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Normal and genetically engineered skeletal muscle cells (myoblasts) show promise as drug delivery vehicles and as therapeutic agents for treating muscle degeneration in congenital myopathies. To date, genetically engineered myoblasts are one of the few cell types capable of long term systemic delivery of proteins at therapeutic levels. Myoblast transplantation poses unique challenges in comparison to organ transplants. In contrast to organs, transplanted myoblasts become incorporated into multinucleated muscle fibers of the host and form stable cell hybrids in which the nuclei remain distinct. However, the immunobiology of myoblast transplantation is poorly understood.
Retrovirally infected myoblasts expressing ß-galactosidase are useful in studies of myoblast transplantation. Myoblast survival can be quantitated and histological analyses of cell fate can be performed. Using these techniques, Pavlath, et al. (1994) showed that allogeneic myoblasts, like organ transplants, are rapidly rejected unless immunosuppressant drugs are administered. In order to make myoblast-mediated gene therapy a practical drug delivery method for the future, the immune response of the host against allogeneic myoblasts must be overcome. As a step toward this goal, Pavlath et al. (1994) demonstrated that two different transient immunosuppressive treatments can lead to long term retention of allogeneic myoblasts in mice. Major Histocompatibility Complex (MHC) Class I and II molecules are molecules with critical roles in immune recognition of allografts. These molecules have been shown to be expressed by muscle cells in vitro. Mouse models provide powerful research tools, such as well-defined genetics, necessary to address hypotheses relevant to basic biological problems inherent in allogeneic myoblast transplantation. Analysis of the expression and role of molecules involved in rejection of transplanted myoblasts should provide insight into the underlying mechanism of allogeneic myoblast rejection. This information will be useful for further trials of transient immunosuppressive treatments to enhance the retention of allogeneic myoblast transplants. Ultimately, it may be possible to engineer a universal donor strain of nonimmunogenic myoblasts deficient in the function or expression of the molecules that mediate myoblast rejection. Transient immunosuppressive techniques, coupled with future advances in rendering cells nonimmunogenic to bypass systemic treatment of the host, may broaden the scope of myoblast-mediated gene therapy for treatment of disease.
- Myoblasts with isolated class I and II MHC disparities are rejected with different kinetics than are fully MHC mismatched myoblasts.
- If isolated MHC disparities effect the kinetics of rejection, one would expect an increase in the number of ß-gal labeled fibers compared to full class I and II disparity.
- If rejection is delayed, compared to fully MHC incompatible myoblasts, a crucial role for the specific MHC gene in the immune recognition of foreign myoblasts is indicated.
Myoblast Isolation: To increase the yield of myoblasts from adult skeletal muscle, the tibialis anterior muscles of each type of mice were pre-damaged with dry ice. Four days later, mononucleated cells were liberated from minced tibialis anterior muscles pooled from 2 mice by mechanical and enzymatic dissociation of the dissected muscle followed by an enrichment of myogenic cells by preplating. Cells were grown in Ham's F10, supplemented with 20% fetal bovine serum, 2.5 ng/ml basic fibroblast growth factor, penicillin G (200 units/ml) and streptomycin (200 µg/ml) in a humidified 5% CO2 incubator at 37 °C on collagen coated dishes. These growth conditions led to a further enrichment of myoblasts to >95% as measured by the percentage of desmin expressing cells, a myoblast specific marker.
Retroviral Infection of Myoblasts: The retroviral vector MFG-lacZ which encodes E. coliß-galactosidase (lacZ) was introduced into ecotropic Bosc 23 packaging cells using a transient transfection system which yields high titer retroviruses. Myoblast cultures were infected with the recombinant retrovirus by spinning at 3500 rpm for 30 mins at 32 °C in the presence of the infectious retroviral supernatant and 8 µg/ml polybrene. The infection procedure was repeated every 6-8 hours for a total of 6 rounds. Greater than 90% of each myoblast type expressed ß-galactosidase (ß-gal). ß-gal expression was determined by fixing the cultures with 4% paraformaldehyde, 0.25% glutaraldehyde, 100 mM NaH2PO4, pH 7.4 for 4 mins at 4 °C. After rinsing with phosphate buffered saline, the cultures were incubated with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) in a solution of 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2 in phosphate buffered saline for 8-12 hrs at 37 °C. Cells expressing ß-gal form a blue reaction product from the substrate X-gal.
Transplantation: Cultured cells were trypsinized, washed several times in sterile F10 and 0.5% bovine serum albumin (transplantation buffer), resuspended at a density of 2 x 107 cells/ml in transplantation buffer, and kept on ice. Animals were anesthetized with an i.p. injection of a cocktail of 35 mg/kg ketamine, 5 mg/kg rompun xylazine, and 0.1 mg/kg butorphanol. The fur on the anterior portion of the lower leg was clipped and an incision approximately 3 mm was made in the skin overlying the tibialis anterior muscle of the leg. One injection of 5 µL was made into the belly of the tibialis anterior along its longitudinal axis under microscopic visualization using a 25 µl Hamilton syringe fitted with a 27 gauge needle. The skin was sutured shut with a 6-0 suture. €
Tissue Preparation: Animals were sacrificed at two and four weeks after transplantation in accord with all rules and regulations set forth by the Division of Animal Resources at Emory University. The tibialis anterior muscles were dissected out and embedded in OCT mounting media. Samples were frozen in 2-methylbutane cooled in liquid nitrogen and stored at -70 °C. Serial cross sections of 14 µm for histology staining and 30 µm for ß-gal analysis were collected along the length of each muscle sample at regular intervals of approximately 360 µm. ß-gal expression was analyzed by incubation with X-gal. Histology was examined using standard hematoxylin and eosin (H&E) staining.
Analysis: All samples were examined under a Zeiss Axiophot microscope and the number of ß-gal expressing fibers and the degree of infiltration scored. The number of ß-gal fibers for each sample was the value obtained from the cross section containing the greatest number of ß-gal expressing fibers. A fiber was counted as positive for ß-gal expression if more than half of the myofiber was stained blue and the staining did not appear to be due to the bleeding of color by adjacent fibers. The mean number of ß-gal fibers + the standard error of mean (SEM) was calculated for each type of myoblast transplanted. For each type of myoblast, 2-4 samples were analyzed. Data was normalized to the number of ß-gal expressing fibers obtained when each myoblast type was transplanted into control immunodeficient (Scid) mice and is expressed as mean ± SEM. The number (mean ± SEM) of ß-gal expressing fibers in nude mice for the different donor myoblasts was: C57 = 92, C3H = 161 ± 2, bm 7 = 161 ± 24, bm 11 = 120, bm 12 = 121 ± 12, bm 14 = 220. The degree of infiltration was determined by the amount of lymphocytes present in the H/E area corresponding to the location of the ß-gal expressing fibers. Infiltration was represented by clusters of small blue nuclei and was rated as either small, medium, or large. Medium amounts of infiltration were consistently seen in hosts injected with bm 11 or bm 14 myoblasts at two weeks. All other mutant myoblast transplants into C57 hosts resulted in small levels of infiltration, and all Scid mice exhibited normal histology after transplantation.
Naturally occurring mouse mutants exist in specific genes at the class I and II MHC loci. Each mutant strain, in theory, differs from the wild type C57/BL6 only in the expression of the mutant molecule.
Slower kinetics of myoblast rejection occurs in isolated donor-host MHC disparities than in fully mismatched MHC donor/host combinations.
In the rejection of bm 14 and bm 11 myoblasts, the infiltrating lymphocytes have disappeared by 4 weeks, and the number of ß-gal-expressing fibers is low.
When mutant myoblasts are retained, the histology is similar to that seen in syngeneic C57 transplants, and the number of ß-gal-expressing fibers is high. Conclusions Isolated MHC I and MHC II disparities result in slower kinetics of rejection as compared to fully mismatched myoblasts. This is evident by the increase in the number of ß-gal labeled fibers expressed by isolated donor-host disparities compared to full class I and II donor-host disparities. Disparities in the D and K regions of the Class I MHC locus (bm 11 and 14 mutants) appear to cause a faster rate of rejection than other mutants, as indicated by the low number of ß-gal labeled fibers compared to other mutants. Thus, specific MHC genes, notably the D gene and a region of the K gene of Class I MHC, appear to play a crucial role in the immune recognition of foreign myoblasts. Thus, coupling transient immunosuppressive techniques with isolated donor-host MHC disparities in myoblasts may broaden the scope of myoblast-mediated gene therapy for the treatment of disease.
- Pavlath GK. Rando TA. Blau HM. 1994. Transient immunosuppressive treatment leads to long-term retention of allogeneic myoblasts in hybrid myofibers. J. of Cell Biol. 127(6 Pt 2):1923-32.
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