|ognizant Communication Corporation|
VOLUME 9, NUMBER 4, 2000
Cell Transplantation, Vol. 9, pp. 445-451, 2000
0963-6897/00 $20.00 + 00
Copyright © 2000 Cognizant Comm. Corp.
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M. G. McAlinden1 and D. J. Wilson2
1Department of Orthopaedics, School of Medicine, The Queen's
University of Belfast, Musgrave Park Hospital, Stockman's Lane, Belfast
UK, BT9 7JB
2Department of Anatomy, School of Medicine, The Queen's University of Belfast, Medical Biology Centre, 97, Lisburn Road, Belfast, UK, BT9 7BL
Conventionally, culture medium is supplemented with fetal bovine serum (FBS): such serum presents potential risks of foreign protein contamination and transmission of viral or prion-related disease if used in culture of cells intended for human reimplantation. As it has been suggested that a composite of cultured human cancellous bone-derived cells and a bone graft substitute may present a solution to the well-recognized complications and limited availability associated with harvest of fresh bone graft, this study aimed to compare the proliferative response of human cancellous bone-derived cells supplemented with FBS or autologous human serum (AHS) to determine whether AHS is a practical alternative. Explant cultures were established using greater trochanter trabecular bone from 10 consenting patients (aged 57-84) undergoing total hip arthroplasty. At the same time, serum was harvested. The cells were characterized by alkaline phosphatase expression and by in vitro mineralization in enhanced medium. At confluence, cells were aliquoted into multiwell plates and grown for 9 days in medium supplemented with 5%, 10%, 15%, or 20% AHS or 10% FBS. Proliferative response was determined by a crystal violet dye binding assay. There was no significant difference between proliferation in 5% AHS and 10% FBS. However, 10%, 15%, and 20% AHS all produced significantly greater proliferation than 10% FBS. The proliferative response was dose related. FBS is said to be rich in growth and attachment factors, which is why it is widely used in tissue culture. These results suggest that species specificity, even when using adult serum, outweighs these advantages. It should therefore be considered as a prerequisite for any program involving reimplantation of cultured human cells. Clinical trials of cultured human cancellous bone-derived cells have now begun.
Keywords: Bone; Cell culture; Serum; Human
Address correspondence to Dr. D. J. Wilson, Department of Anatomy, The Queen's University of Belfast, Medical Biology Centre, 97, Lisburn Road, Belfast, UK, BT9 7BL. Tel: +44 (2890) 335797; E-mail: firstname.lastname@example.org
Robert J. Ketchum,1 Shaoping Deng,2 Marcus Weber,2 Henning Jahr,2 and Kenneth L. Brayman2
1Department of Anatomy & Cell Biology, Oklahoma State
University College of Osteopathic Medicine, Tulsa, OK 74107-1898
2Harrison Department of Surgical Research, Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104
Isolated canine islets transplanted to hyperglycemic rats fail to restore euglycemia in almost all cases, although the grafted islet tissue appears to be morphologically intact for up to 48 h following transplantation. Cytokines typically produced in the xenograft environment (e.g., IL-1 and TNF) inhibit insulin biosynthesis and secretion from isolated pancreatic islets, and are associated with the production of nitric oxide (NO). To further define the relationship between NO production and islet xenotransplantation, the inhibition of NO in a splenocyte/islet coculture system, and the in vivo effect of this inhibition on canine islet xenotransplantation, was investigated. Splenocytes (SPLC) from Lewis rats were cocultured with canine islets (freshly isolated or cultured 7 days), supernatant removed, and NO concentration (NO2) determined by optical density (Griess reaction, 550 nm, expressed as nmol nitrite/106 cells/18 h). Lipopolysaccharide (LPS) was used as a positive control of SPLC production of NO. Stimulation by LPS resulted in maximal NO production (2.20 ± 0.16 nmol/106 cells/18 h, p < 0.001 compared to baseline values of 0.73 ± 0.04 nmol/106 cells/18 h). In the presence of NO inhibitors (NMA, polymyxin B, hydrocortisone, aminoguanidine, DMSO), nitrite levels did not significantly rise above unstimulated values. Freshly isolated canine islets did stimulate NO production (1.26 ± 0.12 nmol/106 cells/18 h, p < 0.001). In contrast, cultured canine islets did not stimulate NO production (0.84 ± 0.09 nmol/106 cells/18 h). Transplantation of freshly isolated canine islets to STZ-diabetic recipient Lewis rats resulted in amelioration of hyperglycemia in only 50% (n = 6) of recipients 12 h posttransplant, with a return to hyperglycemia at all subsequent time points. Transplantation of 7-day cultured canine islets resulted in amelioration of hyperglycemia in 88% of recipients 12 h posttransplant and 63% of recipients 24 h posttransplant [p = 0.028, mean survival time (MST) = 1.0 days, n = 8]. Transplantation of canine islet xenografts with aminoguanidine therapy (BID, n = 11) resulted in amelioration of hyperglycemia in 100% of recipients at 12 h posttransplant, decreasing to 82% by 24 h following transplantation (p = 0.002, MST = 0.9 days). These results demonstrate that freshly isolated canine islets are potent stimulators of NO production by rat SPLC in vitro, and that culture of canine islets, or addition of NO inhibitors, abrogates stimulated NO production. These results also demonstrate a statistically significant improvement (p < 0.001) in early function of canine islet xenografts following 7 days of islet culture prior to transplant, and following recipient treatment with aminoguanidine. These studies suggest that the production of NO in the microenvironment of the graft site may adversely affect engraftment and function of canine islets, and suggest that the abrogation of islet-stimulated NO production may improve engraftment following islet xenotransplantation.
Key words: Islet transplantation; Xenotransplantation; Nitric oxide; Canine islets; Islet/splenocyte coculture
Address correspondence to Robert J. Ketchum, Ph.D., Department of Anatomy & Cell Biology, College of Osteopathic Medicine, Oklahoma State University, 1111 West 17th Street, Tulsa, OK 74107-1898. Tel: (918) 561-8256; Fax: (918) 561-8419; E-mail: email@example.com
Patrick Bosch,1 Doug Musgrave,1 Steven Ghivizzani,2 Christian Latterman,1 Charles S. Day,1 and Johnny Huard1,2
1Department of Orthopaedic Surgery, University of Pittsburgh,
Pittsburgh, PA 15213
2Department of Molecular Genetics and Biochemistry, University of Pittsburgh Medical Center, Pittsburgh, PA 15213
The development of new clinically applicable methods for the delivery of bone morphogenic protein (BMP) is an area of intensive research. Cell-mediated gene therapy approaches are being explored as a potential delivery vehicle. Primary muscle-derived cells isolated from an adult mouse were transduced with an adenoviral-BMP-2 construct. These cells were injected into the triceps surae of severe combined immune deficient (SCID) mice where they induced heterotopic bone formation. BMP-2 expression by these muscle-derived cell constructs was measured in vitro to estimate in vivo BMP-2 delivery. In vitro expression of BMP-2 by 3 x 105 muscle-derived cells was 87.89 ng/72 h. These results suggest that the efficiency of muscle cell-based gene delivery of BMP-2 exceeds the direct delivery of recombinant BMP-2 protein.
Key words: Muscle-derived cells; BMP-2; Gene therapy; Bone; Adenovirus
Address correspondence to Johnny Huard, Ph.D., Director: Growth and Development Laboratory, Rm. 4151 Rangos Research Center, 3705 Fifth Avenue, Pittsburgh, PA 15213-2583. Tel: (412) 692-7822; Fax: (412) 692-7095; E-mail: firstname.lastname@example.org
Evaluation of an Intrathecal Immune Response in Amyotrophic Lateral Sclerosis Patients Implanted With Encapsulated Genetically Engineered Xenogeneic Cells
A. D. Zurn,1 H. Henry,2 M. Schluep,3 V. Aubert,4 L. Winkel,1 B. Eilers,2 C. Bachmann,2 and P. Aebischer1
1Gene Therapy Center and Division of Surgical Research, Centre
Hospitalier Universitaire Vaudois, Lausanne University Medical School,
1011 Lausanne, Switzerland
2Clinical Chemistry Laboratory, Centre Hospitalier Universitaire Vaudois, Lausanne University Medical School, 1011 Lausanne, Switzerland.
3Department of Neurology, Centre Hospitalier Universitaire Vaudois, Lausanne University Medical School, 1011 Lausanne, Switzerland.
4Division of Immunology and Allergology, Centre Hospitalier Universitaire Vaudois, Lausanne University Medical School, 1011 Lausanne, Switzerland.
A phase I/II clinical trial has been performed in 12 amyotrophic lateral sclerosis (ALS) patients to evaluate the safety and tolerability of intrathecal implants of encapsulated genetically engineered baby hamster kidney (BHK) cells releasing human ciliary neurotrophic factor (CNTF). These patients have been assessed for a possible intrathecal or systemic immune response against the implanted xenogeneic cells. Hundreds of pg CNTF/ml could be detected for several weeks in the cerebrospinal fluid (CSF) of 9 out of 12 patients, in 2 patients up to 20 weeks after capsule implantation. Slightly elevated leukocyte counts were observed in 6 patients. Clear evidence for a delayed humoral immune response was found in the CSF of only 3 patients out of 12 (patients #4, #6, and #10). Characterization of the antigen(s) recognized by the antibodies present in these CSF samples allowed to identify bovine fetuin as the main antigenic component. The defined medium used for maintaining the capsules in vitro before implantation contains bovine fetuin. Fetuin may therefore still be adsorbed to the surface of the cells and/or the polymer membrane, or be present in the medium surrounding the encapsulated cells at the time of implantation. Because of the insufficient availability of CSF samples, as well as the relatively poor sensitivity of the assays used, a weak humoral immune response against components of the implanted cells themselves cannot be excluded. However, the present study demonstrates that encapsulated xenogeneic cells implanted intrathecally can survive for up to 20 weeks in the absence of immunosuppression and that neither CNTF nor the presence of antibodies against bovine fetuin elicit any adverse side effects in the implanted patients.
Key words: Amyotrophic lateral sclerosis; Xenotransplantation; Encapsulated cells; Immune response
Address correspondence to A. Zurn, Gene Therapy Center and Division of Surgical Research, CHUV, Lausanne University Medical School, CH-1011 Lausanne, Switzerland. Tel: 41 21 314 24 62; Fax: 41 21 314 24 68; E-mail: Anne.Zurn@chuv.hospvd.ch
Stuart I. Hodgetts,1 Manfred W. Beilharz,2 Anthony A. Scalzo,2 and Miranda D. Grounds1
Departments of 1Anatomy and Human Biology and 2Microbiology, The University of Western Australia, Nedlands, Perth, Western Australia, 6907
Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 105 cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+-depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.
Key words: Myoblast transfer therapy; Survival; CD4+/CD8+/NK1.1 depletion; DNA quantification
Address correspondence to Dr. Stuart I. Hodgetts, Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, Western Australia, 6907. Tel: 618 9380 7127; Fax: 618 380 1051; E-mail: email@example.com
The Influence of Muscle Fiber Type in Myoblast-Mediated Gene Transfer to Skeletal Muscles
Zhuqing Qu Petersen and Johnny Huard
Department of Orthopaedic Surgery and Molecular Genetics & Biochemistry, Musculoskeletal Research Center, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, PA 15261
Myoblast transplantation has been hindered by immune rejection problems, as well as the poor survival and spread of transplanted cells. Our recent study has shown that the poor survival of the injected cells can be totally overcome by the use of specific populations of muscle-derived cells. In the present study, we have investigated whether a relationship exists between the fate of transplanted cells and the muscle fiber types. Four kinds of myogenic cells [primary myoblasts at a high purity (PMb), myoblasts isolated from fast single fibers (FMb), mdx (MCL), and MtMd-1 cell lines] were infected with an adenoviral vector carrying a LacZ reporter gene and injected into mdx hindlimb muscle. The LacZ transduced myofibers formed by the fusion of the injected myoblasts at 2-10 days postinjection were colocalized with MyHC stainings. The PMb cells, which expressed both slow and fast MyHCs in vitro, displayed the same phenotypes when injected into the m. soleus and m. gastrocnemius (white) muscles, which contained 70% and 0% of slow myofibers, respectively, and showed a high degree of fusion with host muscle fibers. In contrast, the FMb cells only expressed fast MyHCs in vitro and fused exclusively with each other or with host fast muscle fibers when injected in the m. gastrocnemius. Injected MCL and MtMd-1 fused predominantly with each other and displayed a similar expression of MyHCs to those they expressed in vitro. Just a few host myofibers were found to express the reporter gene product following implantation of both cell lines, indicating that these myogenic cell lines display an intrinsic potential to fuse together rather than with host myofibers. Based on the data, we concluded that 1) the essential key to survival is the ability of the donor cells to fuse with the host myofibers, and 2) the most successful combination is achieved between donor primary muscle cells that express both fast and slow MyHC and a host muscle type that facilitates fusion.
Key words: Myoblast transplantation; MyHC isoforms; Duchenne muscular dystrophy; Gene transfer; Ex vivo approach
Address correspondence to Dr. Johnny Huard, Director: Growth & Development Laboratory, Department of Orthopaedic Surgery, and Molecular Genetics and Biochemistry, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, PA, 15213. Tel: (412) 692-7807; Fax: (412) 692-7095; E-mail: firstname.lastname@example.org
K. Goldring,1,2 G. E. Jones,2 and D. J. Watt1
1Department of Neuromuscular Diseases, Division of Neuroscience
and Psychological Medicine, Imperial College School of Medicine, Charing
Cross Campus, St Dunstan's Road, London W6 8RP, UK
2The Randall Institute, King's College London, 26-29 Drury Lane, London WC2B 5RL, UK
Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57Bl/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment. It was important to determine the characteristics of the converting cell. A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture. In the present study we cloned cells derived from the dermis to identify the converting cells. Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones. We investigated whether the protein b-galactoside binding protein (bGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts, could be a candidate factor responsible for conversion. Medium harvested from COS-1 cells infected with a construct containing bGBP has been used for this investigation. Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.
Key words: Dermal fibroblast; bGBP; Myoblasts; Desmin; Duchenne muscular dystrophy
Address correspondence to Dr Diana Watt, Department of Neuromuscular Diseases, Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Charing Cross Campus, St Dunstan's Road, London W6 8RP, UK. Tel: 44-181-846-7029; Fax: 44-181-846-7025; E-mail: email@example.com
C. N. Pagel, J. E. Morgan, J. G. Gross, and T. A. Partridge
Muscle Cell Biology, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
Transplantation of disaggregated myoblasts from normal donor to the muscles of a diseased host, or reimplantation of genetically modified host myoblasts, has been suggested as a possible route to therapy for inherited myopathies such as Duchenne muscular dystrophy, or to supply missing proteins that are required systemically in diseases such as hemophilia. With two exceptions, studies of myoblast transfer in the mouse have involved transplantation of donor myoblasts isolated from adult or neonatal skeletal muscle satellite cells. In this study we present evidence that thymic myoid cells are capable of participating in the regeneration of postnatal skeletal muscle, resulting in the expression of donor-derived proteins such as dystrophin and retrovirally encoded proteins such as b-galactosidase within host muscles. This leads us to conclude that thymic myoid cells may provide an alternative to myoblasts derived from skeletal muscle as a source of myogenic cells for myoblast transfer.
Key words: Myoblast transplantation; Thymic myoid cell; Dystrophin; Muscle regeneration
Address correspondence to Jennifer E. Morgan, Muscle Cell Biology, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. Tel: 0208 383 8265; Fax: 0208 383 8264; E-mail: firstname.lastname@example.org
Y. Torrente,1 E. El Fahime,2 N. J. Caron,2 N. Bresolin,3,4 and J. P. Tremblay2
1Centro Dino Ferrari, Institute of Clinical Neurology, University
of Milan, Milan, Italy
2Unité de recherche en Génétique humaine, Centre hospitalier de l'Université Laval, Ste-Foy, Québec, Canada
3IRCCS Ospedale Maggiore Policlinico, Italy
4IRCCS Eugenio Medea, Bosisio Parini, Italy
The effect of pretreatments of host muscles with metalloproteinases (MMPs) or with notexin on the migration of transplanted myoblasts was investigated. Transgenic TnILacZ mice in which the b-galactosidase gene is under the control of a quail fast skeletal troponin I gene promoter were used as donors. A polyethylene microtube with four perforations was inserted in the tibialis anterior (TA) of CD1 mice. Both pretreatment substances and cells were slowly injected through that microtube. Muscles were pretreated 2 days before myoblast injection either with a mixture of collagenase, matrilysin, and notexin or with only collagenase and matrilysin or only notexin. As control for our experiments, TnILacZ and C2C12 myoblasts were also injected in TA muscles not pretreated. Comparison of short and long-term myoblast radial migration was performed using a dye (PKH26) and X-gal staining, respectively. The recipient mice were immunosuppressed with FK506. Two days after myoblast transplantation, the cell movement in muscles pretreated with collagenase, matrilysin, and notexin was slightly greater than in muscles pretreated only with collagenase and matrilysin but was about twice that observed in muscles treated with notexin alone. Almost no radial migration of TnILacZ myoblasts was observed in untreated muscles. The C2C12 myoblasts showed a four- to fivefold higher migration capacity than TnILacZ myoblasts. At 15 days after TnILacZ myoblast transplantation, the farthest positive b-gal muscle fibers show a two- to threefold extension of the initial migration observed at 2 days, demonstrating the ability of myoblasts to continue the migration following all pretreatments and even in the untreated muscles. In addition, more muscle fibers expressed the b-gal reporter gene in muscles pretreated only with MMPs. Our results clearly demonstrate that muscle pretreatments with MMPs increase myoblast migration and fusion with host muscle fibers after transplantation and that the C2C12 cell line producing MMPs has a higher migratory capacity.
Key words: Myoblast transplantation; Transgenic mice; Metalloproteinase
Address correspondence to Jacques P. Tremblay, Ph.D., Unité de recherche en Génétique humaine, Centre hospitalier de l'Université Laval, 2705, boul. Laurier, RC-9300, Ste-Foy, Québec, Canada G1V 4G2. Tel: (418) 654-2186; Fax: (418) 654-2207; E-mail: Jacques-P.Tremblay@crchul.ulaval.ca
J. E. Anderson, M. Weber, and C. Vargas
Department of Human Anatomy & Cell Science, University of Manitoba, Canada
Deflazacort slows the progress of Duchenne muscular dystrophy (DMD) with fewer side effects than prednisone. In mdx mice, deflazacort treatment augments repair and growth of new muscle fibers. We tested the hypothesis that deflazacort improves muscle function and promotes repair by increasing myogenic cell proliferation and fiber differentiation. mdx mice (3.5 weeks old) were treated with deflazacort (1.2 mg/kg) or vehicle for 4 weeks. Forelimb grip strength was measured. After 4 weeks, the right tibialis anterior muscle (TA) was crush injured to induce synchronous regeneration. DNA was labeled using different markers 24 and 2 h before collecting tissues 4 days after injury. The expression of creatine kinase (CK) isoforms, laminin-2 (merosin) mRNA and protein, and proliferation by myogenic cells were measured and satellite cells were identified by immunolocalization of c-met receptor. Peak grip strength increased 15% within 10 days of treatment, and was maintained up to 6 weeks after the end of treatment in a second experiment. Expression of CK MM in the regenerating TA rose from 46% to 55% of total CK activity after deflazacort treatment. Satellite cells were more numerous and appeared earlier on new fibers, in concert with a threefold increase in proliferation by myogenin+ (but not MyoD+) myoblasts. a2-Laminin mRNA expression and protein increased 1.3-5.5-fold relative to MM CK in regenerating and dystrophic TA, respectively. These studies support the hypothesis that deflazacort promotes functional gains, myogenic differentiation, myoblast fusion, and laminin expression in regenerating dystrophic muscle. The potential to augment precursor specification, strength, and possible membrane stability may be useful in directing long-term benefits for DMD patients and short-term amplification of precursors prior to myoblast transfer.
Key words: Muscle regeneration; Myogenin; Satellite cell; c-met; Laminin; bFGF
Address correspondence to Dr. Judy E. Anderson, Department of Human
Anatomy & Cell Science, University of Manitoba, 730 William Avenue,
Winnipeg, MB, Canada R3E 0W3. Tel: 204-789-3716; Fax: 204-789-3920; E-mail: