|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 17, NUMBERS 10/11, 2008
Cell Transplantation, Vol. 17, pp. 1103-1113, 2008
0963-6897/08 $90.00 + 00
Copyright © 2008 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Mesenchymal Stem Cell Transplantation for Neurodegenerative Diseases
Yvan Torrente1 and Elio Polli2
1Stem Cell Laboratory, Department of Neurological Science,
Fondazione IRCCS Ospedale Maggiore Policlinico, Centro Dino Ferrari, University
of Milan, Milan, Italy
2Laboratory of Matarelli Foundation for Blood Diseases, Department of Pharmacology and Pharmacology Chemotherapy and Medical Toxicology, University of Milan, Milan, Italy
Neurodegenerative diseases are characterized by a progressive degeneration of selective neural populations. The lack of effective treatment and the characteristic of their pathology make these diseases appropriate candidates for cell therapy. Mesenchymal stem cells (MSCs) are multipotent stem-like cells that are capable of differentiating into mesenchymal and nonmesenchymal lineages. Their regenerative capacity after in vivo transplantation into animal models of neurodegenerative diseases has suggested that they could be useful against human diseases. Human bone marrow-derived MSCs (hMSCs) can be easily amplified in vitro and their transdifferentiation has been claimed in vitro and in vivo in neural cells. There are some doubts concerning the exact mechanisms responsible for the beneficial outcome observed after MSC transplantation into neurodegenerating tissues. Possible interpretations include cell replacement, trophic factor delivery, and immunomodulation. This review mainly concerns hMSCs transplantation in neurodegenerative diseases, because it has proven to be feasible, safe, and potentially effective. Although they have been used in hundreds of clinical trials, mixed results and no functional and long-lasting integration have so far been observed. hMSCs transplantations therefore still have their "dark side." However, the challenge in well-planned clinical trials merits discussion.
Key words: Mesenchymal stem cells; Transplantation; Cell fusion; Regenerative medicine
Address correspondence to Prof. Elio Polli, Fondazione A. e A. Matarelli, Via Aldo Lusardi 7, 20122 Milano, Italy. Te: +39 0258328399 or +39 0258328385; Fax: +39 0258328403; E-mail: firstname.lastname@example.org
Long-Term Survival, Robust Neuronal Differentiation, and Extensive Migration of Human Forebrain Stem/Progenitor Cells Transplanted to the Adult Rat Dorsal Root Ganglion Cavity
Elisabet Åkesson,1* Martin Sandelin,2* Nadezda Kanaykina,2 Håkan Aldskogius,2 and Elena N. Kozlova1
1Division of Neurodegeneration, Department of Neurobiology,
Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden
2Department of Neuroscience, Neuroanatomy, Uppsala University Biomedical Center, Uppsala, Sweden
Neurons in dorsal root ganglia (DRGs) transmit sensory information from peripheral tissues to the spinal cord. This pathway can be interrupted, for example, as the result of physical violence, traffic accidents, or complications during child delivery. As a consequence, the patient permanently loses sensation and often develops intractable neuropathic pain in the denervated area. Here we investigate whether human neural stem/progenitor cells (hNSPCs) transplanted to the DRG cavity can serve as a source for repairing lost peripheral sensory connections. We found that hNSPCs robustly differentiate to neurons, which survive long-term transplantation. The neuronal population in the transplants was tightly surrounded by astrocytes, suggesting their active role in neuron survival. Furthermore, 3 months after grafting hNSPCs were found in the dorsal root transitional zone (DRTZ) and within the spinal cord. The level of differentiation of transplanted cells was high in the core of the transplants whereas cells that migrated to the DRTZ and spinal cord were undifferentiated, nestin-expressing precursors. These data indicate that peripherally transplanted hNPSCs can be used for repair of dorsal root avulsion or spinal cord injuries; however, additional factors are required to guide their differentiation to the desired type of neurons. Furthermore, hNPSCs that migrate from the DRG cavity graft site to the DRTZ and spinal cord may provide trophic support for regenerating dorsal root axons, thereby allowing them to reenter the host spinal cord.
Key words: Neural stem cell; Sensory neuron; Neuronal degeneration; Neuronal regeneration; Cell migration; Plasticity
Address correspondence to Elena N. Kozlova, Department of Neuroscience, Uppsala University Biomedical Center, PO Box 587, SE-751 23 Uppsala, Sweden. Tel: +46-(0)18-471 4968; Fax: +46-(0)18-51 15 40; E-mail: Elena.Kozlova@anatomi.uu.se
*These authors contributed equally.
Environmental Housing and Duration of Exposure Affect Striatal Graft Morphology in a Rodent Model of Huntington's Disease
Máté D. Döbrössy1,2 and Stephen B. Dunnett1
1Brain Repair Group, School of Biosciences, Cardiff University,
Cardiff CF10 3US, UK
2Laboratory of Molecular Neurosurgery, Universitätsklinikum Freiburg, 79106 Freiburg, Germany
Clinical trials of cell replacement therapy in Huntington's disease have shown its safety, feasibility, and potentially long-lasting effects. However, more needs to be known regarding the conditions that stimulate plasticity and compensation achieved by neural grafts to maximize posttransplantation recovery of such neurorehabilitative therapies. The effects of enriched environment (EE), behavioral experience, and transplantation can each separately influence neuronal plasticity and recovery of function after brain damage, and the mechanisms by which these factors interact to modify the survival, integration, or function of grafted tissues are at present unknown. To investigate the effects of variable housing conditions and duration on morphological and cellular changes within embryonic striatal transplants, rats received unilateral excitotoxic lesions of the striatum, followed by E15 whole-ganglionic eminence suspension grafts. The rats were divided into three groups according to housing: full-time EE, 1 h/day exposure to EE, or standard laboratory cages. The experimental design included "early" (7 weeks postgrafting) and "late" (13 weeks postgrafting) survival time points to explore the effects of exposure lengths to the three housing conditions. The morphological and cellular effects on the grafts were analyzed using immunohistochemistry, cell morphology, image analysis, and enzyme-linked immunoassay. Both the duration of the exposure and the housing conditions were seen to influence multiple parameters of grafted cell morphology. The factors acted either independently (e.g., on graft size), complementarily (e.g., on spine density), or had no distinctive effect (e.g., on lesion size) on graft development. Features of embryonic striatal grafts and their trophic milieu were influenced both by the complexity of the environmental conditions and by the length of exposure to them. The data suggest that neurorehabilitation should be a feature of clinical trials of cell transplantation in order to exploit the underlying mechanisms that promote anatomical integration of the grafted cells and maximize transplantmediated functional recovery.
Key words: Environmental enrichment; Striatal grafts; Huntington's disease; Rat
Address correspondence to Dr. Máté D. Döbrössy, Stereotactic Neurosurgery, Laboratory of Molecular Neurosurgery, Universitätsklinikum Freiburg, Breisacher Str. 64, 79106 Freiburg, Germany. Tel.: (+49) 0761/270-5036; Fax: (+49) 0761/270-9303; E-mail: email@example.com
Sertoli Cells Enhance Formation of Capillary-Like Structures In Vitro
Brian T. Golat1 and Don F. Cameron1,2,3
1Department of Pathology & Cell Biology, College of Medicine,
University of South Florida, Tampa, FL, USA
2Department of Neurosurgery, College of Medicine, University of South Florida, Tampa, FL, USA
3Biomedical Engineering, College of Engineering, University of South Florida, Tampa, FL, USA
Sertoli cells isolated from the testis (referred to as extratesticular Sertoli cells) have been shown to facilitate allo- and xenogeneic cell transplantations. It appears likely that the ability of these cells to enhance the success of cell engraftment is due, in part, to the retention of their intratesticular functions of trophic support and immunoprotection. Sertoli cells also are involved in the regulation of angiogenesis in the testis, which may also contribute to enhanced cell engraftment success facilitated by extratesticular Sertoli cells. Because the maintenance of the cell's intratesticular angiogenic function has not yet been evaluated for extratesticular Sertoli cells, this study examined the cell's ability to enhance angiogenesis in vitro. Sertoli cell conditioned media were derived from isolated rat Sertoli cell cultures and used in a rat aortic model of induced angiogenesis, in endothelial and smooth muscle cell monocultures, and in endothelial smooth muscle cocultures. An angiogenic rat cytokine array identified angiogenic factors in the control and conditioned media. Aorta sections incubated with Sertoli cell conditioned media showed a marked increase in the formation of capillary-like structures when compared to controls. Likewise, endothelial cells incubated in conditioned media organized into capillary-like structures not observed when incubated in control media. In coculture, smooth muscle cells were associated with endothelial cell-derived capillary-like structures only when incubated in conditioned media. Cytokine arrays indicated the presence and a qualitative increase of specific angiogenic growth factors in Sertoli cell conditioned media not observed in control media. Results indicate that extratesticular Sertoli cells retain their intratesticular angiogenic function in vitro.
Key words: Extratesticular Sertoli cells; Angiogenesis; In vitro
Address correspondence to Don F. Cameron, Ph.D., Department of Pathology & Cell Biology, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA. E-mail: firstname.lastname@example.org
Transplantation of Mesenchymal Stem Cells Improves Atrioventricular Conduction in a Rat Model of Complete Atrioventricular Block
Miki Yokokawa,1 Shunsuke Ohnishi,2 Hatsue Ishibashi-Ueda,3 Hiroaki Obata,2 Kentaro Otani,2 Yoshinori Miyahara,2 Koichi Tanaka,2 Wataru Shimizu,1 Kazuo Nakazawa,4 Kenji Kangawa,5 Shiro Kamakura,1 Soichiro Kitamura,6 and Noritoshi Nagaya1,2
1Department of Internal Medicine, National Cardiovascular
Center, Osaka, Japan
2Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, Osaka, Japan
3Department of Pathology, National Cardiovascular Center, Osaka, Japan
4Laboratory of Development and Evaluation of Biomedical Instruments and Systems, National Cardiovascular Center Research Institute, Osaka, Japan
5Department of Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan
6Department of Cardiovascular Surgery, National Cardiovascular Center, Osaka, Japan
Mesenchymal stem cells (MSCs) are multipotent cells that differentiate into a variety of lineages including myocytes and vascular endothelial cells. However, little information is available regarding the therapeutic potential of MSCs in patients with atrioventricular block (AVB). We investigated whether local implantation of MSCs improves AV conduction in a rat model of complete AVB. Complete AVB was achieved by injection of ethanol into the AV nodal region of Lewis rats. Five days after ethanol injection, 2 × 106 of MSCs (MSC group) or vehicle (Control group) were injected into the AV nodal region. Animals were monitored by electrocardiograms for 14 days, and physiological and histological examinations were performed. The 1:1 AV conduction was recovered in 5 of 15 rats (33%) in the MSC group during the followup period, whereas no improvement was observed in the control group. MSC transplantation significantly decreased collagen deposition in the AV node, which was associated with a marked decrease in transforming growth factor-b1 expression. In vitro experiments demonstrated that MSCs secreted a large amount of antifibrotic factors such as hepatocyte growth factor and interleukin-10, and MSC conditioned medium inhibited the growth of adult cardiac fibroblasts. In addition, local injection of MSC conditioned medium recovered AV conduction in 2 of 15 rats (13%). MSC transplantation improved AV conduction in a rat model of complete AVB, at least in part through antifibrotic paracrine effects.
Key words: Atrioventricular node; Fibrosis; Atrioventricular block; Mesenchymal stem cell; Transplantation
Address correspondence to Shunsuke Ohnishi, M.D., Ph.D., Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Tel: +81-6-6833-5012; Fax: +81-6-6833-9865; E-mail: email@example.com
Mesenchymal Stem Cells Protective Effect in Adriamycin Model of Nephropathy
Alberto Magnasco,1* Mirko Corselli,2* Roberta Bertelli,3 Adalberto Ibatici,2 Monica Peresi,4 Gabriele Gaggero,4 Valentina Cappiello,3,5 Barbara Chiavarina,2 Girolamo Mattioli,6 Rosanna Gusmano,5 Jean Louis Ravetti,4 Francesco Frassoni,2 and Gian Marco Ghiggeri1,3
1Department of Nephrology, G. Gaslini Children Hospital,
2Stem Cells and Cell Therapy, Department of Hemato-Oncology, San Martino Hospital, Genoa, Italy
3Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Genoa, Italy
4Department of Pathology, San Martin Hospital, Genoa, Italy
5Renal Child Foundation, Genoa, Italy
6Department of Pediatric Surgery, G. Gaslini Children Hospital, Genoa, Italy
Mesenchymal stem cells (MSCs) may be of value in regeneration of renal tissue after damage; however, lack of biological knowledge and variability of results in animal models limit their utilization. We studied the effects of MSCs on podocytes in vitro and in vivo utilizing adriamycin (ADR) as a model of renal toxicity. The in vivo experimental approach was carried out in male Sprague-Dawley rats (overall 60 animals) treated with different ADR schemes to induce acute and chronic nephrosis. MSCs were given a) concomitantly to ADR in tail vein or b) in aorta and c) in tail vein 60 days after ADR. Homing was assessed with PKH26-MSCs. MSCs rescued podocytes from apoptosis induced by ADR in vitro. The maximal effect (80% rescue) was obtained with MSCs/podocytes coculture ratio of 1:1 for 72 h. All rats treated with ADR developed nephrosis. MSCs did not modify the clinical parameters (i.e., proteinuria, serum creatinine, lipids) but protected the kidney from severe glomerulosclerosis when given concomitantly to ADR. Rats given MSCs 60 days after ADR developed the same severe renal damage. Only a few MSCs were found in renal tubule-interstitial areas 1-24 h after injection and no MSCs were detected in glomeruli. MSCs reduced apoptosis of podocytes treated with ADR in vitro. Early and repeated MSCs infusion blunted glomerular damage in chronic ADR-induced nephropathy. MSCs did not modify proteinuria and progression to renal failure, which implies lack of regenerative potential in this model.
Key words: Mesenchymal stem cells (MSCs); Adriamycin; Podocytes; Nephrotic syndrome; Regeneration; Kidney
Address correspondence to Gian Marco Ghiggeri, M.D., Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Largo G. Gaslini, 5. 16148 Genova, Italy. Tel: +39-010-380742; Fax: +39-010-395214; E-mail: firstname.lastname@example.org
*Alberto Magnasco and Mirko Corselli equally contributed as first authors.
Clonal Analysis of Hematopoiesis-Supporting Activity of Human Mesenchymal Stem Cells in Association With Jagged1 Expression and Osteogenic Potential
Satoshi Fujita,1,2 Junya Toguchida,3 Yutaka Morita,4 and Hiroo Iwata1
1Department of Reparative Materials, Institute for Frontier
Medical Sciences, Kyoto University, Kyoto 615-8211, Japan
2Regenerative Medicine Research Center, Itabashi Chuo Medical Center, Kobe 650-0047, Japan
3Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 615-8211, Japan
4Department of Obstetrics and Gynecology, Itabashi Chuo Medical Center, Tokyo 174-0051, Japan
Human mesenchymal stem cells (hMSCs) are promising feeder cells for expanding hematopoietic stem cells (HSCs), but their potential is heterogeneous. We examined the hematopoiesis-supporting activity of hMSC at the clonal level in relation to the osteogenic potential and gene expression. Hematopoiesis-supporting activities of stably immortalized clonal hMSC lines were evaluated by the expansion of CD34+CD38- cells after 7-day coculture with human cord blood-derived CD34+ cells. Six of 16 clones expanded the numbers of CD34+CD38- cells >500-fold. These hematopoiesis-supportive clones also showed high gene expression of Jagged1, a Notch ligand, as well as high potential to deposit calcium after osteogenic induction. Thus, osteogenic hMSC clones may provide proper microenvironments for HSC expansion, ultimately conveying self-renewal signals to HSCs via the Notch pathway. However, they lost hematopoiesis-supporting activity after osteogenic differentiation. The hematopoiesis-supportive clones are potentially useful for hematopoietic microenvironment studies and as components of a coculture system for expansion of HSCs, free from contamination by xenogeneic pathogens.
Key words: Hematopoietic stem cell; Mesenchymal stem cell; Expansion; Umbilical cord blood; Cell transplantation
Address correspondence to Hiroo Iwata, Ph.D., Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel/Fax: +81-75-751-4119; E-mail: email@example.com
Extracellular Matrix Stability of Primary Mammalian Chondrocytes and Intervertebral Disc Cells Cultured in Alginate-Based Microbead Hydrogels
S. A. Abbah,* W. W. Lu, S. L. Peng, D. M. K. Aladin, Z. Y. Li, W. K. Tam, K. M. C. Cheung, K. D. K. Luk, and G. Q. Zhou
Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong SAR, China
Three-dimensional alginate constructs are widely used as carrier systems for transplantable cells. In the present study, we evaluated the chondrogenic matrix stability of primary rat chondrocytes and intervertebral disc (IVD) cells cultured in three different alginate-based microbead matrices to determine the influence of microenvironment on the cellular and metabolic behaviors of chondrogenic cells confined in alginate microbeads. Cells entrapped in calcium, strontium, or barium ion gelled microbeads were monitored with the live/dead dual fluorescent cell viability assay kit and the 1,9-dimethylmethylene blue (DMB) assay designed to evaluate sulfated glycosaminoglycan (s-GAG) production. Expression of chondrogenic extracellular matrix (ECM) synthesis was further evaluated by semiquantitative RT-PCR of sox9, type II collagen, and aggrecan mRNAs. Results indicate that Ca and Sr alginate maintained significantly higher population of living cells compared to Ba alginate (p < 0.05). Production of s-GAG was similarly higher in Ca and Sr alginate microbead cultures compared to Ba alginate microbeads. Although there was no significant difference between strontium and calcium up to day 14 of culture, Sr alginate showed remarkably improved cellular and metabolic activities on long-term cultures, with chondrocytes expressing as much as 31% and 44% greater s-GAG compared to calcium and barium constructs, respectively, while IVD cells expressed 63% and 74% greater s-GAG compared to calcium and barium constructs, respectively, on day 28. These findings indicate that Sr alginate represent a significant improvement over Ca- and Ba alginate microbeads for the maintenance of chondrogenic phenotype of primary chondrocytes and IVD cells.
Key words: Alginate microbeads; Cell encapsulation; Chondrogenic extracellular matrix; Chondrocytes; Intervertebral disc cells
Address correspondence to Dr. Guang-Qian Zhou, Department of Orthopaedics and Traumatology, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China. Tel: 00852 2819 9589; Fax: 00852 2818 9210; E-mail: firstname.lastname@example.org
*Present address: Department of Orthopaedic Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore 117510.
The Fate of Implanted Syngenic Muscle Precursor Cells in Injured Striated Urethral Sphincter of Female Rats
Anne-Sophie Biérinx and Alain Sebille
Faculté de Médecine, Atelier de Régénération Neuromusculaire, Université Pierre et Marie Curie-Paris 6, 75012 Paris, France
We studied the outcome of syngenic skeletal muscle precursor cells (MPCs) implanted in the striated urethral sphincter of the female rat. These cells were injected at the site of a longitudinal sphincterotomy performed 21 days before implantation. MPCs were isolated from the striated hindlimb muscles of syngenic adult rats and were infected with a retrovirus carrying the gene for either the green fluorescent protein (GFP) or the b-galactosidase enzyme (b-gal). MPCs (2 × 105) were injected longitudinally at the site of the lesion in 48 animals using a 10-ml Hamilton syringe. Then the whole urethras were excised from 2 h up to 90 days for cross section immunocytochemistry analysis. All the urethras exhibited connective tissue in place of the injury of the striated fibers. Two hours after injection a cluster of small round basophilic cells was observable at the site of injection and some of them expressed GFP or b-gal. A few GFP- and b-gal-positive cells were already detectable 7 days after injection. A large amount of injected cells probably died after injection. Many striated fibers of the urethra became GFP positive from day 7 until day 21, suggesting that few MPCs were allowed to incorporate the divided extremities of the striated fibers from day 7. Unfortunately, we did not observe centronucleated regenerated fibers in this experiment.
Key words: Muscle precursor cells (MPCs); Urethral sphincter; Sphincteric deficiency; Grafted MPCs
Address correspondence to Anne-Sophie Biérinx at her current address: Randall Division, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK.
Comparison of Autologous Full-Thickness Gingiva and Skin Substitutes for Wound Healing
Abraham P. Vriens,1 Taco Waaijman,1 Henk M. van den Hoogenband,1 Edith M. de Boer,1 Rik J. Scheper,2 and Susan Gibbs1
1Department of Dermatology, VU University Medical Centre,
1081 HV Amsterdam, The Netherlands
2Department of Pathology, VU University Medical Centre, 1081 HV Amsterdam, The Netherlands
Ideally tissue-engineered products should maintain the characteristics of the original tissue. For example, skin represents orthokeratinized epithelium and oral gingiva represents parakeratinized epithelium. The aim of this study was to develop an autologous full-thickness gingiva substitute suitable for clinical applications and to compare it with our autologous full-thickness skin substitute that is routinely used for healing chronic wounds. Autologous full-thickness skin and gingiva substitutes were constructed under identical culture conditions from 3-mm punch biopsies isolated from the upper leg or gingiva tissue, respectively. Both consisted of reconstructed epithelia on acellular dermis repopulated with fibroblasts. To compare the characteristics of the original and reconstructed tissue, differential morphological observations and expression of differentiation markers (keratins 6, 10, and 17 and stratum corneum precursors involucrin, loricrin, and SKALP) were determined. Skin and gingiva substitutes were transplanted onto therapy-resistant leg ulcers or tooth extraction sites in order to determine their effects on wound healing. The tissue-engineered constructs maintained many of the differential histological and immunohistochemical characteristics of the original tissues from which they were derived. The skin substitute was orthokeratinized, and the gingiva substitute was parakeratinized. Transplantation of skin (n = 19) and gingiva substitutes (n = 3) resulted in accelerated wound healing with no adverse effects. As identical culture systems were used to generate both the skin and gingiva substitutes, the differences observed in tissue (immuno)histology can be attributed to intrinsic properties of the tissues rather than to environmental factors (e.g., air or saliva). This study emphasizes the importance of closely matching donor sites with the area to be transplanted. Our results represent a large step forward in the area of clinical applications in oral tissue engineering, which have until now greatly lagged behind skin tissue engineering.
Key words: Tissue engineering; Skin; Gingiva; Clinical application
Address correspondence to Susan Gibbs, Ph.D., Department of Dermatology, VU University Medical Center, De Boelelaan 1118, 1081 HV Amsterdam, The Netherlands. Tel: 0031 20 4442815; Fax: 0031 20 4442816; E-mail: email@example.com
Functional Assessment of the Quality of Human Hepatocyte Preparations for Cell Transplantation
María Teresa Donato,1,2,3 Agustín Lahoz,4 Sandra Montero,4 Ana Bonora,2,3 Eugenia Pareja,5 José Mir,5 José V. Castell,1,2,3 and María José Gómez-Lechón2,3
1Departamento de Bioquímica y Biología Molecular,
Facultad de Medicina, Universidad de Valencia, Valencia, Spain
2Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Valencia, Spain
3CIBERHEPAD, FIS, Spain
4Unidad Analítica Mixta, Hospital La Fe-Advancell, Valencia, Spain
5Unidad de Transplante Hepático, Hospital La Fe, Valencia, Spain
Hepatocyte transplantation is an alternative therapy to orthotopic liver
transplantation for the treatment of liver diseases. Good quality freshly
isolated or cryopreserved human hepatocytes are needed for clinical transplantation.
However, isolation, cryopreservation, and thawing processes can seriously
impair hepatocyte viability and functionality. The aim of the present study
was to develop a fast and sensitive procedure to estimate the quality of
hepatocyte preparations prior to clinical cell infusion. To this end, cell
viability, attachment efficiency, and metabolic competence (urea synthesis
and drug-metabolizing P450 activities) were selected as objective criteria.
Viability of hepatocyte suspension was estimated by trypan blue staining.
DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated
dishes was quantified to estimate adherence capacity. Urea production was
determined after incubating hepatocyte suspensions with 2 mM ClNH4
for 30 min. The cytochrome P450 function was assayed by a 30-min incubation
of hepatocyte suspension with a cocktail mixture containing selective substrates
for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1,
and 3A4). The assay can be applied to both freshly isolated and cryopreserved
hepatocyte suspensions, and the results are available within 1 h, which
could help to make short-term decisions: 1) to assess the suitability for
cell transplantation of a preparation of freshly isolated hepatocytes or
a particular batch of thawed cells, or 2) to estimate the convenience of
particular cell preparation.
Key words: Functional markers; Hepatocyte transplantation; Urea synthesis; P450 activities
Address correspondence to María José Gómez-Lechón, Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Avda de Campanar 21, 46009-Valencia, Spain. Tel: +34 96 1973048; Fax: +34 96 1973018; E-mail: firstname.lastname@example.org
Proliferation of Hepatocyte Progenitor Cells Isolated From Adult Human Livers in Serum-Free Medium
Kazunori Sasaki,1,2 Junko Kon,1 Toru Mizuguchi,2 Qijie Chen,1 Hidekazu Ooe,1 Hideki Oshima,1,2 Koichi Hirata,2 and Toshihiro Mitaka1
1Department of Pathophysiology, Cancer Research Institute,
Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan
2Department of Surgery I, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan
Rat small hepatocytes (SHs) are committed progenitor cells that can differentiate into mature hepatocytes and can selectively proliferate in serum-free medium when they are cultured on hyaluronic acid (HA)-coated dishes. In this study we examined the separation of human SHs from adult human livers. We obtained liver tissues from the resected liver of 16 patients who underwent hepatic resections. Extracted liver specimens were clearly separate from the tumor regions with sufficient margins. Hepatic cells were isolated using the modified method of two-step collagenase perfusion. A low-speed centrifugation was performed and cells in the supernatant were finally cultured on HA-coated dishes in serum-free DMEM/F12 medium including nicotinamide, EGF, and HGF. Small-sized hepatocytes selectively proliferated to form colonies and many colonies continued growing for more than 3 weeks. The average number of cells in a colony was 38.6 ± 18.0, 79.0 ± 54.0, and 101.5 ± 115.7 at day 7, 14, and 21, respectively. About 0.04% of plated cells could form an SH colony. Immunocytochemistry showed that the cells forming a colony were positive for albumin, transferrin, keratin 8, and CD44. The results of RT-PCR showed that colony-forming cells expressed albumin, transferrin, a1-antitrypsin, fibrinogen, glutamine synthetase, many cytochrome P450s, and liver-enriched transcription factors (HNF3a, HNF4a, C/EBPa, and C/EBPb). Furthermore, the cells expressed not only the genes of hepatic differentiated functions but also those of both hepatic stem cell marker (Thy1.1, EpCAM, AFP) and SH marker (CD44, D6.1A, BRI3). Albumin secretion into culture medium was also observed. Our results demonstrate the existence of hepatocyte progenitor cells in human adult livers, and the cells can grow in a serum-free medium on HA-coated dishes. Human SHs may be a useful source for cell transplantation as well as pharmaceutical and toxicological investigations.
Key words: Small hepatocytes; Hyaluronic acid; Hepatic stem cells; CD44; Hepatic differentiated functions
Address correspondence to Toshihiro Mitaka, M.D., Ph.D., Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, S-1, W-17, Chuo-Ku, Sapporo 060-8556, Japan. Tel: +81-11-611-2111, ext. 2390; Fax: +81-11-615-3099; E-mail: email@example.com
A New Experimental Protocol for Preferential Differentiation of Mouse Embryonic Stem Cells Into Insulin-Producing Cells
Ortwin Naujok,1* Flavio Francini,1* Sally Picton,2 Anne Jörns,1,3 Clifford J. Bailey,2 and Sigurd Lenzen1
1Institute of Clinical Biochemistry, Hannover Medical School,
2School of Life and Health Sciences, Aston University, Birmingham, UK
3Center of Anatomy, Hannover Medical School, Hannover, Germany
Mouse embryonic stem (ES) cells have the potential to differentiate into insulin-producing cells, but efficient protocols for in vitro differentiation have not been established. Here we have developed a new optimized four-stage differentiation protocol and compared this with an established reference protocol. The new protocol minimized differentiation towards neuronal progeny, resulting in a population of insulin-producing cells with b-cell characteristics but lacking neuronal features. The yield of glucagon and somatostatin cells was negligible. Crucial for this improved yield was the removal of a nestin selection step as well as removal of culture supplements that promote differentiation towards the neuronal lineage. Supplementation of the differentiation medium with insulin and fetal calf serum was beneficial for differentiation towards monohormonal insulin-positive cells. After implantation into diabetic mice these insulin-producing cells produced a time-dependent improvement of the diabetic metabolic state, in contrast to cells differentiated according to the reference protocol. Using a spinner culture instead of an adherent culture of ES cells prevented the differentiation towards insulin-producing cells. Thus, prevention of cell attachment in a spinner culture represents a means to keep ES cells in an undifferentiated state and to inhibit differentiation. In conclusion, this study describes a new optimized four-stage protocol for differentiating ES cells to insulin-producing cells with minimal neuronal cell formation.
Key words: Embryonic stem cells; Differentiation; Insulin; Diabetes
Address correspondence to Prof. Sigurd Lenzen, Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany.
*These two authors contributed equally to this work.
Prolonged Survival of Microencapsulated Neonatal Porcine Islet Xenografts in Immune-Competent Mice without Antirejection Therapy
Tsunehiro Kobayashi,1,2* Hossein Arefanian,1,3* George Harb,1 Eric B. Tredget,1 Ray V. Rajotte,1 Gregory S. Korbutt,1 and Gina R. Rayat1
1Surgical-Medical Research Institute, Department of Surgery,
University of Alberta, Edmonton, Alberta, Canada
2Department of Surgery, Nara Medical University, Nara, Japan
3Endocrinology and Metabolism Research Center, Dr. Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
Several studies have demonstrated that in vitro culture of islets prolonged islet graft survival in immunecompetent mice without administration of antirejection drugs. However, we recently showed that in vitro cultured microencapsulated neonatal porcine islets (NPI) were rejected in immune-competent mice not receiving antirejection therapy. The aim of this study was to determine whether culture of microencapsulated NPI in vivo could promote long-term survival of microencapsulated NPI in immune-competent mice without administration of antirejection drugs. Microencapsulated NPI that were cultured in vitro for 7 and 50 days or transplanted initially in immune-deficient C.B.-17 SCID-BEIGE mice for 100 days (in vivo cultured) were characterized and transplanted into streptozotocin-induced diabetic immune-competent BALB/c mice. Day 50 in vitro cultured and day 100 in vivo cultured microencapsulated NPI showed significantly higher insulin and DNA content, indicating maturation of NPI compared to day 7 in vitro cultured microencapsulated NPI. Interestingly, in vivo cultured microencapsulated NPI expressed lower levels of porcine antigens compared to day 7 and day 50 in vitro cultured microencapsulated NPI. Transplantation of day 7 in vitro cultured microencapsulated NPI did not reverse diabetes in immune-competent BALB/c mouse recipients. In contrast, transplantation of day 50 in vitro cultured and in vivo cultured microencapsulated NPI into diabetic immune-competent BALB/c mice resulted in the immediate reversal of hyperglycemia within 2 days posttransplantation. However, all recipients of day 50 in vitro cultured microencapsulated NPI eventually rejected their grafts by day 15 posttransplantation, while 6 of 10 BALB/c mouse recipients of in vivo cultured microencapsulated NPI maintained normoglycemia for 100 days posttransplantation. These results show that in vivo culture of NPI in immune-deficient mice results in the modulation of NPI, which allows for their long-term survival in immune-competent mice without antirejection therapy.
Key words: Islet transplantation; Type 1 diabetes; Neonatal porcine islets; Microencapsulation; Immune modulation; Maturation
Address correspondence to Dr. Gina R. Rayat, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Tel: (780) 492-6894; Fax: (780) 492-1627; E-mail: firstname.lastname@example.org
*These authors contributed equally to this work.
Cell Surface Modification by Activated Polyethylene Glycol Prevents Allosensitization After Islet Transplantation
Yu-Mee Wee,1 Dong-Gyun Lim,1 Yang-Hee Kim,1 Jin-Hee Kim,1 Song-Cheol Kim,1 EunSil Yu,2 Myung-Ok Park,3 Monica Young Choi,1 Youn-Hee Park,1 Hyuk-Jai Jang,1 Eun-Young Cho,1 Myung-Hwan Cho,4 and Duck-Jong Han1
1Department of Surgery, Ulsan University College of Medicine
& Asan Medical Center, Seoul, 138-736, Korea
2Department of Pathology, Ulsan University College of Medicine & Asan Medical Center, Seoul, 138-736, Korea
3BiopolyMed Inc, Seoul, 136-701, Korea
4Department of Biological Science, Konkuk University, Seoul, 143-701, Korea
The necessity to transplant islet tissue without the need for immunosuppressant therapy has led to the development of materials for immune modulation. Pegylation makes islets antigenically silent, protecting them from the adsorption of foreign protein and thus avoiding immune injury. The aim of this study is to determine whether pegylation of islets prolongs islet survival and function both during tissue culture and posttransplantation. We used cyanuric chloride-activated methoxy-polyethylene glycol for cell surface modification. To detect the pegylation effect on splenocytes, we measured antibody binding inhibition and abrogation of lymphocyte proliferation. To detect the pegylation effect on islet grafts, we performed rodent islet transplantation. Islet viability and function were maintained after pegylation. Pegylated islets showed a 90% decrease in antibody binding and decreased lymphocyte proliferation in a mixed lymphocyte culture. However, when pegylated islets were transplanted, no prolongation of graft survival was observed. When a subtherapeutic dose of immunosuppressant was given at the time of transplantation of pegylated islets, islet graft survival was significantly prolonged. In addition, when rats were sensitized with donor splenocytes, graft survival was prolonged by pegylation. We observed that pegylation of islets, combined with a subtherapeutic dose of immunosuppressant, protects the graft from rejection. Prolonged graft survival in sensitized recipients showed that pegylation of islets shifted the pattern of rejection from an acute humoral response to a less aggressive cellular alloresponse.
Key words: Islet transplantation; Sensitization; Polyethylene glycol; Immunohistochemistry
Address correspondence to Duck-Jong Han, M.D., Department of Surgery, Ulsan University College of Medicine & Asan Medical Center, 388-1, Pungnab-dong, Songpa-gu, Seoul, 138-736, Korea. Tel: 82-2-3010-3487; Fax: 82-2-3010-4182; E-mail: email@example.com
Beneficial Effect of the Nonpeptidyl Low Molecular Weight Radical Scavenger IAC on Cultured Human Islet Function
Rita Mancarella,1 Silvia Del Guerra,1 Matilde Masini,2 Marco Bugliani,1 Luca Valgimigli,3 Gian Franco Pedulli,3 Moreno Paolini,4 Donatella Canistro,4 Andrea Armando,5 Antonio Soleti,5 Franco Filipponi,6 Franco Mosca,6 Ugo Boggi,6 Stefano Del Prato,1 Piero Marchetti,1 and Roberto Lupi1
1Department of Endocrinology and Metabolism, Metabolic Unit,
University of Pisa, Pisa, Italy
2Department of Experimental Pathology, University of Pisa, Pisa, Italy
3Department of Organic Chemistry "A. Mangini", University of Bologna, Bologna, Italy
4Department of Pharmacology, Molecular Toxicology Unit, Alma Mater Studiorum, University of Bologna, Bologna, Italy
5Medestea Research, Medestea, Torino, Italy
6Department of Oncology and Transplant Surgery, University of Pisa, Pisa, Italy
We examined a possible protective effect of the nonpeptidyl low molecular weight radical scavenger IAC [bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decanedioate di-hydrochloride] on isolated human islet cells against isolation and culture oxidative stress. Islets isolated from pancreases of nondiabetic multiorgan donors by collagenase digestion were purified by density gradient centrifugation. After the isolation, islets were either exposed or not exposed for 7 days to 10 mmol/L IAC. We found that IAC markedly reduced oxidative stress and ameliorated islets function. These results suggest that the use of IAC could be an interesting pharmacological approach for the treatment of the islets before transplantation.
Key words: Human pancreatic islet; Oxidative stress; Antioxidants; Islet transplantation
Address correspondence to Dr. Roberto Lupi, BHS, Department of Endocrinology and Metabolism, Metabolic Unit, Cisanello Hospital, via Paradisa 2, 56100 Pisa, Italy. Tel: +39050995134; Fax: +39050541521; E-mail: firstname.lastname@example.org