ognizant Communication Corporation


VOLUME 12, NUMBER 1, 2003

Cell Transplantation, Vol. 12, pp. 1-11, 2003
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Generation of Hepatocyte-Like Cells From Human Embryonic Stem Cells

Lakshmi Rambhatla, Choy-Pik Chiu, Pratima Kundu, Yun Peng, and Melissa K. Carpenter

Geron Corporation, 230 Constitution Drive, Menlo Park, CA 94025

Use of human hepatocytes for therapeutic and drug discovery applications is hampered by limited tissue source and the inability of hepatocytes to proliferate and maintain function long term in vitro. Human embryonic stem (hES) cells are immortal and pluripotent and may provide a cell source for functional human hepatocytes. We report here that hES cells can be induced to differentiate into hepatocyte-like cells. Treatment with sodium butyrate induced hepatic differentiation as well as significant cell death, resulting in approximately 10-15% yield of a homogeneous population of cells. The differentiated cells have morphological features similar to that of primary hepatocytes and 70-80% of the cells express liver-associated proteins (albumin, alpha-1-antitrypsin, cytokeratin 8 and 18), accumulate glycogen, have inducible cytochrome P450 activity, and do not express alpha-fetoprotein. Because of the inherent proliferative capacity of hES cells, these cells may provide a reliable source of normal human hepatocytes for research and transplantation.

Key words: Human embryonic stem cells; In vitro differentiation; Hepatocytes; Cell therapy

Address correspondence to Melissa K. Carpenter, Geron Corporation, 230 Constitution Drive, Menlo Park, CA 94025. Tel: (650) 473-7771; Fax: (650) 473-7750; E-mail: mcarpenter@geron.com

Cell Transplantation, Vol. 12, pp. 13-25, 2003
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Functional and Immunohistochemical Evaluation of Porcine Neonatal Islet-Like Cell Clusters

T. B. Nielsen,1 K. B. Yderstraede,1 H. D. Schrøder,2 Jens Juul Holst,4 Klaus Brusgaard,3 and H. Beck-Nielsen1

1Department of Medical Endocrinology, 2Department of Pathology, and 3Department of Clinical Biochemistry, Odense University Hospital, Denmark
4Department of Medical Physiology, University of Copenhagen, Denmark

Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transplantation to athymic nude mice. On average we obtained 29,000 NICCs from each pancreas. In a perifusion system, NICCs responded poorly to a glucose challenge alone, but 10 mmol/L arginine elicited a fourfold increase in insulin secretion and 16.7 mmol/L glucose + 10 mmol/L arginine caused a sevenfold increase in insulin secretion, indicating some sensitivity towards glucose. Hormone content as well as the number of hormone-containing cells increased for the first 14 days of culture. When NICCs were stained for hormones, proliferation (Ki67), and duct cells (CK7), some insulin- and glucagon-positive cells co-stained for proliferation. However no co-staining was observed between insulin- and glucagon-positive cells or between hormone- and CK7-positive cells. Following transplantation of 2000 NICCs under the renal capsule of diabetic nude mice, BG levels were normalized within an average of 13 weeks. Oral and IP glucose tolerance tests revealed a normal or even faster clearance of a glucose load compared with normal controls. Immunohistochemical examination of the grafts revealed primarily insulin-positive cells. In summary, in vitro, NICCs responded to a challenge including glucose and arginine. There was a potential for expansion of the b-cell mass of NICCs in vitro as well as in vivo where NICCs eventually may normalize blood glucose of diabetic mice.

Key words: Perifusion; Islet transplantation; Xenotransplantation; Neonatal pigs; Islet-like cell clusters; b-Cell expansion

Address correspondence to Thomas Buschmann Nielsen, Department of Endocrinology M, Odense University Hospital, Sdr. Boulevard 29, DK 5000 Odense C, Denmark. Tel: +45 6591 4538; Fax: + 45 6590 6821; E-mail: thnielsen@health.sdu.dk

Cell Transplantation, Vol. 12, pp. 27-32, 2003
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Activation of Macrophage-Associated Molecules After Brain Death in Islets

Hirochika Toyama, Moriatsu Takada, Yasuyuki Suzuki, and Yoshikazu Kuroda

Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kobe University, Kobe, Japan

Islet transplantation is now established as an optional treatment for type I diabetes. However, rates of insulin independence in islet transplant recipients are still low. Although the major source of allograft is derived from brain-dead patient, the nonphysiologic state of brain death (BD) deteriorates organs such as liver and kidney. To determine the effects of BD on islets, a rodent model of BD has been used. Histologically, islets of BD rats showed decreased permeability and impaired integrity of the cell membranes. Flow cytometric analysis showed that CD11b/c-positive cells within islets were slightly increased in BD. This result suggests that BD induces macrophage infiltration into the islets. Moreover, RT-PCR revealed significant augmentation of macrophages-associated inflammatory molecules (IL-1b, IL-6, TNF-a, and MCP-1) in islets from a BD donor. Inducible nitric oxide synthase (iNOS) was weakly expressed, although not reaching statistical significance compared with control. Our results indicate that islets from a BD donor are immunologically activated and have a potential risk factor for early graft loss and a poor long-term function of grafts in clinical setting of islet transplantation. Immunomodulation, to eliminate intraislet immunocytes and/or activated macrophage-associated molecules, might be necessary for the better outcome after islet graft from BD donors.

Key words: Islet of Langerhans; Brain death; Intraislet cytokines; Islet-infiltrating immunocyte; Immunogenicity

Address correspondence to Moriatsu Takada, M.D., Ph.D., Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kobe University, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel: +81-78-382-5925; Fax: +81-78-382-5939; E-mail: moriatsu@med.kobe-u.ac.jp

Cell Transplantation, Vol. 12, pp. 33-41, 2003
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Areal Density Measurement Is a Convenient Method for the Determination of Porcine Islet Equivalents Without Counting and Sizing Individual Islets

N. Lembert,1,2 J. Wesche,1 P. Petersen,2 M. Doser,3 H. D. Becker,2 and H. P. T. Ammon1

1Department of Pharmacology, Auf der Morgenstelle 8, University of Tübingen, 72076 Tübingen, Germany
2Department of General Surgery, Hoppe-Seyler Straße 3, University of Tübingen, 72076 Tübingen, Germany
3Institute of Textile and Process Engineering (ITV), Stuttgart-Denkendorf, 73770 Denkendorf, Germany

The determination of islet mass is important for the normalization of islet experiments in the laboratory and for the precise dosing of islets for transplantation. The common microscopical analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into islet equivalents (IEQ), which is the volume of a spherical islet with a diameter of 150 mm. However, islets are of irregular form, which makes this determination user dependent, and the analysis is irreproducible once the original sample is discarded. This routine technique of islet quantification was compared with the analysis of areal density measurements. It was assumed that the entire area occupied by islets can be expressed in IEQ without sizing and counting individual islets. Porcine islets were isolated by continuous digestion/filtration and purified by gradient centrifugation. Purified islets were stained with dithizone and were repeatedly pictured under the microscope with random area selection. A total of 51 pictures was taken from 11 different purifications and stained islets were detected by digital image analysis. The correlation coefficient (r) between both analyses was 0.977 with an underestimation of islet yield by areal density detection (slope: 0.75 ± 0.03). Areal density analysis per picture took about 1 min, which is about 10 times faster than the traditional method without increasing the method error (CV 2.1% vs. 2.7%). In summary, areal density measurements allow a rapid and reproducible estimation of IEQ without counting individual islets. It can be performed in a single step analysis without computer programming and is valuable for online determinations of islet yield preceding transplantation.

Key words: Porcine islets; Islet quantification; Islet transplantation; Area density; Bioartificial pancreas

Address correspondence to Dr. Nicolas Lembert, Department of Pharmacology, Auf der Morgenstelle 8, University of Tübingen, 72076 Tübingen, Germany. Tel: +49 7071 29 72471; E-mail: nicolas.lembert@uni-tuebingen.de

Cell Transplantation, Vol. 12, pp. 43-49, 2003
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Hepatocyte Transplantation in the Treatment of Acute Liver Failure: Microencapsulated Hepatocytes Versus Hepatocytes Attached to an Autologous Biomatrix

Giovanni Ambrosino,1 Sergio Varotto,1 Stefano M. M. Basso,1 Attilio Cecchetto,2 Paolo Carraro,3 Agostino Naso,4 Giustina De Silvestro,5 Mario Plebani,3 Giovanni Abatangelo,7 Daniele Donato,8 Andriano Cestrone,8 Gianpiero Giron,6 and Davide F. D'Amico1

1Department of Surgical and Gastroenterologic Sciences, Liver Transplant Unit, School of Medicine, University of Padova, Via Giustiniani 2, 35128 Padova, Italy
2Department of Pathology, University of Padova, Via A. Gabelli 61, 35128 Padova, Italy
3Laboratory of Chemical Analysis, 4Department of Nephrology, and 5Transfusional Unit, University of Padova, Via Giustiniani 2, 35128 Padova, Italy
6Department of Anesthesiology, University of Padova, Via C. Battisti 267, 35128 Padova, Italy
7Department of Histology and Tissue Engineering, University of Padova, Viale G. Colombo 3, 35128 Padova, Italy
8Medical Office, Padova University Hospital

A liver transplant is considered today to be the only effective therapeutic solution for many otherwise intractable hepatic disorders. However, liver transplantation is beset by shortage of donors. Over the years, many liver support systems have been developed to supply the liver functions, mostly as a bridge to transplantation. Transplantation of isolated hepatocytes (HcTx) instead of whole liver has constituted one of the most appealing possibilities to treat several diseases. We compared two different models of HcTx in a surgical model of acute liver failure in pigs, using microencapsulated hepatocytes (MHcTx) and hepatocytes attached to a porcine biomatrix (PBMHcTx), both transplanted into peritoneum. The collected data were survival, laboratory findings, hemodynamic parameters, light microscopy, histology, MTT, and glycogen content. The group with PBMHcTx has a better outcome than the group with MHcTx (p < 0.05). Histology showed normal morphology of the hepatocytes, high glycogen content, 75% viability, positive MTT, and 95% adhesion of the hepatocytes to the biomatrix. Our biomatrix (PBM) provides cell-to-cell contact and interaction with extracellular matrix, which have been shown to play major roles in hepatocyte survival and physiologic regulation of gene expression, and guarantee a prompt engraftment and an adequate neovascularization. PBMHcTx is a useful method to treat acute liver failure and it indicates a possible liver-direct gene therapy in the treatment of inherited and acquired disorders.

Key words: Hepatocytes transplantation; Model of acute liver failure; Microencapsulated hepatocytes; Porcine autologous biomatrix; Biomatrix; Bioreactor

Address correspondence to Giovanni Ambrosino, M.D., Department of Surgical and Gastroenterologic Sciences, Liver Transplantation Unit, School of Medicine, University of Padova, via Giustiniani 2, 35128 Padova, Italy. E-mail: giovanni.ambrosino@unipd.it

Cell Transplantation, Vol. 12, pp. 51-58, 2003
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Efficacy of a Polyurethane Foam/Spheroid Artificial Liver by Using Human Hepatoblastoma Cell Line (Hep G2)

J. Fukuda,1 K. Okamura,1 K. Nakazawa,3 H. Ijima,1 Y. Yamashita,2 M. Shimada,2 K. Shirabe,2 E. Tsujita,2 K. Sugimachi,2 and K. Funatsu1

1Department of Chemical Engineering, Faculty of Engineering, and 2Department of Surgery and Science, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan
3Department of Chemical Processes and Environments, The University of Kitakyushu, Kitakyushu, Japan

We investigated the availability of human hepatoblastoma cell line (Hep G2), compared with human primary hepatocytes (HH) and porcine primary hepatocytes (PH), as a cell source for the hybrid artificial liver support system (HALSS) by using polyurethane foam (PUF). All three kinds of hepatocytes spontaneously formed spherical multicellular aggregates (spheroids) of 100-200 mm diameter in the pores of PUF within 3 days of culture. In a PUF stationary culture, Hep G2 spheroids recovered the ammonia removal activity that was lost in monolayer culture, although the removal for each unit cell number was about one tenth that of HH spheroids and about one eighth of PH spheroids. The synthesis activities of albumin and fibrinogen of each unit cell number of Hep G2 were also upregulated by PUF spheroid culture, and were about twice as high as in monolayer culture. The albumin secretion activity of Hep G2 spheroids was almost the same as that of PH spheroids. HH scarcely secreted these proteins in this experiment, probably because they were cultured in a serum-free medium. In the PUF module in a circulation culture, HH had high ammonia removal and low synthesis activities similar to stationary culture. Hep G2 proliferated to a high cell density, such as about 4.8 x 107 cells/cm3-module at 10 days of culture. Although Hep G2 spheroids had low ammonia removal activity in each cell, the removal rate in the PUF module was almost the same as for PH at 7 days of culture because of the high cell density culture by cell proliferation. The albumin secretion rate by Hep G2 in the PUF module also increased with cell proliferation and was about 10 times higher than the initial rate for PH at 7 days of culture. These results suggest that Hep G2 is a potential cell source for the PUF-HALSS.

Key words: Hybrid artificial liver; Spheroids; Polyurethane foam; Hep G2; Human primary hepatocytes; Porcine primary hepatocytes

Address correspondence to Kazumori Funatsu, Ph.D., Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel: 81-92-642-3512; Fax: 81-92-642-3513; E-mail: funatsu@apex.chem-eng.kyushu-u.ac.jp

Cell Transplantation, Vol. 12, pp. 59-68, 2003
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Induction of Necrosis and DNA Fragmentation During Hypothermic Preservation of Hepatocytes in UW, HTK, and Celsior Solutions

Salomon L. Abrahamse,1 Pieter van Runnard Heimel,1 Robin J. Hartman,1 Rob A. F. M. Chamuleau,2 and Thomas M. van Gulik1

Departments of 1Surgery (Surgical Laboratory) and 2Experimental Hepatology, Academic Medical Center, The University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4°C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not enhanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.

Key words: Hepatocytes; Transplantation; Preservation; Liver; Necrosis; Apoptosis

Address correspondence to Prof. Dr. Thomas M. van Gulik, Surgical Laboratory IWO 1-153, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: ++-31205666653; Fax: ++-31206976621; E-mail: t.m.vangulik@amc.uva.nl

Cell Transplantation, Vol. 12, pp. 69-74, 2003
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Human Hepatocyte Isolation and Relationship of Cell Viability to Early Graft Function

Ragai R. Mitry, Robin D. Hughes, Marion M. Aw, Claire Terry, Giorgina Mieli-Vergani, Raffaele Girlanda, Paolo Muiesan, Mohamed Rela, Nigel D. Heaton, and Anil Dhawan

Institute of Liver Studies, Guy's, King's and St. Thomas' School of Medicine, and King's College Hospital, Denmark Hill, London, UK

Hepatocyte transplantation is emerging as an additional modality of treatment for patients with acute liver failure or liver-based metabolic disorders. The procedure requires isolation of high-quality hepatocytes from unused donor livers. Hepatocytes were isolated from 20 donor livers (11 right lobes, 3 left lateral segments, 6 whole livers) using a collagenase perfusion technique. Cell viability (median 56%, range 13-95%) and yield (median 1.4 x 109 cells, range 2.0 x 106-1.8 x 1010 cells) varied according to the tissue available. Fatty livers rejected for transplantation gave lower cell viability (median 45%, range 25-59%). There was a significant correlation between age of donor (median 21 years, range 7-66 years) and viability of isolated hepatocytes in vitro (r = -0.683, p = 0.001). The 13 segments of livers were from reduced/split grafts used for clinical transplantation in 9 children and 4 adults. There was no significant correlation between in vitro cell viability and clinical parameters including intensive care stay, serum aspartate aminotransferase, and international normalized ratio (in the first 7 days), and allograft rejection or other early posttransplant complications, in patients transplanted with the corresponding tissue.

Key words: Hepatocyte transplantation; Collagenase perfusion; Steatosis; Graft function

Address correspondence to Dr. Anil Dhawan, Paediatric Liver Service, King's College Hospital, Denmark Hill, London SE5 9RS, UK. Tel: +44-20-73463214; Fax: +44-20-73463564; E-mail: anil.dhawan@kcl.ac.uk

Cell Transplantation, Vol. 12, pp. 75-82, 2003
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Tolerance and Immunity Following In Utero Transplantation of Allogeneic Fetal Liver Cells: The Cytokine Shift*

H. Sefrioui, J. Donahue, E. A. Gilpin, A. S. Srivastava, and E. Carrier

Department of Medicine, Pediatrics, and Family and Preventive Medicine, University of California San Diego School of Medicine

Although in utero transplantation (IUT) has resulted in donor-specific tolerance to postnatal solid organ transplantation, the mechanisms of this tolerance remain poorly understood. Our recent findings demonstrate that under specific conditions prenatal injection of allogeneic cells may lead to allosensitization instead of tolerance. These laboratory observations were supported by clinical findings as well, and therefore suggested that, depending on the conditions of prenatal transplantation, tolerance or immunity may develop. The present study explored the role of CD4 cells, cytokines, and I-E superantigen in developing tolerance vs. immunity after in utero transplantation. Sixteen animals survived IUT (40-60% survival rate) and were free from any signs of graft-versus-host disease (GVHD). Mice were considered tolerant when their antidonor and antihost CTL responses were similar, sensitized when antidonor responses were significantly higher than antihost and anti-third-party responses, and nontolerant when antidonor responses in transplanted and control mice were similar. The TH1->TH2 shift was associated with tolerance and TH2->TH1 shift with allosensitization. Our results showed that tolerant BALB/c (H-2d, I-E+)->C57BL/6 (H-2b, I-E-) (2/7) mice showed higher IL-4 (p < 0.05) in antidonor MLR, and partial deletion of recipient I-E-reactive T cells (CD3Vb11) (p < 0.045). On the other hand, nontolerant animals (5/7) demonstrated high production of IFN-g (p < 0.05) without deletion of CD3Vb11 T cells. In C57CBL/6 (H-2b, I-E-)->C3H (H-2k, I-E+) mice CD3Vb11 T cells do not play any role in tolerance induction because they are deleted in the C3H background. Tolerant mice (4/9) showed an overproduction of IL-4 (p < 0.05) in antidonor MLR whereas allosensitized animals (5/9) demonstrated high level of IFN-g (p < 0.05). Suppressor cells seem to play no role in tolerant C57BL/6->C3H as demonstrated by suppressor assay. Hence, a shift from TH1->TH2 or TH2->TH1 cytokines may determine whether tolerance or immunity develops.

Key words: In utero transplantation; Fetal liver cells; Tolerance; Allosensitization; I-E-reactive cells; Cytokines

Address correspondence to Ewa Carrier, M.D., University of California San Diego Blood and Marrow Transplantation Division, 9500 Gilman Drive, 0062, La Jolla, CA 92093-0062. Tel: (858) 657-6790; Fax: (858) 534-7340; E-mail: ecarrier@ucsd.edu.

*A part of this study was presented at the 2002 Tandem BMT meetings, February 22-25 in Orlando, FL.

Cell Transplantation, Vol. 12, pp. 83-90, 2003
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Donor Age and Gender Are the Strongest Predictors of Marrow Recovery From Cadaveric Vertebral Bodies

Helen Newman,1,4 Jo Anna Reems,1,2 Theodore H. Rigley,1 Daniel Bravo,1 and D. Michael Strong1,3,4

1Northwest Tissue Center & Puget Sound Blood Center, 921 Terry Avenue, Seattle, WA 98104
University of Washington, Division of Medicine, 2Department of Hematology, 3Department of Surgery, and 4Department of Orthopaedics, 1959 NE Pacific St., Seattle, WA 98195

The purpose of this retrospective analysis was to determine whether there were donor factors that were useful for predicting the yield of nucleated cells from marrow derived from cadaveric vertebral bodies. An analysis of 132 donors over a 6-year period was performed. The average number of vertebral bodies procured from each donor was 10.2 ± 1.6 (range 5-14). The total number of nucleated cells recovered per donor ranged from 24 x 109 to 160 x 109 with an average recovery of 69 ± 28 x 109 cells. The cell viability of the recovered cells was >95%. The average age of the donors was 33 ± 14 years (mean ± SD; range 12-65) with an average weight of 169 ± 41 lb (range 82-308 lb). Males comprised 68% of the donor population. The average number of days from admission to death was 1.9 ± 1.7 with a range of 1-11.4 days and the interval between asystole and procurement averaged 3.1 ± 2.3 h (range 0.1-14.7 h). The majority of donors died from head trauma due to an intracranial bleed, gunshot wound, or closed head injury. Regression analysis of the data indicated that the total nucleated cell yield tended to decrease with increasing time between hospital admission and death. The data also indicated that in general female donors yielded lower cell numbers independent of age and male donors under 30 years of age yielded the highest number of cells.

Key words: Organ donor marrow; Vertebral bodies; Stem cells

Address correspondence to Jo Anna Reems, Ph.D., Northwest Tissue Center, Puget Sound Blood Center, 921 Terry Avenue, Seattle, WA 98104. Tel: (206) 292-2317; Fax: (206) 343-1776; E-mail: joannar@psbc.org

Cell Transplantation, Vol. 12, pp. 91-100, 2003
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Tumor Necrosis Factor-a (TNF-a) Stimulates Chemotactic Response in Mouse Myogenic Cells

Y. Torrente,1,3* E. EL Fahime,2* N. J. Caron,2 R. Del Bo,1 M. Belicchi,1 F. Pisati,1 J. P. Tremblay,2 and N. Bresolin3,4

1Centro Dino Ferrari, Institute of Clinical Neurology, University of Milan, Milan, Italy
2Unité de recherche en Génétique humaine, Centre hospitalier de l'Universit, Laval, Ste-Foy, Québec, Canada
3IRCCS Ospedale Maggiore Policlinico, Milano, Italy
4IRCCS Eugenio Medea, Bosisio Parini, Italy

Migration of transplanted myogenic cells occurs during both embryogenesis and regeneration of skeletal muscles and is important for successful myoblast transplantation, but little is known about factors that promote chemotaxis of these cells. Tumor necrosis factor-a (TNF-a) is known to induce chemotactic effect on several cell types. In this study, we investigated its influence on the in vitro and in vivo motility of C2C12 and primary myoblasts. In the in vitro test performed in the blind-well Boyden chambers, we showed that TNF-a (50-400 U/ml) significantly enhanced the ability of myogenic cells to migrate. The dose-response curve for this factor was bell shaped, with maximum activity in the 200 U/ml range. In the in vivo test, intramuscular administration of TNF-a was performed by an Alzet pump connected to a perforated polyethylene microtube inserted in the tibialis anterior (TA) of CD1 mice. In these experiments, myoblasts were injected under the muscle epimysium. The recipient mice were immunosuppressed with FK506. Our results showed that, 5 days after myoblast transplantation, cells migrated further in the muscles infused with TNF-a than in the muscles not exposed to TNF-a. TNF-a not only has a chemotactic activity but may also modify cell migration via its action on matrix metalloproteinase (MMP) expression. The proteolytic activities of the MMPs secreted in the muscles were thus also assessed by gelatin zymography. The results showed an increased of MMP-2 and MMP-9 transcripts in the TNF-a-infused muscles injected with myogenic cells. Myoblast migration during transplantation may be enhanced by overlapping gradients of several effector molecules such as TNF-a, interferon-g (INF-g), and interleukins, released at the site of muscle injury. We propose that TNF-a may promote myoblast migration directly through chemotactic activity and indirectly by enhancing MMP activity at the site of muscle injury.

Key words: Myoblast transplantation; Tumor necrosis factor-a; Transgenic mice; Metalloproteinase; Chemotactic

Address correspondence to Jacques P. Tremblay, Ph.D., Unité de Génétique humaine, Centre Hospitalier de l'Universite Laval, 2705 boul. Laurier, RC-9300, Ste-Foy, G1V 4G2, Québec Canada. Tel: 418-654-2186; Tel: 418-656-4141, ext. 7307; Fax: 418-654-2207; Jacques-P.Tremblay@crchul.ulaval.ca

*These authors contributed equally to the manuscript.