, Tampa, FL, USA

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of b-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation b-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Key words: Neonatal porcine cell clusters (NPCCs); Sertoli cells; Simulated microgravity coculture; Structure; Function

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, USA. Tel: (813) 974-9434; Fax: (813) 974-2058; E-mail: dcameron@hsc.usf.edu

*These two authors contributed equally to this work.

Cell Transplantation, Vol. 15, pp. 67-74, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Engineered Cell Therapy for Sustained Local Myocardial Delivery of Nonsecreted Proteins

Jing Bian,1,3 Matt Kiedrowski,3 Niladri Mal,3 Farhad Forudi,2 and Marc S. Penn1,2,3

1Department of Chemical and Biomedical Engineering, Cleveland State University, Cleveland, OH 44115-2425, USA
2Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, OH 44195, USA
3Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA

Novel strategies for the treatment of congestive heart failure have taken the form of gene and cell therapy to induce angiogenesis, optimize calcium handling by cardiac myocytes, or regenerate damaged myocardial tissue. Arguably both gene- and cell-based therapies would be benefited by having the ability to locally deliver specific transcription factors and other usually nonsecreted proteins to cells in the surrounding myocardial tissue. The herpes simplex virus type 1 (HSV-1) tegument protein VP22 has been shown to mediate protein intercellular trafficking to mammalian cells and finally localize into the nucleus, which makes it a useful cargo-carrying functional protein in cell-based gene therapy. While VP22 has been studied as a means to modulate tumor growth, little is known about the distribution and transport kinetics of VP22 in the heart and its potential application in combination with autologous cell transplantation for the delivery of proteins to myocardial tissue. The aim of this study was to evaluate the efficacy of VP22 fusion protein intercellular trafficking combined with autologous cell transplantation in the heart. In an in vitro study untransfected rat heart cells were cocultured with stably transfected rat cardiac fibroblasts (RCF) with fusion constructs of VP22. The control experiment was untransfected rat heart cells co-plated with RCF stably transfected with enhanced green fluorescence protein (eGFP). The Lewis rat model was selected for in vivo study. In the in vitro studies there was a 14-fold increase in the number of GFP-positive cells 48 h after initiating coculture with VP22-eGFP RCF compared to eGFP RCF. In the rat model, transplantation of VP22-eGFP expressing RCF led to VP22-eGFP fusion protein delivery to an area of myocardial tissue that was 20-fold greater than that observed when eGFP RCF were transplanted. This area appeared to reach a steady state between 7 and 10 days after transplantation. The VP22-eGFP area consisted of eGFP-positive endothelium, smooth muscle cells, and cardiac myocytes with delivery to an area of approximately 1 mm2 of myocardial tissue. Our data suggest a viable strategy for the delivery of proteins that are not naturally secreted or internalized, and provide the first insight into the feasibility and effectiveness of cell-penetrating proteins combined with cell transplantation in the heart.

Key words: Cell-based therapy; Intercellular trafficking; Protein delivery; VP22

Address correspondence to Marc S. Penn, M.D., Ph.D., Departments of Cardiovascular Medicine and Cell Biology, NC10, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Tel: (216) 444-7122; Fax: (216) 444-9404; E-mail: pennm@ccf.org

Cell Transplantation, Vol. 15, pp. 75-82, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Molecular Imaging Reveals Skeletal Engraftment Sites of Transplanted Bone Marrow Cells

Philipp Mayer-Kuckuk,1,3 Mikhail Doubrovin,1 Luc Bidaut,1 Tulin Budak-Alpdogan,3 Shangde Cai,1 Vanessa Hubbard,2 Onder Alpdogan,2 Marce van den Brink,1,2 Joseph R. Bertino,3 Ronald G. Blasberg,1 Debabrata Banerjee,1,3 and Juri Gelovani4

1In Vivo Cellular Molecular Imaging Center, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
2Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
3The Cancer Institute of New Jersey, RWJMS, UMDNJ, New Brunswick, NJ, USA
4Department of Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

Molecular imaging holds great promise for the in vivo study of cell therapy. Our hypothesis was that multimodality molecular imaging can identify the initial skeletal engraftment sites post-bone marrow cell transplantation. Utilizing a standard mouse model of bone marrow (BM) transplantation, we introduced a combined bioluminescence (BLI) and positron emission tomography (PET) imaging reporter gene into mouse bone marrow cells. Bioluminescence imaging was used for monitoring serially the early in vivo BM cell engraftment/expansion every 24 h. Significant cell engraftment/expansion was noted by greatly increased bioluminescence about 1 week posttransplant. Then PET was applied to acquire three-dimensional images of the whole-body in vivo biodistribution of the transplanted cells. To localize cells in the skeleton, PET was followed by computed tomography (CT). Co-registration of PET and CT mapped the sites of BM engraftment. Multiple, discrete BM cell engraftment sites were observed. Taken together, this multimodality approach may be useful for further in vivo characterization of various therapeutic cell types.

Key words: Molecular imaging; Bioluminescence imaging; Positron emission tomography (PET); Computed tomography (CT); Skeleton; Transplantation

Address correspondence to Debabrata Banerjee, Ph.D., Departments of Medicine and Pharmacology, Room 3035, Cancer Institute of New Jersey, RWJMS, UMDNJ, 195 Little Albany Street, New Brunswick, NJ 08903, USA. Tel: 732-235-6458; Fax: 732-235-8181; E-mail: banerjed@umdnj.edu