|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 15, NUMBERS 8/9, 2006
Cell Transplantation, Vol. 15, pp. 675-687, 2006
0963-6897/06 $90.00 + 00
Copyright © 2006 Cognizant Comm. Corp.
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Autologous Bone Marrow Transplantation in Patients With Subacute and Chronic Spinal Cord Injury
Eva Syková,1,2 Ales Homola,1,2 Radim Mazanec,3 Hynek Lachmann,3 Simona Langkramer Konrádová,4 Petr Kobylka,4 Radek Pádr,5 Jirí Neuwirth,5 Vladimír Komrska,6 Vladimír Vávra,6 Jan Stulík,7 and Martin Bojar3
1Center for Cell Therapy and Tissue Repair and Department
of Neuroscience, 2nd Medical Faculty, Charles University, Prague, Czech
2Institute of Experimental Medicine, ASCR, Prague, Czech Republic
3Department of Neurology, University Hospital Motol and 2nd Medical Faculty, Charles University, Prague, Czech Republic
4Institute of Hematology and Blood Transfusion, Prague, Czech Republic
5Department of Pediatric Hematology and Oncology, University Hospital Motol and 2nd Medical Faculty, Charles University, Prague, Czech Republic
6Department of Imaging Methods, University Hospital Motol and 2nd Medical Faculty, Charles University, Prague, Czech Republic
7Department of Spondylosurgery, University Hospital Motol, Prague, Czech Republic
Stem cell transplants into spinal cord lesions may help to improve regeneration and spinal cord function. Clinical studies are necessary for transferring preclinical findings from animal experiments to humans. We investigated the transplantation of unmanipulated autologous bone marrow in patients with transversal spinal cord injury (SCI) with respect to safety, therapeutic time window, implantation strategy, method of administration, and functional improvement. We report data from 20 patients with complete SCI who received transplants 10 to 467 days postinjury. The follow-up examinations were done at 3, 6 ,and 12 months after implantation by two independent neurologists using standard neurological classification of SCI, including the ASIA protocol, the Frankel score, the recording of motor and somatosensory evoked potentials, and MRI evaluation of lesion size. We compared intra-arterial (via catheterization of a. vertebralis) versus intravenous administration of all mononuclear cells in groups of acute (10-30 days post-SCI, n = 7) and chronic patients (2-17 months postinjury, n = 13). Improvement in motor and/or sensory functions was observed within 3 months in 5 of 6 patients with intra-arterial application, in 5 of 7 acute, and in 1 of 13 chronic patients. Our case study shows that the implantation of autologous bone marrow cells appears to be safe, as there have been no complications following implantation to date (11 patients followed up for more than 2 years), but longer follow-ups are required to determine that implantation is definitively safe. Also, we cannot yet confirm that the observed beneficial effects were due to the cell therapy. However, the outcomes following transplantation in acute patients, and in one chronic patient who was in stable condition for several months prior to cell implantation, are promising. It is evident that transplantation within a therapeutic window of 3-4 weeks following injury will play an important role in any type of stem cell SCI treatment. Trials involving a larger population of patients and different cell types are needed before further conclusions can be drawn.
Key words: Evoked potentials; Magnetic resonance imaging; Mesenchymal stem cells; Regeneration; Spinal cord injury; Stem cells
Address correspondence to Prof. Eva Syková M.D., D.Sc., Institute of Experimental Medicine ASCR, Vídenská 1083, 142 20 Prague 4, Czech Republic. Tel: +420-241062230; Fax: +420-241062783; E-mail: email@example.com
Transplantation of Bioreactor-Produced Neural Stem Cells Into the Rodent Brain
M. McLeod,1 M. Hong,1 A. Sen,2 D. Sadi,1 R. Ulalia,1 L. A. Behie,2 and I. Mendez1
1Cell Restoration Laboratory, Brain Repair Centre, Dalhousie
University, Halifax, Nova Scotia, Canada
2Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada
The development of new cell replacement strategies using neural stem cells (NSC) may provide an alternative and unlimited cell source for clinical neural transplantation in neurodegenerative diseases such as Parkinson's and Huntington's disease. The clinical application of neural transplantation using NSC will therefore depend upon the availability of clinical grade NSC that are generated in unlimited quantities in a standardized manner. In order to investigate the utility of NSC in clinical neural transplantation, undifferentiated murine NSC were first expanded for an extended period of time in suspension bioreactors containing a serum-free medium. Following expansion in suspension bioreactors, NSC were still able to differentiate in vitro into both astrocytes and neurons after exposure to brain-derived neurotrophic factor (BDNF), suggesting that bioreactor expansion does not alter cell lineage potentiality. Undifferentiated bioreactor-expanded NSC were then transplanted into the rodent striatum. Immunohistochemical examination revealed undifferentiated bioreactor-expanded NSC survived transplantation for up to 8 weeks and expressed the astrocytic immunohistochemical marker glial fibrillary acidic protein (GFAP), suggesting that the host striatal environment influences NSC cell fate upon transplantation. Moreover, no tumor formation was observed within the graft site, indicating that NSC expanded in suspension bioreactors for an extended period of time are a safe source of tissue for transplantation. Future studies should focus on predifferentiating NSC towards specific neuronal phenotypes prior to transplantation in order to restore behavioral function in rodent models of neurodegenerative disease.
Key words: Neural stem cells; Suspension bioreactor; Neural transplantation; Differentiation; Glial fibrillary acidic protein
Address correspondence to Dr. Mark McLeod, Ph.D., Cell Restoration Laboratory, Brain Repair Centre, 12th Floor, Department of Anatomy and Neurobiology and Surgery (Neurosurgery), Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5. Tel: (902) 494 8896; Fax: (902) 494 1212; E-mail: firstname.lastname@example.org
Murine Brain Progenitor Cells Have the Ability to Differentiate Into Functional Neurons and Integrate Into the CNS
Shinghua Ding,1* Conrad A. Messam,1* Peiying Li,1 Michael E. Selzer,2 Marc A. Dichter,2 and Philip G. Haydon1
1Department of Neuroscience, University of Pennsylvania School
of Medicine, Philadelphia, PA 19104, USA
2Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
Although neural stem and progenitor cells have been shown to differentiate into neurons, few studies have examined the physiological properties of the differentiated neurons derived from stem cells. Here we show that mouse brain progenitor cells (mBPCs) differentiated in culture by removal of mitogenic factors or addition of BDNF or GDNF express neuronal-specific proteins including MAP-2 and synaptobrevin II. However, these cells demonstrate small voltage-gated Na+ currents and are not able to generate action potentials. When the mBPCs are cocultured with developing rat hippocampal neurons, the stem cells differentiate into neurons expressing MAP-2, develop large voltage-gated Na+ currents, and are able to generate action potentials. To investigate the influence of a mature CNS environment on survival, differentiation, migration, and morphological integration, mBPCs were transplanted into the spinal cord of adult mice. Undifferentiated cells transplanted into the spinal cord exhibited limited migration and expressed NG2, but did not differentiate to express MAP-2. Predifferentiated cells migrated to both gray and white matter with about 23% cells developing MAP-2 immunoreactivity after 8 weeks. These results suggest that both the environment and state of differentiation may dictate migration and the differentiation pathway of stem cells after transplantation.
Key words: Growth factors; Electrophysiology; Oligodendrocyte; Neuronal markers; Transplantation
Address correspondence to Dr. Shinghua Ding, Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. Tel: (215) 746-6789 or (215) 746-6547; Fax: (215) 746-6792; E-mail: email@example.com
*These authors contributed equally to this work.
Immunologic Consequences of Multiple, High-Dose Administration of Allogeneic Mesenchymal Stem Cells to Baboons
Kirstin J. Beggs,1 Alex Lyubimov,2 Jade N. Borneman,1 Amelia Bartholomew,2 Annemarie Moseley,1 Robert Dodds,1 Michael P. Archambault,1 Alan K. Smith,1 and Kevin R. McIntosh1
1Osiris Therapeutics, Inc., Baltimore, MD, USA
2University of Illinois, Chicago, IL, USA
Mesenchymal stem cells (MSCs) express low immunogenicity and demonstrate immunomodulatory properties in vitro that may safely allow their transplantation into unrelated immunocompetent recipients without the use of pharmacologic immunosuppression. To test this hypothesis, three groups of baboons (three animals per group) were injected as follows: group 1 animals were injected with vehicle; group 2 animals were injected IV with DiI-labeled MSCs (5 x 106 MSCs/kg body weight) followed 6 weeks later by IM injections of DiO-labeled MSCs (5 x 106 MSCs/kg) from the same donor; and group 3 animals were treated similarly as group 2 except that MSCs were derived from two different donors. Muscle biopsies, performed 4 weeks after the second injection of MSCs, showed persistence of DiO-labeled MSCs in 50% of the recipients. Blood was drawn at intervals for evaluation of basic immune parameters (Con A mitogen responsiveness, PBMC phenotyping, immunoglobulin levels), and to determine T-cell and alloantibody responses to donor alloantigens. Host T-cell responses to donor alloantigens were decreased in the majority of recipients without suppressing the overall T-cell response to Con A, or affecting basic parameters of the immune system. All recipient baboons produced alloantibodies that reacted with donor PBMCs. Two of six animals produced alloantibodies that reacted with MSCs. We conclude that multiple administrations of high doses of allogeneic MSCs affected alloreactive immune responses without compromising the overall immune system of recipient baboons. The induction of host T-cell hyporesponsiveness to donor alloantigens may facilitate MSC survival.
Key words: Mesenchymal stem cells; Baboons; Immunogenicity; Allogeneic; T-cell hyporesponsiveness; Alloantibody
Address correspondence to Kevin R. McIntosh, Ph.D., Cognate BioServices, Inc., 1448 S. Rolling Road, Baltimore, MD 21227, USA. Tel: 410-455-5550; Fax: 410-455-5551; E-mail: firstname.lastname@example.org
Human Bone Marrow Mesenchymal Stem Cells Seeded on Modified Collagen Improved Dermal Regeneration In Vivo
Marta Markowicz,1 Eva Koellensperger,1 Sabine Neuss,2,3 Sarah Koenigschulte,1 Christine Bindler,1 and Norbert Pallua1
1Department of Plastic Surgery, Hand and Burn Surgery, RWTH
Aachen University, Pauwelsstrasse 30, 52057 Aachen, Germany
2Institute of Pathology, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen, Germany
3Interdisciplinary Center for Clinical Research IZKF BIOMAT 3, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen, Germany
In the correction of functional and aesthetic impairments, loss of soft connective tissue creates the need for adequate implant material. The reconstruction of defects resulting from radical excisions, trauma, or hereditary diseases has seen the use of combined grafts and flaps. With the aim of minimizing donor site morbidity, new methods have been evaluated. Because of a low rate of vascularization, with artificial dermal templates the take has only been poor. As shown in previous studies, improved angiogenetic potency and epidermal formation has been obtained in modified, cell-seeded collagen matrices. We have now investigated the suitability of adult bone marrow mesenchymal stem cells (hMSC) for soft tissue engineering. In this study, hMSC were isolated and expanded. Cells (106) were seeded onto EDC cross-linked collagen sponges and implanted in 30 immunodeficient mice. Collagen sponges without cells were used as controls. The grafts were evaluated after 2 and 6 weeks. After explantation, macroscopic appearance, weights, and histology (scaffold degradation, cellularity, and invasion depth of the seeded cells) were all assessed. After 2 and 6 weeks in vivo, new vessels were found macroscopically on all cell-seeded collagen grafts. The control grafts appeared to be degraded with a lower rate of vessel ingrowth. In the experimental group, weight gain was significant after 2 and 6 weeks in vivo compared to the same grafts after 72 h in vitro, while weight increased only slightly in the control group. Histologically, populated scaffolds showed a high density of vascularization under a capsule. The control sponges showed single capillaries and a thicker capsule. Compared to the controls, cellularity (cells/field) was greater in cell-containing collagen grafts after 2 and 6 weeks. The results obtained demonstrate that in vitro cultured human mesenchymal stem cells seeded on modified collagen sponges may be able to act as a replacement for soft tissue.
Key words: Human bone marrow mesenchymal stem cells; Soft tissue engineering; Collagen matrices; Scaffolds; Animal model
Address correspondence to Dr. med. Marta P. Markowicz, Department of Plastic Surgery, Hand and Burn Surgery, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen, Germany. Tel: +49-241-8089701; Fax: +49-241-8082634; E-mail: email@example.com
Establishment of Cocultures of Osteoblasts, Schwann Cells, and Neurons Towards a Tissue-Engineered Approach for Orofacial Reconstruction
Kirsten Haastert,1,2 Nicole Semmler,1 Maike Wesemann,1 Martin Rücker,3 Nils-Claudius Gellrich,3 and Claudia Grothe1,2
1Department of Neuroanatomy, Hannover Medical School, Hannover,
2Center of Systems Neuroscience (ZSN) Hannover, Hannover, Germany
3Department of Oral and Maxillofacial Surgery, Hannover Medical School, Hannover, Germany
In orofacial reconstruction not only the osseous structures themselves but also neighboring cranial nerves need to be regenerated. To replace autologous bone implants, biocompatible tissue-engineered scaffolds are under investigation at least for bone replacement but until now these studies have not focused on parallel reconstruction of injured cranial nerves. The present study contributes to the development of optimized tissue-engineered products that will enable regeneration of both bone and nervous tissue. For the first time, cocultures of primary osteoblasts (rat or human) and primary Schwann cells (rat or human) were established. The suitability of monocultures of osteoblasts and cocultures of osteoblasts plus Schwann cells as substrate for sensory neurons as well as motoneurons was tested here. The results suggest that whereas osteoblasts provide a good substrate for sensory neurons, motoneurons depend on the presence of Schwann cells for survival and neurite outgrowth. For prolonged availability of regeneration-promoting growth factors at the site of the graft, those proteins should be delivered by the transplanted cells themselves. To enable this, we established electroporation-based nonviral transfection of osteoblasts as well as Schwann cells. Our new cell culture system will enable investigations of the effect of graft-derived growth factors on osteoblasts and Schwann cells as well as on neurite outgrowth from cocultured neurons of the sensory and motor system.
Key words: Orofacial reconstruction; Tissue engineering; Osteoblast; Schwann cells; Bone tissue; Nervous tissue
Address correspondence to Dr. med. vet. Kirsten Haastert. Department of Neuroanatomy, OE 4140, Hannover Medical School, Carl-Neuberg-Str.1, D-30623 Hannover, Germany. Tel: +49-511-532-2891; Fax: +49-511-532-2880; E-mail: firstname.lastname@example.org
The Use of the BD Oxygen Biosensor System to Assess Isolated Human Islets of Langerhans: Oxygen Consumption as a Potential Measure of Islet Potency
Chris Fraker,1 Mark R. Timmins,2 Richard D. Guarino,3 Perry D. Haaland,3 Hirohito Ichii,1 Damaris Molano,1 Antonello Pileggi,1 Raffaella Poggioli,1 Sharon C.Presnell,3 Luca Inverardi,1 Mitra Zehtab,1 and Camillo Ricordi1
1Diabetes Research Institute, University of Miami Miller
School of Medicine, Miami, FL, USA
2BD Biosciences-Discovery Labware, New Bedford, MA, USA
3BD Technologies, Research Triangle Park, NC, USA
The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding ("dO2 slope"). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized "dO2 slope" in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.
Key words: Oxygen consumption rate; Islets; Islet potency; Transplantation
Address correspondence to Camillo Ricordi, M.D., Stacy Joy Goodman Professor of Surgery and Medicine, Chief, Division of Cellular Transplantation, Scientific Director and Chief Academic Officer, Diabetes Research Institute, Senior Associate Dean for Research, Miller School of Medicine at the University of Miami, 1450 NW 10th Avenue, Miami, FL 33136, USA. Tel: (305) 243-6913; Fax: (305) 243-4404; E-mail: email@example.com
Adenovirus-Mediated Expression of the Anticoagulant Hirudin in Human Islets: A Tool to Make the Islets Biocompatible to Blood
Sanja Cabric, Graciela Elgue, Bo Nilsson, Olle Korsgren, and Peter Schmidt
Division of Clinical Immunology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden
Human islets induce an injurious clotting reaction at the time of transplantation. A potential strategy to counteract this reaction would be to allow the islets to express hirudin, a protein with direct anticoagulative activity. Human islets were transduced with an adenoviral vector encoding hirudin, an empty corresponding vector, or left untreated. Islet culture supernatants were analyzed for hirudin using an ELISA, a chromogenic substrate assay based on the thrombin-binding properties of hirudin and in a whole blood viscosimetry assay. Immunohistochemical evaluation and determination of hirudin content revealed an abundant expression of hirudin after transduction. Hirudin content in transduced islets was in the range of the insulin content levels. A delay in human whole blood clotting time could be observed after addition of supernatants taken from islet cultures expressing hirudin. However, transduced islets showed an impaired glucose-stimulated insulin release, but could readily be retrieved 6 weeks after transplantation to athymic mice. A marked expression and secretion of hirudin with functional capacity can be induced in human islets using an adenoviral vector. The impairment in glucose-stimulated insulin release in hirudin-secreting islets, compared to controls, indicates that the additional protein synthesis affects the functional capacity of the islets.
Key words: Transplantation; Coagulation; Human islets of Langerhans; Hirudin; Adenoviral vector
Address correspondence to Sanja Cabric, The Rudbeck Laboratory C11, Dag Hammarskölds väg 20, SE-751 85 Uppsala, Sweden, Tel: +46 18 611 3984; Fax: +46 18 611 0222; E-mail: firstname.lastname@example.org
Survival of an Islet Allograft Deficient in iNOS After Implantation Into Diabetic NOD Mice
Andreas Börjesson, Annika K. Andersson, and Stellan Sandler
Department of Medical Cell Biology, Uppsala University Uppsala, Sweden
Proinflammatory cytokines play a major role in rejection of pancreatic islet allografts and in type 1 diabetes (T1D). In rodent islets, exposure to IL-1β alone or combined with IFN-γ induces expression of inducible nitric oxide synthase (iNOS). Inhibition of iNOS or a deletion of the iNOS gene has been shown to be protective in animal models of T1D. In the present study we tested the hypothesis that transplantation of pancreatic islets deficient in iNOS (iNOS-/-) would permit increased graft survival. Pancreatic islets isolated from wild-type (wt) mice and iNOS-/- mice were allogeneically transplanted beneath the kidney capsule of spontaneously diabetic NOD mice. When blood glucose increased above 12.0 mM after preceding normalization of hyperglycemia, animals were sacrificed. Histological examinations of grafts were performed and graft gene expression was analyzed by real-time PCR. Transplantations of the two types of islets could reverse hyperglycemia and the grafts functioned for on average 1 week posttransplantation. Morphological examination of both types of islet grafts showed immune cell infiltration around and within the grafts. Remaining endocrine cells could be observed in wt and iNOS-/- islet grafts. In the removed grafts iNOS-/- islet tissue contained higher mRNA levels of insulin, proinsulin convertases (PC-1 and PC-2), and IL-1b compared to transplanted wt islets. The assessments of insulin, PC-1 and PC-2 mRNAs of the grafts suggest that the iNOS-/- islets may be more resistant to destruction in the transplantation model used; however, this was not sufficient to prolong the period of normoglycemia posttransplantation.
Key words: Islets; Cytokines; iNOS; NOD mice
Address correspondence to Andreas Börjesson, Department of Medical Cell Biology, BMC, Box 571, SE-751 23 Uppsala, Sweden. Tel: 46-18-471-44-11; Fax: 46-18-471-40-59; E-mail: Andreas.Borjesson@medcellbiol.uu.se
The Effects of Immunosuppressive Agents on the Function of Human Hepatocytes In Vitro
Trinidad Serrano,* Ragai R. Mitry, Claire Terry, Sharon C. Lehec, Anil Dhawan, and Robin D. Hughes
King's College London School of Medicine at King's College Hospital, Institute of Liver Studies, London, UK
Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/106 cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from <20-year-old donors (687 ± 59 pg/106 cells/24 h) was significantly greater than from older donors (61 ± 7 pg/106 cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.
Key words: Hepatocytes; Tacrolimus; Methylprednisolone; Transplantation; Vascular endothelial growth factor
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 3299 3137; Fax: +44 (0)20 3299 3760; E-mail: email@example.com
*Present address: Transplant Unit, Lozano Blesa University Hospital, Zaragoza, Spain.
Microencapsulation of Engineered Cells to Deliver Sustained High Circulating Levels of Interleukin-6 to Study Hepatocellular Carcinoma Progression
Diarmuid M. Moran,1 Leonidas G. Koniaris,2 Elizabeth M. Jablonski,1 Paul A. Cahill,3 Craig R. Halberstadt,4 and Iain H. McKillop1
1Department of Biology, University of North Carolina at Charlotte,
Charlotte, NC, USA
2Dewitt Daughtry Department of Surgery & The Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA
3School of Biotechnology, Dublin City University, Dublin, Ireland
4Department of General Surgery, Carolinas Medical Center, Charlotte, NC, USA
Interlukin-6 (IL-6) is a pleitropic cytokine that plays a central role in normal and abnormal hepatic function and response. The aims of the current study were to determine the viability of using cell encapsulation technology to introduce a genetically modified xenogeneic (CHO) cell population to elevate circulating levels of rhIL-6 in a rat model and determine the effects of sustained high rhIL-6 levels on hepatocellular carcinoma (HCC) progression in vivo. An alginate matrix was combined with transfected CHO cells, selected for their ability to synthesize rhIL-6, and used to generate uniform alginate-cell beads. Once encapsulated transfected cells continued to undergo replication, formed colonies within the bead, and synthesized/released large quantities of rhIL-6 into culture medium in vitro. Intraperitoneal implantation of beads into rats resulted in significantly increased circulating and intrahepatic levels of rhIL-6 up to 4 days postimplantation. Prolonged implantation led to the escape of CHO cells from the bead, resulting in a host response and CHO cell death within the bead. Subsequently CHO-IL-6 encapsulated cells were implanted into rats previously inoculated intrahepatically with the H4IIE HCC cell line. These studies demonstrated the maintenance of high circulating/intrahepatic rhIL-6 levels in this model. Despite significantly increased rhIL-6, this technique did not significantly alter the rate of net tumor progression. However, Stat3 activity was significantly increased in both normal liver and HCC tissue resected from animals implanted with CHO-IL-6 cells. Collectively these data demonstrate the short-term viability of using cell encapsulation technology to generate high levels of active circulating and intrahepatic cytokines and raise the possibility of modifying specific signal transduction cascades identified to be important during tumor progression.
Key words: Interleukin-6; Hepatocellular carcinoma; Cell encapsulation; Signal transducers and activators of transcription (STAT)
Address correspondence to Iain H. McKillop, Department of Biology, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, NC 28223, USA. Tel: (704) 687-4050; Fax: (704) 687-3128; E-mail: firstname.lastname@example.org
Enhanced Functional Maturation of Fetal Porcine Hepatocytes in Three-Dimensional Poly-L-lactic Acid Scaffolds: A Culture Condition Suitable for Engineered Liver Tissues in Large-Scale Animal Studies
Hongyun Huang,1 Sanshiro Hanada,1 Nobuhiko Kojima,1 and Yasuyuki Sakai1,2
1Institute of Industrial Science, University of Tokyo, 4-6-1
Komaba, Meguro-ku, Tokyo 153-8505, Japan
2Center for Disease Biology and Integrative Medicine, Gradate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
To engineer liver tissues with a clinically significant size, in vivo evaluation of performance using large-scale animal studies are necessary before proceeding to human clinical trials. As pigs are the most suitable candidates, the development of culture conditions suitable for porcine hepatocyte progenitors is very important to engineer pig liver tissue equivalents. We therefore investigated the efficacy of poly-L-lactic acid (PLLA) three-dimensional (3D) scaffolds on the functional maturation of fetal porcine hepatocytes in the presence of various combinations of biofactors. Cells were isolated from pig fetuses obtained from a local slaughterhouse, and cultured for 15 days both in monolayer and PLLA scaffolds. Although 15 days of culture resulted in almost the same ratio of proliferation (about fivefold) in both monolayer and 3D PLLA culture, the PLLA culture with hepatocyte growth factor (HGF, 10 ng/ml) and sodium butylate (Sb, 1 mM) remarkably enhanced various liver-specific functions of fetal porcine hepatocytes. The final attained functions based on the numbers of immobilized cells on day 1 compared with those of day 1 monolayers; 191-fold increase in albumin secretion, 70.5-fold increase in cytochrome P450 IA1/2 capacity, 20.9-fold increase in ammonia removal, and 18.0-fold increase in urea synthesis were obtained. These functions were 2.0-3.3-fold higher than those obtained by the same period of monolayer culture. In addition, final attained unit cell-based functions on day 15 were almost comparable to the levels reported for cultures of adult porcine hepatocytes in both monolayer and 3D spheroid cultures. These results demonstrate that the use of a biodegradable polymer-based 3D culture with an appropriate combination of biofactors is a promising approach to maximize functional maturation of hepatocyte progenitors from large animals. In addition, the established culture conditions are worth using to engineer large liver tissue equivalents for pigs in large-animal-based preclinical studies.
Key words: Fetal porcine hepatocytes; Three-dimensional culture; Biofactors; Poly-L-lactic acid scaffold; Liver-specific functions; Liver tissue engineering
Address correspondence to Hongyun Huang, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan. Tel: +81-3-5452-6349; Fax: +81-3-5452-6348; E-mail: email@example.com
In Vitro Functionality of Human Fetal Liver Cells and Clonal Derivatives Under Proliferative Conditions
Tanja Deurholt,1 Lysbeth ten Bloemendaal,1,2 Aniska A. Chhatta,1 Albert C. W. A. van Wijk,1,2 Kees Weijer,3 Jurgen Seppen,1 Ronald P. J. Oude Elferink,1 Robert A. F. M. Chamuleau,1 and Ruurdtje Hoekstra1,2
1AMC Liver Center, Academic Medical Center, Amsterdam, The
2Department of Surgery (Surgical Laboratory), Academic Medical Center, Amsterdam, The Netherlands
3Department of Cell Biology and Histology, Academic Medical Center, Amsterdam, The Netherlands
Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we evaluated the hepatic function of HFLCs under proliferative conditions, to determine whether HFLCs can replace mature hepatocytes for in vitro applications. HFLCs were isolated from fetal livers of 16 weeks gestation. Hepatic functions of HFLCs were determined in primary culture and after expansion in vitro. Clonal derivatives were selected and tested for hepatic functionality. Results were compared to primary mature human hepatocytes in vitro. No differences were observed between primary HFLCs and mature human hepatocytes in albumin production and mRNA levels of various liver-specific genes. Ureagenesis was 4.4-fold lower and ammonia elimination was absent in HFLCs. Expanding HFLCs decreased hepatic functions and increased cell stretching. In contrast, clonal derivatives had stable functionality and morphology and responded to differentiation stimuli. Although their hepatic functions were higher than in passaged HFLCs, functionality was at least 20 times lower compared to mature human hepatocytes. HFLCs cannot replace mature human hepatocytes in in vitro applications requiring extensive in vitro expansion, because this is associated with decreased hepatic functionality. Selecting functional subpopulations can, at least partly, prevent this. In addition, defining conditions that support hepatic differentiation is necessary to obtain HFLC cultures suitable for in vitro hepatic applications.
Key words: Hepatocyte; Fetal; Proliferation; Liver function; In vitro
Address correspondence to R. Hoekstra, Department of Surgery (Surgical Laboratory), IWO-1-172, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: 0031-20-5666683; Fax: 0031-20-6976621; E-mail: firstname.lastname@example.org
Development, Characterization, and Use of a Fetal Skin Cell Bank for Tissue Engineering in Wound Healing
Anthony S. De Buys Roessingh,1 Judith Hohlfeld,1 Corinne Scaletta,2 Nathalie Hirt-Burri,1 Stefan Gerber,3 Patrick Hohlfeld,3 Jan-Olaf Gebbers,4 and Lee Ann Applegate2
1Department of Pediatric Surgery, University Hospital Lausanne,
2Orthopedic Cell Therapy Unit, University Hospital Lausanne, Lausanne, Switzerland
3Department of Obstetrics, University Hospital Lausanne, Lausanne, Switzerland
4Department of Pathology, Cantonal Hospital, Lucern, Switzerland
Wound healing in fetal skin is characterized by the absence of scar tissue formation, which is not dependent on the intrauterine environment and amniotic fluid. Fetal cells have the capacity of extraordinary expansion and we describe herein the development of a fetal skin cell bank where from one organ donation (2-4 cm2) it is possible to produce several hundred million fetal skin constructs of 9 x 12 cm2. Fetal cells grow three to four times more rapidly than older skin cells cultured in the same manner and these banked fetal cells are very resistant against physical and oxidative stress when compared to adult skin cells under the same culture conditions. They are up to three times more resistant to UVA radiation and two times more resistant towards hydrogen peroxide treatment. This mechanism may be of major importance for fetal cells when they are delivered to hostile wound environments. For fetal cell delivery to patients, cells were associated with a collagen matrix to form a three-dimensional construct in order to analyze the capacity of these cells for treating various wounds. We have seen that fetal cells can modify the repair response of skin wounds by accelerating the repair process and reducing scarring in severe burns and wounds of various nature in children. Hundreds of thousands of patients could potentially be treated for acute and chronic wounds from one standardized and controlled cell bank.
Key words: Fetal cell therapy; Tissue engineering; Oxidative stress
Address correspondence to Dr. Lee Ann Laurent-Applegate, Orthopedic Cell Therapy Unit, University Hospital, CHUV, PAV 03, CH-1011 Lausanne, Switzerland. Tel: (++41) 21 314 3510; Fax: (++41) 21 887 8414; E-mail: Lee.Laurent-Applegate@chuv.ch
Induction of Tolerance Across Fully Mismatched Barriers by a Nonmyeloablative Treatment Excluding Antibodies or Irradiation Use
Lionel Stephan,1 Christophe Pichavant,1 Manaf Bouchentouf,1 Philippe Mills,1 Geoffrey Camirand,2 Saloua Tagmouti,1 David Rothstein,2 and Jacques P. Tremblay1
1Human Genetic, CHUQ-CHUL, Laval University, Ste-Foy, G1V4G2,
2Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520-8029, USA
A mixed-chimerism approach is a major goal to circumvent sustained immunosuppression, but most of the proposed protocols need antibody treatment or host irradiation. Another promising experience involves busulfan combined with cyclophosphamide treatment. Additionally, recent publications demonstrated that, differing from busulfan, treosulfan administration does not present severe organ or hemato toxicities. Currently, Duchenne muscular dystrophy (DMD) patients are treated with chronic immunosuppression for muscle precursor cell transplantation (MT). We have developed a safe tolerance approach within this cellular allotransplantation therapy background. Thus, we have conditioned, prior to a donor BALB/c MT, the dystrophic mouse model C57Bl10J mdx/mdx, with our treatment based on a donor-specific transfusion, then a treosulfan treatment combined with single cyclophosphamide dose, and finally a donor bone marrow transplantation (TTCB). A first MT was performed in all mixed chimeric mice resulting from the TTCB treatment in the left tibialis anterior (TA) muscles. A second MT from the same donor strain was performed 100 days later in the right TA without any additional therapy. Results show that all treated mice developed permanent mixed chimerism. Long-lasting donor-positive fibers were present in both TAs of the mice, which received MT after the TTCB treatment. Only a basal level of infiltration was observed around donor fibers and mixed chimeric mice rejected third-party haplotype skin grafts. Thus, mixed chimerism development with this TTCB conditioning regimen promotes donor-specific stable tolerance, avoiding costimulatory blockade antibodies or irradiation use and side effects of sustained immunosuppressive treatments. This protocol could be eventually applied for MT to DMD patients or others tissue transplantations.
Key words: Treosulfan; Chimerism; Tolerance; Transplantation; Muscular dystrophy
Address correspondence to Dr. Jacques P. Tremblay, Human Genetic,
CHUQ-CHUL, Laval University, 2705 boulevard Laurier, Ste-Foy, G1V4G2, Canada.
Tel: 418-654-2186; Fax: 418-654-2207; E-mail: Jacques-P.email@example.com