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Chapter 12 Methods for Myoblast Transplantation
CHAPTER 12
Methods for Myoblast Transplantation Thomas A. Rando* and Helen M. Blaut Department of Neurology and Neurological Sciences of Molecular Pharmacology Stanford University School of Medicine Stanford. California 94305
t Department
I. Introduction 11. Preparation of Cells A. Preparation of Primary Cultures B. Growth and Enrichment of Myoblasts from Primary Cultures 111. Labeling of Myoblasts in Vitro by Retroviral Mediated Gene Transfer A. Retroviral Infection B. Assessment of Efficiency of Retroviral Labeling C. Selection of Labeled Cells IV. Transplantation Techniques V. Evaluation of Transplant A. Anatomical Assay B. Biochemical Assay VI. Concluding Remarks References
I. Introduction The transplantation of muscle arose in studies of muscle regeneration (Carlson, 1973) in which transplanted muscle cells have served as models for the behavior of endogenous muscle stem cells, or satellite cells, that are responsible for the regenerative capacity of muscle. However, muscle transplantation as a therapeutic approach to hereditary muscle diseases was soon recognized (Partridge et al., 1978). This approach is based on the ability of transplanted muscle cells to fuse with existing muscle fibers (which are multinucleated syncytia), thereby contributing a normal gene to a cell in which a crucial gene product is defective or absent (Partridge, 1991). This principle has been extended to the use of transplanted myoblasts as cellular delivery vehicles for therapeutic approaches METHODS IN CELL BIOLOGY, VOL. 52
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to systemic diseases as well (Barr and Leiden, 1991;Dhawan et al., 1991). Finally, myoblast transplantation is another technique in the armamentarium to effect in vivo gene transfer to study regulation of gene expression in vivo. This chapter focuses on techniques of transplanting myoblasts derived from primary cultures of mouse muscle. Although much more difficult to maintain in culture and to engineer genetically than are myoblasts of established cell lines, myoblasts from primary cultures offer many advantages for transplantation studies. First, primary cultures can be derived from any mouse strain, including naturally occurring mutants, transgenic strains, and strains with gene deletions. Thus, the potential phenotypes for study in tissue culture and for transplantation are vastly expanded. Second, the use of primary myoblasts overcomes a common problem of transplantation immunology, namely histoincompatibility. With the limited repertoire of myogenic cell lines available, it is often necessary to cross major histocompatibility barriers when transplanting myoblasts into specific hosts, thus requiring the use of immunosuppressants. For any host, primary myoblasts from that strain provide sygeneic cells for transplantation, thus obviating the need to use immunosuppressants with their inherent toxicities and other diverse effects that could affect myoblast behavior following transplantation. Following are detailed a variety of techniques for the preparation of primary myoblasts for transplantation, as well as some methods for execution and analysis of the transplantation procedure itself, that should be helpful to anyone interested in applying this approach to a wide range of biological and therapeutic questions.
II. Preparation of Cells A. Preparation of Primary Cultures
Primary cultures can be derived from postnatal mouse muscle by standard mechanical and enzymatic dissociation techniques (Rando and Blau, 1994). Cultures are best derived from neonatal mice because the yield of myogenic cells decreases with age, especially within the first 3 weeks after birth. Dissect out the skeletal muscles from the limbs and place them in a glass dish, maintaining tissue culture sterility once the muscle is removed. Add just enough PBS to keep the muscle moist, and mince finely with razor blades. Enzymatic dissociation is then best carried out with a combination of a neutral protease, such as dispase and a colla11 (Boehringer Mannheim Corp., Indianapolis, Indiana) at 2.4 U/ml, genase, such as collagenase D (Boehringer Mannheim Corp.), at 1.5 Ulml.Add approximately 2 mi of the enzyme mixture (adjusted to the appropriate pH and Ca2+concentration for the specific enzymes) per gram of tissue and continue mincing for a few minutes. Transfer the contents to a sterile tube and incubate at 37°C until the mixture is a fine slurry, usually about 20-40 min. Filter the slurry through 80-pm nylon mesh to remove large pieces of tissue, centrifuge the filtrate to pellet the cells, aspirate off the supernatant, resuspend the cells in 5 ml of growth medium (see later discussion) per gram of tissue dissected,
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and plate on dishes coated with type I collagen (100 pgml applied for 6-8 hr at 37°C). Place in an incubator with 5% C02/95% air, and allow the cells to attach and begin proliferating for 2 days before changing the medium. B. Growth and Enrichment of Myoblasts from Primary Cultures
Primary cultures are mixtures of cell types, mostly myoblasts and fibroblasts. For myoblast transplantation, it is preferable to have as pure a population of myogenic cells as possible. Myoblast-selective growth conditions, although not rapid, yield pure and stable populations of myoblasts from primary cultures (Rando and Blau, 1994). Different combinations of nutrient mixtures and additives to the medium provide different stimuli either for cell growth or for differentiation. The combination listed next is an effective selection and growth medium for primary myoblasts in that there is preferential growth of myoblasts over fibroblasts and a very low rate of spontaneous differentiation of myoblasts. This growth medium is as follows: Ham’s F-10 nutrient mixture (Gibco BRL, Gaithersburg, Maryland) 20% fetal calf serum (FCS) (HyClone Laboratories, Inc., Logan, Utah) 2.5 ngml basic fibroblast growth factor (Promega Corp., Madison, Wisconsin) Penicillin (200 U/ml) and streptomycin (200 U/ml) During the first several passages, the technique of “pre-plating” removes a portion of the fibroblasts from the cultures (Richler and Yaffe, 1970) and is especially useful with primary cultures derived from older mice in which the proportion of fibroblasts tends to be higher than in those from neonatal muscle. Using collagen-coated dishes for pre-plating is much more rapid (as little as 5 min) and even more effective. The cultures should be maintained in growth medium at cell densities of between 30 and 70% confluence. After 2 to 3 weeks in culture, nearly 100% of the cells are myogenic as judged by staining for the intermediate filament protein desmin (Rando and Blau, 1994). Such populations have a remarkable proliferative capacity and can be maintained in culture for more than 30 population doublings. Using the same culture conditions, myoblasts can be cloned from primary cultures. It may be that using 1:1mixtures of growth medium and “conditioned medium” (e.g., growth medium collected from a confluent dish of C2 myoblasts or fibroblasts after 24 hr) is more effective than growth medium alone for growing cells at clonal density. This has not been rigorously tested. Cloning cells is more time-consuming and results in a more homogeneous population of myoblasts, but for some applications it may be preferable to have a clonal population. The most significant problem of growing mouse myoblasts is their propensity to detach partially from the dish and round up. Extended coating of dishes with collagen (e.g., 24 hr), not leaving cells on a dish for more than 5 days, and plating cells on dishes coated with laminin (which is much more expensive than type I collagen) have all helped to maintain the population in an adherent state.
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III. Labeling of Myoblasts in Ktro by Retroviral Mediated Gene Transfer For most applications of myoblast transplantation, it is essential to have a method of differentiating the donor cells from the host cells, whether to determine the fate of the transplanted cells, to assess the effect on host fibers of the incorporation of donor cells, or simply to assess the efficiency of the transplantation procedure. Retroviral-mediated gene transfer offers a rapid and efficient method of introducing a hereditable marker into dividing cells (Temin, 1986). The lac2 gene is widely used as a marker because of the available histochemical and biochemical assays of its product, the bacterial P-galactosidase (@gal) enzyme, but certainly other reporter gene markers would be useful as well (Alam and Cook, 1990). A. Retroviral Infection Replication-defectiveretrovirusescontaining the ZucZ gene have been effective for labeling primary mouse myoblasts (Rando and Blau, 1994). At the time of infection, it is useful to have several plates of retroviral producer cells at different densities so that when the myoblasts are ready to be infected, there is always a plate of producer cells at the appropriate confluence. Myoblasts should be 3040% confluent and producer cells should have just reached confluence at the time of infection. Remove the medium from the producer cells, filter it through a 0.45-pm filter, supplement it with FCS to 10% and with polybrene to 10 pg/ ml, and replace the myoblast growth medium with this virus-containing medium. Incubate the myoblasts in this medium for 4-6 hr, then replace it with fresh growth medium. To increase the percentage of myoblasts infected, this procedure can be repeated every 24 hr with little detrimental effect on myoblast growth. B. Assessment of mciency of Retroviral Labeling Typically, retroviral-mediated gene transfer is successful in only a subset of myoblasts (Fig. 1;see Color Plates). Depending on the specific properties of the retrovirus, the viral titer in the producer cell medium, and the infectivity of the myoblasts, the efficiency of gene transfer can range from less than 1%to greater than 50%. By staining a portion of the retrovirally infected cultures with X-gal (5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside), a chromogenic substrate for @-gal(Pearson et ul., 1963),one can determine the efficiency of gene transduction. This compound yields a blue reaction product in cells expressing high levels of P-gal; thus, lacZ+ cells can be distinguished from ZucZ- cells. The staining of cells is done as follows:
1. Reagents Fixative; 4% paraformaldehyde, 0.25% glutaraldehyde in 100 mM Napi, pH 7.2
Ch. 1, Fig. 4 Appearance of quail and chick embryo during surgery. (A) The 16-somite quail embryo has been removed from the egg, rinsed free of contaminating debris, and staked out in a black-bottomed Sylgard-coated dish with insect pins, as described in the text. The embryo is located centrally within the clear area pellucida and is surrounded by the densely white area opaca, the margins of which already contain rust-colored blood islands. Other features of the embryo are indicated. (B) The 17-somite chick embryo is viewed through a window in the eggshell after black ink has been injected under the blastoderm, as described in the text. Somite 3, the first clearly demarcated somite in an embryo of this age, is indicated. The surgical target area is schematically represented with the somite number and somite stages indicated.
Ch. 1,Fig. 6 Histological discrimination between quail and chick nuclei in chimeric embryos. (A) Feulgen-stained cross section through a chimeric chick embryo 1 day after surgically replacing the lateral halves of wing level somites with those of quail as described in the text. The neural tube and wing bud are out of the field to the left and right, respectively. Under light microscopy, the quail nuclei contain bright, magenta-colored nucleoli (black arrows labeled Q-I, 4-2, and 4-3). whereas the staining of chick nuclei is pale and diffuse (white arrows labeled, C-I, C-2, and C-3). Nuclei marked C-1 and C-2 reside in cells of the dermomyotome and myotome, respectively. C-3 marks the nucleus of a sclerotome cell. Q-I marks the nucleus of a cell in the lateral part of the dermomyotome. 4-2 marks the nucleus of a dermomyotome-derived cell that has dissociated from the lateral edge of the nucleus of a cell in the lateral sclerotome. Note that the medial-lateral boundary between chick and quail cells extends from the dermomyotome through the entire sclerotome. Note also that the cells in the lateral most margin of the myotome are chick derived, whereas all of the cells of the lateral dermomuotome and migratory cells are quail derived. (B) Anti-quail antibody-stained cross section through a chimeric chick embryo 1 day after surgically replacing the dorsal half of a wing level somite with that of a quail as described in the text. The neural tube (nt) can be seen on the left-hand margin, whereas the wing bud is out of the field to the right. Under light microscopy, the quail nuclei contain densely stained, dark nucleoli (black arrows labeled Q-I, Q-2, and Q-3), whereas the staining of chick nuclei is pale and diffuse (white arrows labeled C-I, C-2, and C-3). Nuclei marked C-I, C-2, and C-3 are those of chick cells in the dorsomedial, ventromedial, and lateral sclerotome, respectively. Nuclei marked Q-I and Q-2 reside in the medial and lateral myotome regions, respectively. The nucleus marked 4-3 resides within a cell of the dermomyotome. Note that quail nuclei are restricted to dorsal somite derivatives, dermomyotome and myotome, and that chick nuclei are restricted to selertome, a derivative of the ventral somite half.
Ch. 2, Fig. 4 Examples of in situ hybridization performed after P-galactosidase staining on sections (A) or in whole mount (B).
(A) Ten-and-a-half-daychimeric mouse embryo generated with ES cells containing the nlucZ gene in the Msxll locus was fixed in 4% paraformaldehydefor 2 hr stained for P-gal activity overnight, refixed, embedded in paraffin, and sectioned. I n situ hybridization was carried out with an antisense MyoD probe, but using two radioisotopes, and the section counterstained with eosin (0.1%). In darkfield !.%gal+areas such as the limb (star) appear as pink, whereas intense areas of p-gal staining such as the dorsal neural tube (arrowhead) appear blue. Note expression of the skeletal muscle-specific, myogenic factor gene MyoD in the myotomes (arrowheads), where silver grains appear as white. (B) Nine-and-a-half-dayheterozygote embryo containing nlucZ in one allele of the myogenic factor gene myf-5. fixed in 4% paraformaldehyde for 3 hr stained for p-galactosidase activity. Positive nuclei are detected in the forming myotomes (arrow). The embryo was then hybridized with digoxigenin-labeled antisense Msxl. Note Msxlspecific expression in the neural tube (arrowhead) and limb bud (star), which appear as purple. Bar: 100 pm.
Ch. 2, Fig. 5 Double immunofluorescence of a 3-day-old organ culture of segmental plate from MLC3F/nIs-LacZ embryos, reconstituted with neural tubes isolated from nontrangenic siblings. The culture was first stained for P-galactosidase activity and then with an anti-sarcomeric myosin, MyHC polyclonal antibody (A), and an anti-PI11 tubulin monoclonal antibody (B). A MyHCpositive, P-gal-positive myogenic cell is indicated by an arrowhead. Bar: 10 pm.
Ch. 6 , Fig. 3 Analysis of cardiomyocyte differentiation by immunohistochemistry. (A) Phase contrast of a stage 1 1 embryo fixed for immunohistochemistry. (B) MF20-positive differentiated cardiomyocytes are located within the tubular heart. (C) Explant of cardiogenic mesoderm plus ectoderm removed from the embryo at stage 7 and cultured for 48 hr in MI99 minimal medium. Differentiation has occurred in culture as detected by the MF20 antibody.
Ch. 9, Fig. 7 ClAKE 1494 transgenic mice infected with RCASBP/AP(A). Mice were infected at day 5 and sacrified 7 days later. Leg whole mounts were prepared and stained for alkaline phosphatase (AP) acitivity (see Felderspiel er al., 1994). Panel A shows a leg that was infected with viral supernatant (0.024.03ml) containing 5000-7500 infectious units. RepresentativeAP stained with muscle fibers (f) that appear as purple streaks and satellite cells (s) that appear as purple dots are indicated. The experiment was repeated with RCASBP/AP(A) infected chicken embryo fibroblasts (CEFs) (panels B-D). Panel B shows a leg from a nontransgenic animal infected in parallel with infected CEFs. Panels C and D show the AP-positive fibers and cells on both sides of a leg whole mount from a transgenic animal. Reprinted in part, with permission from Felderspiel et al. (1994).
Ch. 12, Fig. 1 The efficiency of retroviral-mediated transfer of the lacZ gene can be determined by staining with X-gal. A culture of primary mouse myoblasts was infected with a retrovirus containing the lacZ gene and then fixed and stained with the P-gal substrate X-gal. The cells that contain the blue reaction product are those that have been successfully transduced. The percentage of the transduced cells in the culture is a measure of the efficiency of retroviral-mediated gene transfer. Bar = 60 pm.
Ch. 12, Fig. 4 Primary myoblasts fused to host fibers after transplantation. A suspension of primary myoblasts that were
selected by FACS to be virtually 100% labeled with the 0-gal marker were transplanted into the tibialis anterior muscle of an adult mouse. Three weeks later, the muscle was analyzed for the presence of hybrid myofibers; that is, host myofibers that have incorporated donor nuclei, by staining with X-gal. (A) The hybrid fibers as normal caliber fibers that stain blue; also present within the field are normal (not hybrid) host fibers that do not stain blue. (B) An adjacent section was stained with hematoxylin and eosin to demonstrate the normal histological appearance of the transplant. Bar = 3@m.
Ch. 13, Fig. 1 (A) Schematic depicting myotube culture in Matrigel. Note that the fibroblasts form a connective sheet on top of the Matrigel that can be physically removed after approximately X days in culture. Panels: ( I ) Cells are suspended in quail growth medium and plated on a dish that is first coated with collagen and then Matrigel. (2) Cells attach to the Matrigel surface by 12 hr after plating. (3) twenty-four to forty-eight hr after plating, the presumptive myoblasts move through the Matrigel in order to attach to the collagen. The fibroblasts remain on the Matrigel surface. (4) Six days after plating, fine forceps are used to peel off the sheet of fibroblasts, leaving the myotubes and presumptive myoblasts embedded in the Matrigel. (B) Demonstration of absence of nonmyogenic cells. After 21 days in culture, cells were stained with rhodamine-phalloidin to visualize actin filaments (red), and DAPI to visualize nuclei (blue). Note that all nuclei are associated with myotubes and that no fibroblasts are visible.
Ch. 13, Fig. 2 (A) Myotubes showing sarcomeric structures, visualized with anti-myosin heavy-chain primary antibodies and rhodamine-conjugated secondary antibodies. (B) Myotubes with structures incorporating overexpressed myosin essential light chains, visualized with anti-myosin essential light-chain primary antibodies and fluorescein-conjugated secondary antibodies. (C) Myotubes containing disrupted structures resulting from overexpression of myosin heavy chains, visualized with anti-myosin heavy-chain primary antibodies and rhodamine-conjugated secondary antibodies. (D) Myotubes with structures incorporating overexpressed troponin T,visualized with anti-troponin T primary antibodies and rhodamine-conjugated secondary antibodies.
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Staining solution: 5 mM K3Fe(CN)6,5 mM We(CN),, 2 mM MgC12in PBS X-gal stock solution: 40 mg/ml X-gal (Sigma Chemical Co., St. Louis, Missouri) in dimethylformamide (store at -2O"C, protected from light) 2. Protocol Fix cells for 4 min in cold (4°C) fixative. Wash cells in PBS for 5 min, 2 times. Dilute X-gal stock in staining solution to a final concentration of 1 mg/ml before use (1/40 dilution of X-gal stock). Apply diluted X-gal solution to cells; incubate at 37°C overnight. Examine microscopically for presence of "blue" cells (Fig. 1). In this assay, the efficiency of retroviral infection, and thus gene transfer, is determined as the number of labeled cells as a percentage of the total. For retroviruses that express very high levels of @-gal, shorter incubation times (4 hr) with X-gal will be sufficient.
C. Selection of Labeled Cells It is desirable to have as close to 100%of the cells expressing the marker as possible, and thus some method of selecting the labeled cells is needed. Cloning cells is straightforward but time-consuming. Since some retroviruses contain genes whose products confer drug resistance, growth of the population in the presence of the drug results in the survival only of cells infected with retrovirus. The gene for neomycin phosphotransferase, confering neomycin resistance, is useful because mouse myoblasts are sensitive to low concentrations (50 pg/ml) of the neomycin analogue, G418. The @-galenzyme itself can be used as a means of selecting infected cells by fluorescence activated cell sorting (FACS). In the presence of @-gal,the substrate FDG (fluorescein di-@-D-galactopyranoside)is cleaved to yield fluorescein. Thus, @-galexpressing cells have a high fluorescence intensity in the presence of FDG and can be separated from cells with low @-galactivity (Nolan et al., 1988). 1. Reagents
Staining medium: 4% FCS in PBS FDG solution: 2 mM FDG (Molecular Probes, Inc., Eugene, Oregon) in H 2 0 (make up as lOOX stock in 1:1mixture of H20and DMSO; store at -20°C, protected from light) Stop solution: 1mM phenylethyl-@-D-thiogalactoside (PETG), 1 pg/ml propidium iodide, in staining medium, on ice.
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2. Protocol
Trypsinize cells, centrifuge, and resuspend at lo7celldm1in staining medium. If necessary, pass cell suspension through 80 pm nylon mesh to filter out cell clumps. Transfer cell suspension to 4-ml FACS tube, reserving 100 pl of the suspension in a separate tube as control cells, to be analyzed in the absence of FDG. Place cell suspensions at 37°C for 10 min. Add 100 pl of FDG solution (warmed to 37°C) for every 100 p1 of cell suspension. For the control cell suspension, add 100 p1 of H 2 0 (37°C). Mix thoroughly and place the tubes at 37°C for 1 min. To stop the reaction and inhibit diffusion of the product out of cells, add to each tube 1.8 ml of ice-cold stop buffer for every 200 pl in the tube, and place the tubes immediately on ice. Set up cell sorter to detect fluorescein and propidium iodide; set autofluorescence compensation using the control cell suspension. Gate out the cells positive for propidium iodide (dead cells). Analyze control cells. Analyze FDG-treated cells and sort, selecting cells with highest fluorescence intensity (see next paragraph). Upon analysis of the control cells, a single peak of low fluorescence intensity should be seen (Fig. 2A). Analysis of the FDG-treated cells should reveal two peaks, both at higher fluorescence than the control cells (Fig. 2B). The cells represented in the peak with highest fluorescence are those expressing lac2 and should be selected by gating out cells in the lower peak. When cells obtained from this kind of sort are stained with X-gal, routinely >99% are found to be lacZ+. It is, of course, imperative to maintain sterile conditions during cell sorting. Adding gentamicin (50 pg/ml) to the culture medium for 2-3 days after sorting may diminish the likelihood of contamination from handling outside the hood. After remaining in suspension for a prolonged period, mouse myoblasts do not plate efficiently, so they should be plated at twice the usual density.
IV.Transplantation Techniques Cells thus enriched to be purely myogenic and selected to be virtually 100% labeled are ideal for transplantation. The following suggestions for the handling of the cells before transplantation are more anecdotal than rigorously tested Cells should be passaged no less than 48 hr before transplantation, should be provided with fresh medium between 12 and 24 hr before transplantation, and should be about 50% confluent at the time of transplantation. Harvest the cells by trypsinization and estimate cell number and viability using a hemacytometer
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Fig. 2 p-gal labeled myoblasts can be selected by FACS. Primary myoblasts were labeled with p-
gal by retroviral mediated gene transfer (see Fig. 1, Color Plates). Since only a portion of the cells were labeled, the labeled cells were selected by FACS using the substrate FDG. (A) Labeled myoblasts in the absence of FDG have a characteristic fluorescence intensity about a single peak. (B) In the presence of FDG, the cells fall into two peaks of fluorescence intensity: Cells that have not been infected by the retrovirus form a peak at low intensity; cells that have been infected and express high levels of @-galdisplay a much higher fluorescence intensity and form a second peak to the right.
and trypan blue exclusion. Wash the cells twice in serum-free medium, resuspend them at a predetermined density (e.g., 2 X lo7 cells/ml) in the buffer to be used for transplantation, and place them on ice. The buffer should be a nutrient mixture with protein added (e.g., 0.5% bovine serum albumin in F-10). Just before transplantation, resuspend the cells gently, draw up -100 p1 into a round, capillary pipet tip, and inject the suspension into the barrel of a Hamilton syringe with a 27-gauge needle attached. Insert the plunger into the barrel of the syringe, and the cells are ready to be injected. Approximately 70 p1 of cell suspension is sufficient to fill the barrel of the syringe and the needle. While the cells are being prepared for injection, anesthetize the mouse, shave the skin over the muscles to be injected, and secure the limbs on a dissecting board. Make an incision in the skin overlying the muscle to be injected and retract the skin to reveal the muscle. The tibialis anterior is particularly useful because it has well-defined borders and is easily accessible. With the aid of a dissecting microscope, insert the needle along the axis of the muscle fibers to the mid-belly of the muscle (Fig. 3). Slowly inject 5 or 10 p1 of the cell suspension, slowly withdraw the needle, and suture the skin closed. One of the critical aspects of successful transplantations is keeping cells viable before injection. Suspending the cells in PBS and keeping them at room tempera-
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Fig. 3 A representation of the injection procedure for myoblast transplantation. The tibialis anterior is revealedand is shown with a needle inserted into the midbelly region where the suspension of myoblasts will be injected.
ture leads to a loss of viability (as judged by trypan blue exclusion) of greater than 50% of the cells over a 6-hrperiod. Using the transplantation buffer listed earlier and maintaining the cells on ice maintains greater than 90% viability during the same time. Also for large-scale procedures involving dozens of mice, it is useful to have several dishes of cells and harvest them as needed such that “fresh” cells are being used throughout the day. Allowing the suspension to sit in the syringe leads to cell clumping and uneven cell injections.
V. Evaluation of Transplant The final assessment following myoblast transplantation will of course depend on the specific goals of the transplantation, whether it is for studies of muscle regeneration, for therapeutic gene transfer, or for analysis of gene expression in viva However, regardless of the goal, a measure of the outcome of the transplantation itself will be critical to any experiment. Such a measure or measures will allow assessment of transplantation efficiency, reproducibility, and
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stability, without which comparisons between experiments would be fraught with uncertainty.
A. Anatomical Assay After myoblast transplantation, an anatomical assessment is perhaps the most informative. The transplantation of cells into a complex, highly organized tissue such as muscle results in local disruption of the tissue architecture. The final histology after the transplant has matured is a combination of the regenerative capacity of the tissue and the interaction of the transplanted cells with that tissue. Thus, it is important to determine the fate of the transplanted cells, which may be quite dissimilar in host strains that have different regenerative capabilities of muscle. In particular, it is important to determine whether the transplanted cells have become incorporated into host fibers, have fused with other mononucleated cells to form new myotubes and myofibers, or have remained as mononucleated cells (which may be quiescent as satellite cells or may differentiate as myocytes; myoblasts from established cell lines may proliferate to form tumors). Additionally, a significant portion of the implanted cells die after transplantation, and the extent of this death can be assessed qualitatively in different transplants over time, as well as quantitatively using a biochemical analysis of implanted cell numbers (see later discussion). With P-gal as a marker, the anatomic fate of surviving myoblasts can be readily determined by staining frozen sections of transplanted muscle with X-gal. Muscles should be processed for sectioning using standard techniques, and cross-sections of muscle should be obtained. The thickness of sections should be between 15 and 30 pm for X-gal staining and less than 10 pm for routine histochemistry. To stain frozen sections with X-gal, use the same protocol described earlier for staining cultured cells. After staining, rinse the slides in PBS, coverslip them using an aqueous mounting medium, and examine them microscopically for the presence of @gal labeled fibers (Fig. 4A; see Color Plates). Stain adjacent sections with hematoxylin and eosin to evaluate the various cellular components of the tissue (Fig. 4B; see Color Plates). Hybrid fibers formed by the fusion of implanted myoblasts with host fibers are identified as @-gallabeled fibers of normal caliber (Fig. 4A). The fusion of implanted myoblasts with other myoblasts usually results in small-diameter pgal labeled fibers that are usually clustered at the site of cell injection. As a way of quantifying the data, the number of P-gal labeled fibers can be counted in a cross-section. From a single injection of 105myoblasts, up to 200 hybrid myofibers can be formed (Rando and Blau, 1994). The number of labeled fibers will vary along the length of the muscle and will diminish with increasing distance from the injection site, as will the intensity of P-gal staining. Thus, it appears that myoblasts do not move significantly from the site of injection, but remain relatively localized.
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B. Biochemical Assay In addition to an anatomical assessment, one can quantitate the transplantation results by measuring the total @-galactivity of the muscle in a tissue homogenate. Although no information can be obtained about the nature of the cells expressing the @-gal(i.e., whether they are unfused myoblasts, host fibers that have incorporated donor cells, etc.), this is a rapid way of comparing transplantation efficiencies under different conditions and over time. There are a variety of quantitative assays of &gal, but the following chemiluminescence assay using the P-gal substrate AMPGD (3-(4-methoxyspiro(l,2-dioxetane-3,2’-tricyclo[3..3.1.1 3~7]decan)4-yl)phenyl-@-~-galactopyranoside)is particularly sensitive (Jain and Magrath, 1991).
1. Reagents Extraction buffer: 100mMP-mercaptoethanol,0.1% Triton, 90 pg/ml phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory units/ml aprotinin, 100p M leupeptin in 100 m M Napi, pH 7.0 Assay buffer: 1 m M MgC12, 100 mM NaPi, pH 8.0 AMPGD stock solution: 10 mg/ml in a 1:1 mixture of H 2 0 and methanol (Tropix Inc., Bedford, Massachusetts) Emerald luminescence enhancer stock (Tropix Inc.), diluted 1 : l O in 0.2 N NaOH PETG solution: 26 mM PETG in H20
2. Protocol Sacrifice the animals and dissect out the injected muscles; mince each muscle briefly with a razor blade. Place minced muscles in tubes for homogenization and add 1 ml of ice-cold extraction buffer to each. Homogenize the tissue and then centrifuge at 4OOOg to pellet insoluble material. Transfer a determined volume (e.g., 10 pl) of the supernatant from each tube to a luminometer tube, and bring each sample up to 100pl with assay buffer. Dilute AMPGD stock solution into assay buffer 1:625. Add 200 p1 of diluted AMPGD solution to each tube and begin timing the reaction. After 15 min, add 25 p1 of PETG solution to each tube. Immediately before placing a tube in luminometer to read, add 300 p1 of diluted Emerald luminescence enhancer solution to the tube. Place tube in luminometer and integrate luminescent signal over 10 sec.
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As a standard, an extract of the cells used for transplantation can be used to relate the &gal activity of the muscle to the implanted cells. Once the muscle is minced, it can be snap-frozen and stored at -80°C for more than a year with no significant loss of @gal activity, and the &gal activity in the tissue homogenate is stable at 4°C for at least several days, probably longer. The results of this assay give a measure of total &gal activity in the muscle as the chemiluminescent signal is proportional to the amount of &gal enzyme. By extrapolation Prom a cell standard, one obtains an estimate of the number of persisting transplanted cells. Such an estimate is useful for comparing techniques to optimize survival of transplanted cells and as a baseline to compare transplants under different experimental conditions. This estimate, however, assumes a constant relationship between a donor cell nucleus and @gal activity, a relationship that may change with time, especially after differentiation and fusion. Changes in cell biochemical activity that affect transcription, translation, mRNA stability, protein stability, and enzymatic efficiency could all alter the relationship between a donor-cell nucleus and its resulting P-gal activity. An independent measure of implanted cell nuclei, one that is conserved during replication and not dependent on cell biochemical activity, is needed to determine the accuracy to the estimate of implanted cell number by &gal activity.
VI. Concluding Remarks Myoblast transplantation provides a unique means to effect in vivo gene transfer because of the ability of implanted myoblasts to fuse with host myofibers. Among the limitations to more widespread use of myoblast transplantation is the time required for growth, purification, and labeling of cells and for detailed transplant analysis. Future advances will occur through improvement of the techniques described here, in particular more rapid methods of purifymg and labeling donor cells, more efficient methods of delivering myoblasts to the tissue, and, ideally, noninvasive methods of determining the fate of the implanted cells. References Alam, J., and Cook, J. L. (1990). Reporter genes: Application to the study of mammalian gene transcription. Anal. Biochem. 188, 245-254. Barr, E., and Leiden, J. M. (1991). Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 254, 1507-1509. Carlson, B. M. (1973). The regeneration of skeletal muscle. A review. Am. J. Anat. 137, 119-150. Dhawan, J., Pan, L. C., Pavlath, G. K., Travis, M. T., Lanctot, A. M., and Blau, H. M. (1991). Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 254, 1509-1512. Jain, V. K., and Magrath, I. T. (1991). A chemiluminescentassay for quantitationof beta-galactosidase in the femtogram range:Application to quantitationof beta-galactosidase in lacZ-transfected cells. Anal. Biochem. 199,119-124.
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Nolan, G. P., Fiering, S.,Nicolas, J. F., and Henenberg, L. A. (1988).Fluorescence-activated cell analysisand sorting of viable mammalian cellsbased on beta-D-galactosidaseactivity after transduction of Escherichia coli lacZ. Proc. Natl. Acad. Sci. USA 85, 2603-2607. Partridge, T. A. (1991). Myoblast transfer: A possible therapy for inherited myopathies? Muscle Nerve 14, 197-212. Partridge, T. A., Grounds, M., and Sloper, J. C. (1978).Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Narure 273, 306-308. Pearson, B., Wolf,P. L., and Vazquez, B. S. (1963). Lab. Invest. 12, 1249-1259. Rando, T. A., and Blau, H. M. (1994).Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J. Cell Biol. 125, 1275-1287. Richler, C.,and Yaffe, D. (1970).The in vitro cultivation and differentiation capacities of myogenic cell lines. Dev. Biol. 23, 1-22. Temin, H. M. (1986).Retrovirus vectors for gene transfer: Efficient integration into and expression of exogenous DNA in vertebrate cell genomes. In “Gene Transfer” (R. Kucherlapat, ed.), pp. 149-187. New York Plenum Press.
Methods for Myoblast Transplantation Thomas A. Rando* and Helen M. Blaut Department of Neurology and Neurological Sciences of Molecular Pharmacology Stanford University School of Medicine Stanford. California 94305
t Department
I. Introduction 11. Preparation of Cells A. Preparation of Primary Cultures B. Growth and Enrichment of Myoblasts from Primary Cultures 111. Labeling of Myoblasts in Vitro by Retroviral Mediated Gene Transfer A. Retroviral Infection B. Assessment of Efficiency of Retroviral Labeling C. Selection of Labeled Cells IV. Transplantation Techniques V. Evaluation of Transplant A. Anatomical Assay B. Biochemical Assay VI. Concluding Remarks References
I. Introduction The transplantation of muscle arose in studies of muscle regeneration (Carlson, 1973) in which transplanted muscle cells have served as models for the behavior of endogenous muscle stem cells, or satellite cells, that are responsible for the regenerative capacity of muscle. However, muscle transplantation as a therapeutic approach to hereditary muscle diseases was soon recognized (Partridge et al., 1978). This approach is based on the ability of transplanted muscle cells to fuse with existing muscle fibers (which are multinucleated syncytia), thereby contributing a normal gene to a cell in which a crucial gene product is defective or absent (Partridge, 1991). This principle has been extended to the use of transplanted myoblasts as cellular delivery vehicles for therapeutic approaches METHODS IN CELL BIOLOGY, VOL. 52
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to systemic diseases as well (Barr and Leiden, 1991;Dhawan et al., 1991). Finally, myoblast transplantation is another technique in the armamentarium to effect in vivo gene transfer to study regulation of gene expression in vivo. This chapter focuses on techniques of transplanting myoblasts derived from primary cultures of mouse muscle. Although much more difficult to maintain in culture and to engineer genetically than are myoblasts of established cell lines, myoblasts from primary cultures offer many advantages for transplantation studies. First, primary cultures can be derived from any mouse strain, including naturally occurring mutants, transgenic strains, and strains with gene deletions. Thus, the potential phenotypes for study in tissue culture and for transplantation are vastly expanded. Second, the use of primary myoblasts overcomes a common problem of transplantation immunology, namely histoincompatibility. With the limited repertoire of myogenic cell lines available, it is often necessary to cross major histocompatibility barriers when transplanting myoblasts into specific hosts, thus requiring the use of immunosuppressants. For any host, primary myoblasts from that strain provide sygeneic cells for transplantation, thus obviating the need to use immunosuppressants with their inherent toxicities and other diverse effects that could affect myoblast behavior following transplantation. Following are detailed a variety of techniques for the preparation of primary myoblasts for transplantation, as well as some methods for execution and analysis of the transplantation procedure itself, that should be helpful to anyone interested in applying this approach to a wide range of biological and therapeutic questions.
II. Preparation of Cells A. Preparation of Primary Cultures
Primary cultures can be derived from postnatal mouse muscle by standard mechanical and enzymatic dissociation techniques (Rando and Blau, 1994). Cultures are best derived from neonatal mice because the yield of myogenic cells decreases with age, especially within the first 3 weeks after birth. Dissect out the skeletal muscles from the limbs and place them in a glass dish, maintaining tissue culture sterility once the muscle is removed. Add just enough PBS to keep the muscle moist, and mince finely with razor blades. Enzymatic dissociation is then best carried out with a combination of a neutral protease, such as dispase and a colla11 (Boehringer Mannheim Corp., Indianapolis, Indiana) at 2.4 U/ml, genase, such as collagenase D (Boehringer Mannheim Corp.), at 1.5 Ulml.Add approximately 2 mi of the enzyme mixture (adjusted to the appropriate pH and Ca2+concentration for the specific enzymes) per gram of tissue and continue mincing for a few minutes. Transfer the contents to a sterile tube and incubate at 37°C until the mixture is a fine slurry, usually about 20-40 min. Filter the slurry through 80-pm nylon mesh to remove large pieces of tissue, centrifuge the filtrate to pellet the cells, aspirate off the supernatant, resuspend the cells in 5 ml of growth medium (see later discussion) per gram of tissue dissected,
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and plate on dishes coated with type I collagen (100 pgml applied for 6-8 hr at 37°C). Place in an incubator with 5% C02/95% air, and allow the cells to attach and begin proliferating for 2 days before changing the medium. B. Growth and Enrichment of Myoblasts from Primary Cultures
Primary cultures are mixtures of cell types, mostly myoblasts and fibroblasts. For myoblast transplantation, it is preferable to have as pure a population of myogenic cells as possible. Myoblast-selective growth conditions, although not rapid, yield pure and stable populations of myoblasts from primary cultures (Rando and Blau, 1994). Different combinations of nutrient mixtures and additives to the medium provide different stimuli either for cell growth or for differentiation. The combination listed next is an effective selection and growth medium for primary myoblasts in that there is preferential growth of myoblasts over fibroblasts and a very low rate of spontaneous differentiation of myoblasts. This growth medium is as follows: Ham’s F-10 nutrient mixture (Gibco BRL, Gaithersburg, Maryland) 20% fetal calf serum (FCS) (HyClone Laboratories, Inc., Logan, Utah) 2.5 ngml basic fibroblast growth factor (Promega Corp., Madison, Wisconsin) Penicillin (200 U/ml) and streptomycin (200 U/ml) During the first several passages, the technique of “pre-plating” removes a portion of the fibroblasts from the cultures (Richler and Yaffe, 1970) and is especially useful with primary cultures derived from older mice in which the proportion of fibroblasts tends to be higher than in those from neonatal muscle. Using collagen-coated dishes for pre-plating is much more rapid (as little as 5 min) and even more effective. The cultures should be maintained in growth medium at cell densities of between 30 and 70% confluence. After 2 to 3 weeks in culture, nearly 100% of the cells are myogenic as judged by staining for the intermediate filament protein desmin (Rando and Blau, 1994). Such populations have a remarkable proliferative capacity and can be maintained in culture for more than 30 population doublings. Using the same culture conditions, myoblasts can be cloned from primary cultures. It may be that using 1:1mixtures of growth medium and “conditioned medium” (e.g., growth medium collected from a confluent dish of C2 myoblasts or fibroblasts after 24 hr) is more effective than growth medium alone for growing cells at clonal density. This has not been rigorously tested. Cloning cells is more time-consuming and results in a more homogeneous population of myoblasts, but for some applications it may be preferable to have a clonal population. The most significant problem of growing mouse myoblasts is their propensity to detach partially from the dish and round up. Extended coating of dishes with collagen (e.g., 24 hr), not leaving cells on a dish for more than 5 days, and plating cells on dishes coated with laminin (which is much more expensive than type I collagen) have all helped to maintain the population in an adherent state.
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III. Labeling of Myoblasts in Ktro by Retroviral Mediated Gene Transfer For most applications of myoblast transplantation, it is essential to have a method of differentiating the donor cells from the host cells, whether to determine the fate of the transplanted cells, to assess the effect on host fibers of the incorporation of donor cells, or simply to assess the efficiency of the transplantation procedure. Retroviral-mediated gene transfer offers a rapid and efficient method of introducing a hereditable marker into dividing cells (Temin, 1986). The lac2 gene is widely used as a marker because of the available histochemical and biochemical assays of its product, the bacterial P-galactosidase (@gal) enzyme, but certainly other reporter gene markers would be useful as well (Alam and Cook, 1990). A. Retroviral Infection Replication-defectiveretrovirusescontaining the ZucZ gene have been effective for labeling primary mouse myoblasts (Rando and Blau, 1994). At the time of infection, it is useful to have several plates of retroviral producer cells at different densities so that when the myoblasts are ready to be infected, there is always a plate of producer cells at the appropriate confluence. Myoblasts should be 3040% confluent and producer cells should have just reached confluence at the time of infection. Remove the medium from the producer cells, filter it through a 0.45-pm filter, supplement it with FCS to 10% and with polybrene to 10 pg/ ml, and replace the myoblast growth medium with this virus-containing medium. Incubate the myoblasts in this medium for 4-6 hr, then replace it with fresh growth medium. To increase the percentage of myoblasts infected, this procedure can be repeated every 24 hr with little detrimental effect on myoblast growth. B. Assessment of mciency of Retroviral Labeling Typically, retroviral-mediated gene transfer is successful in only a subset of myoblasts (Fig. 1;see Color Plates). Depending on the specific properties of the retrovirus, the viral titer in the producer cell medium, and the infectivity of the myoblasts, the efficiency of gene transfer can range from less than 1%to greater than 50%. By staining a portion of the retrovirally infected cultures with X-gal (5-bromo-4-chloro-3-indolyl-~-D-galactopyranoside), a chromogenic substrate for @-gal(Pearson et ul., 1963),one can determine the efficiency of gene transduction. This compound yields a blue reaction product in cells expressing high levels of P-gal; thus, lacZ+ cells can be distinguished from ZucZ- cells. The staining of cells is done as follows:
1. Reagents Fixative; 4% paraformaldehyde, 0.25% glutaraldehyde in 100 mM Napi, pH 7.2
Ch. 1, Fig. 4 Appearance of quail and chick embryo during surgery. (A) The 16-somite quail embryo has been removed from the egg, rinsed free of contaminating debris, and staked out in a black-bottomed Sylgard-coated dish with insect pins, as described in the text. The embryo is located centrally within the clear area pellucida and is surrounded by the densely white area opaca, the margins of which already contain rust-colored blood islands. Other features of the embryo are indicated. (B) The 17-somite chick embryo is viewed through a window in the eggshell after black ink has been injected under the blastoderm, as described in the text. Somite 3, the first clearly demarcated somite in an embryo of this age, is indicated. The surgical target area is schematically represented with the somite number and somite stages indicated.
Ch. 1,Fig. 6 Histological discrimination between quail and chick nuclei in chimeric embryos. (A) Feulgen-stained cross section through a chimeric chick embryo 1 day after surgically replacing the lateral halves of wing level somites with those of quail as described in the text. The neural tube and wing bud are out of the field to the left and right, respectively. Under light microscopy, the quail nuclei contain bright, magenta-colored nucleoli (black arrows labeled Q-I, 4-2, and 4-3). whereas the staining of chick nuclei is pale and diffuse (white arrows labeled, C-I, C-2, and C-3). Nuclei marked C-1 and C-2 reside in cells of the dermomyotome and myotome, respectively. C-3 marks the nucleus of a sclerotome cell. Q-I marks the nucleus of a cell in the lateral part of the dermomyotome. 4-2 marks the nucleus of a dermomyotome-derived cell that has dissociated from the lateral edge of the nucleus of a cell in the lateral sclerotome. Note that the medial-lateral boundary between chick and quail cells extends from the dermomyotome through the entire sclerotome. Note also that the cells in the lateral most margin of the myotome are chick derived, whereas all of the cells of the lateral dermomuotome and migratory cells are quail derived. (B) Anti-quail antibody-stained cross section through a chimeric chick embryo 1 day after surgically replacing the dorsal half of a wing level somite with that of a quail as described in the text. The neural tube (nt) can be seen on the left-hand margin, whereas the wing bud is out of the field to the right. Under light microscopy, the quail nuclei contain densely stained, dark nucleoli (black arrows labeled Q-I, Q-2, and Q-3), whereas the staining of chick nuclei is pale and diffuse (white arrows labeled C-I, C-2, and C-3). Nuclei marked C-I, C-2, and C-3 are those of chick cells in the dorsomedial, ventromedial, and lateral sclerotome, respectively. Nuclei marked Q-I and Q-2 reside in the medial and lateral myotome regions, respectively. The nucleus marked 4-3 resides within a cell of the dermomyotome. Note that quail nuclei are restricted to dorsal somite derivatives, dermomyotome and myotome, and that chick nuclei are restricted to selertome, a derivative of the ventral somite half.
Ch. 2, Fig. 4 Examples of in situ hybridization performed after P-galactosidase staining on sections (A) or in whole mount (B).
(A) Ten-and-a-half-daychimeric mouse embryo generated with ES cells containing the nlucZ gene in the Msxll locus was fixed in 4% paraformaldehydefor 2 hr stained for P-gal activity overnight, refixed, embedded in paraffin, and sectioned. I n situ hybridization was carried out with an antisense MyoD probe, but using two radioisotopes, and the section counterstained with eosin (0.1%). In darkfield !.%gal+areas such as the limb (star) appear as pink, whereas intense areas of p-gal staining such as the dorsal neural tube (arrowhead) appear blue. Note expression of the skeletal muscle-specific, myogenic factor gene MyoD in the myotomes (arrowheads), where silver grains appear as white. (B) Nine-and-a-half-dayheterozygote embryo containing nlucZ in one allele of the myogenic factor gene myf-5. fixed in 4% paraformaldehyde for 3 hr stained for p-galactosidase activity. Positive nuclei are detected in the forming myotomes (arrow). The embryo was then hybridized with digoxigenin-labeled antisense Msxl. Note Msxlspecific expression in the neural tube (arrowhead) and limb bud (star), which appear as purple. Bar: 100 pm.
Ch. 2, Fig. 5 Double immunofluorescence of a 3-day-old organ culture of segmental plate from MLC3F/nIs-LacZ embryos, reconstituted with neural tubes isolated from nontrangenic siblings. The culture was first stained for P-galactosidase activity and then with an anti-sarcomeric myosin, MyHC polyclonal antibody (A), and an anti-PI11 tubulin monoclonal antibody (B). A MyHCpositive, P-gal-positive myogenic cell is indicated by an arrowhead. Bar: 10 pm.
Ch. 6 , Fig. 3 Analysis of cardiomyocyte differentiation by immunohistochemistry. (A) Phase contrast of a stage 1 1 embryo fixed for immunohistochemistry. (B) MF20-positive differentiated cardiomyocytes are located within the tubular heart. (C) Explant of cardiogenic mesoderm plus ectoderm removed from the embryo at stage 7 and cultured for 48 hr in MI99 minimal medium. Differentiation has occurred in culture as detected by the MF20 antibody.
Ch. 9, Fig. 7 ClAKE 1494 transgenic mice infected with RCASBP/AP(A). Mice were infected at day 5 and sacrified 7 days later. Leg whole mounts were prepared and stained for alkaline phosphatase (AP) acitivity (see Felderspiel er al., 1994). Panel A shows a leg that was infected with viral supernatant (0.024.03ml) containing 5000-7500 infectious units. RepresentativeAP stained with muscle fibers (f) that appear as purple streaks and satellite cells (s) that appear as purple dots are indicated. The experiment was repeated with RCASBP/AP(A) infected chicken embryo fibroblasts (CEFs) (panels B-D). Panel B shows a leg from a nontransgenic animal infected in parallel with infected CEFs. Panels C and D show the AP-positive fibers and cells on both sides of a leg whole mount from a transgenic animal. Reprinted in part, with permission from Felderspiel et al. (1994).
Ch. 12, Fig. 1 The efficiency of retroviral-mediated transfer of the lacZ gene can be determined by staining with X-gal. A culture of primary mouse myoblasts was infected with a retrovirus containing the lacZ gene and then fixed and stained with the P-gal substrate X-gal. The cells that contain the blue reaction product are those that have been successfully transduced. The percentage of the transduced cells in the culture is a measure of the efficiency of retroviral-mediated gene transfer. Bar = 60 pm.
Ch. 12, Fig. 4 Primary myoblasts fused to host fibers after transplantation. A suspension of primary myoblasts that were
selected by FACS to be virtually 100% labeled with the 0-gal marker were transplanted into the tibialis anterior muscle of an adult mouse. Three weeks later, the muscle was analyzed for the presence of hybrid myofibers; that is, host myofibers that have incorporated donor nuclei, by staining with X-gal. (A) The hybrid fibers as normal caliber fibers that stain blue; also present within the field are normal (not hybrid) host fibers that do not stain blue. (B) An adjacent section was stained with hematoxylin and eosin to demonstrate the normal histological appearance of the transplant. Bar = 3@m.
Ch. 13, Fig. 1 (A) Schematic depicting myotube culture in Matrigel. Note that the fibroblasts form a connective sheet on top of the Matrigel that can be physically removed after approximately X days in culture. Panels: ( I ) Cells are suspended in quail growth medium and plated on a dish that is first coated with collagen and then Matrigel. (2) Cells attach to the Matrigel surface by 12 hr after plating. (3) twenty-four to forty-eight hr after plating, the presumptive myoblasts move through the Matrigel in order to attach to the collagen. The fibroblasts remain on the Matrigel surface. (4) Six days after plating, fine forceps are used to peel off the sheet of fibroblasts, leaving the myotubes and presumptive myoblasts embedded in the Matrigel. (B) Demonstration of absence of nonmyogenic cells. After 21 days in culture, cells were stained with rhodamine-phalloidin to visualize actin filaments (red), and DAPI to visualize nuclei (blue). Note that all nuclei are associated with myotubes and that no fibroblasts are visible.
Ch. 13, Fig. 2 (A) Myotubes showing sarcomeric structures, visualized with anti-myosin heavy-chain primary antibodies and rhodamine-conjugated secondary antibodies. (B) Myotubes with structures incorporating overexpressed myosin essential light chains, visualized with anti-myosin essential light-chain primary antibodies and fluorescein-conjugated secondary antibodies. (C) Myotubes containing disrupted structures resulting from overexpression of myosin heavy chains, visualized with anti-myosin heavy-chain primary antibodies and rhodamine-conjugated secondary antibodies. (D) Myotubes with structures incorporating overexpressed troponin T,visualized with anti-troponin T primary antibodies and rhodamine-conjugated secondary antibodies.
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Staining solution: 5 mM K3Fe(CN)6,5 mM We(CN),, 2 mM MgC12in PBS X-gal stock solution: 40 mg/ml X-gal (Sigma Chemical Co., St. Louis, Missouri) in dimethylformamide (store at -2O"C, protected from light) 2. Protocol Fix cells for 4 min in cold (4°C) fixative. Wash cells in PBS for 5 min, 2 times. Dilute X-gal stock in staining solution to a final concentration of 1 mg/ml before use (1/40 dilution of X-gal stock). Apply diluted X-gal solution to cells; incubate at 37°C overnight. Examine microscopically for presence of "blue" cells (Fig. 1). In this assay, the efficiency of retroviral infection, and thus gene transfer, is determined as the number of labeled cells as a percentage of the total. For retroviruses that express very high levels of @-gal, shorter incubation times (4 hr) with X-gal will be sufficient.
C. Selection of Labeled Cells It is desirable to have as close to 100%of the cells expressing the marker as possible, and thus some method of selecting the labeled cells is needed. Cloning cells is straightforward but time-consuming. Since some retroviruses contain genes whose products confer drug resistance, growth of the population in the presence of the drug results in the survival only of cells infected with retrovirus. The gene for neomycin phosphotransferase, confering neomycin resistance, is useful because mouse myoblasts are sensitive to low concentrations (50 pg/ml) of the neomycin analogue, G418. The @-galenzyme itself can be used as a means of selecting infected cells by fluorescence activated cell sorting (FACS). In the presence of @-gal,the substrate FDG (fluorescein di-@-D-galactopyranoside)is cleaved to yield fluorescein. Thus, @-galexpressing cells have a high fluorescence intensity in the presence of FDG and can be separated from cells with low @-galactivity (Nolan et al., 1988). 1. Reagents
Staining medium: 4% FCS in PBS FDG solution: 2 mM FDG (Molecular Probes, Inc., Eugene, Oregon) in H 2 0 (make up as lOOX stock in 1:1mixture of H20and DMSO; store at -20°C, protected from light) Stop solution: 1mM phenylethyl-@-D-thiogalactoside (PETG), 1 pg/ml propidium iodide, in staining medium, on ice.
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2. Protocol
Trypsinize cells, centrifuge, and resuspend at lo7celldm1in staining medium. If necessary, pass cell suspension through 80 pm nylon mesh to filter out cell clumps. Transfer cell suspension to 4-ml FACS tube, reserving 100 pl of the suspension in a separate tube as control cells, to be analyzed in the absence of FDG. Place cell suspensions at 37°C for 10 min. Add 100 pl of FDG solution (warmed to 37°C) for every 100 p1 of cell suspension. For the control cell suspension, add 100 p1 of H 2 0 (37°C). Mix thoroughly and place the tubes at 37°C for 1 min. To stop the reaction and inhibit diffusion of the product out of cells, add to each tube 1.8 ml of ice-cold stop buffer for every 200 pl in the tube, and place the tubes immediately on ice. Set up cell sorter to detect fluorescein and propidium iodide; set autofluorescence compensation using the control cell suspension. Gate out the cells positive for propidium iodide (dead cells). Analyze control cells. Analyze FDG-treated cells and sort, selecting cells with highest fluorescence intensity (see next paragraph). Upon analysis of the control cells, a single peak of low fluorescence intensity should be seen (Fig. 2A). Analysis of the FDG-treated cells should reveal two peaks, both at higher fluorescence than the control cells (Fig. 2B). The cells represented in the peak with highest fluorescence are those expressing lac2 and should be selected by gating out cells in the lower peak. When cells obtained from this kind of sort are stained with X-gal, routinely >99% are found to be lacZ+. It is, of course, imperative to maintain sterile conditions during cell sorting. Adding gentamicin (50 pg/ml) to the culture medium for 2-3 days after sorting may diminish the likelihood of contamination from handling outside the hood. After remaining in suspension for a prolonged period, mouse myoblasts do not plate efficiently, so they should be plated at twice the usual density.
IV.Transplantation Techniques Cells thus enriched to be purely myogenic and selected to be virtually 100% labeled are ideal for transplantation. The following suggestions for the handling of the cells before transplantation are more anecdotal than rigorously tested Cells should be passaged no less than 48 hr before transplantation, should be provided with fresh medium between 12 and 24 hr before transplantation, and should be about 50% confluent at the time of transplantation. Harvest the cells by trypsinization and estimate cell number and viability using a hemacytometer
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Fig. 2 p-gal labeled myoblasts can be selected by FACS. Primary myoblasts were labeled with p-
gal by retroviral mediated gene transfer (see Fig. 1, Color Plates). Since only a portion of the cells were labeled, the labeled cells were selected by FACS using the substrate FDG. (A) Labeled myoblasts in the absence of FDG have a characteristic fluorescence intensity about a single peak. (B) In the presence of FDG, the cells fall into two peaks of fluorescence intensity: Cells that have not been infected by the retrovirus form a peak at low intensity; cells that have been infected and express high levels of @-galdisplay a much higher fluorescence intensity and form a second peak to the right.
and trypan blue exclusion. Wash the cells twice in serum-free medium, resuspend them at a predetermined density (e.g., 2 X lo7 cells/ml) in the buffer to be used for transplantation, and place them on ice. The buffer should be a nutrient mixture with protein added (e.g., 0.5% bovine serum albumin in F-10). Just before transplantation, resuspend the cells gently, draw up -100 p1 into a round, capillary pipet tip, and inject the suspension into the barrel of a Hamilton syringe with a 27-gauge needle attached. Insert the plunger into the barrel of the syringe, and the cells are ready to be injected. Approximately 70 p1 of cell suspension is sufficient to fill the barrel of the syringe and the needle. While the cells are being prepared for injection, anesthetize the mouse, shave the skin over the muscles to be injected, and secure the limbs on a dissecting board. Make an incision in the skin overlying the muscle to be injected and retract the skin to reveal the muscle. The tibialis anterior is particularly useful because it has well-defined borders and is easily accessible. With the aid of a dissecting microscope, insert the needle along the axis of the muscle fibers to the mid-belly of the muscle (Fig. 3). Slowly inject 5 or 10 p1 of the cell suspension, slowly withdraw the needle, and suture the skin closed. One of the critical aspects of successful transplantations is keeping cells viable before injection. Suspending the cells in PBS and keeping them at room tempera-
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Fig. 3 A representation of the injection procedure for myoblast transplantation. The tibialis anterior is revealedand is shown with a needle inserted into the midbelly region where the suspension of myoblasts will be injected.
ture leads to a loss of viability (as judged by trypan blue exclusion) of greater than 50% of the cells over a 6-hrperiod. Using the transplantation buffer listed earlier and maintaining the cells on ice maintains greater than 90% viability during the same time. Also for large-scale procedures involving dozens of mice, it is useful to have several dishes of cells and harvest them as needed such that “fresh” cells are being used throughout the day. Allowing the suspension to sit in the syringe leads to cell clumping and uneven cell injections.
V. Evaluation of Transplant The final assessment following myoblast transplantation will of course depend on the specific goals of the transplantation, whether it is for studies of muscle regeneration, for therapeutic gene transfer, or for analysis of gene expression in viva However, regardless of the goal, a measure of the outcome of the transplantation itself will be critical to any experiment. Such a measure or measures will allow assessment of transplantation efficiency, reproducibility, and
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stability, without which comparisons between experiments would be fraught with uncertainty.
A. Anatomical Assay After myoblast transplantation, an anatomical assessment is perhaps the most informative. The transplantation of cells into a complex, highly organized tissue such as muscle results in local disruption of the tissue architecture. The final histology after the transplant has matured is a combination of the regenerative capacity of the tissue and the interaction of the transplanted cells with that tissue. Thus, it is important to determine the fate of the transplanted cells, which may be quite dissimilar in host strains that have different regenerative capabilities of muscle. In particular, it is important to determine whether the transplanted cells have become incorporated into host fibers, have fused with other mononucleated cells to form new myotubes and myofibers, or have remained as mononucleated cells (which may be quiescent as satellite cells or may differentiate as myocytes; myoblasts from established cell lines may proliferate to form tumors). Additionally, a significant portion of the implanted cells die after transplantation, and the extent of this death can be assessed qualitatively in different transplants over time, as well as quantitatively using a biochemical analysis of implanted cell numbers (see later discussion). With P-gal as a marker, the anatomic fate of surviving myoblasts can be readily determined by staining frozen sections of transplanted muscle with X-gal. Muscles should be processed for sectioning using standard techniques, and cross-sections of muscle should be obtained. The thickness of sections should be between 15 and 30 pm for X-gal staining and less than 10 pm for routine histochemistry. To stain frozen sections with X-gal, use the same protocol described earlier for staining cultured cells. After staining, rinse the slides in PBS, coverslip them using an aqueous mounting medium, and examine them microscopically for the presence of @gal labeled fibers (Fig. 4A; see Color Plates). Stain adjacent sections with hematoxylin and eosin to evaluate the various cellular components of the tissue (Fig. 4B; see Color Plates). Hybrid fibers formed by the fusion of implanted myoblasts with host fibers are identified as @-gallabeled fibers of normal caliber (Fig. 4A). The fusion of implanted myoblasts with other myoblasts usually results in small-diameter pgal labeled fibers that are usually clustered at the site of cell injection. As a way of quantifying the data, the number of P-gal labeled fibers can be counted in a cross-section. From a single injection of 105myoblasts, up to 200 hybrid myofibers can be formed (Rando and Blau, 1994). The number of labeled fibers will vary along the length of the muscle and will diminish with increasing distance from the injection site, as will the intensity of P-gal staining. Thus, it appears that myoblasts do not move significantly from the site of injection, but remain relatively localized.
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B. Biochemical Assay In addition to an anatomical assessment, one can quantitate the transplantation results by measuring the total @-galactivity of the muscle in a tissue homogenate. Although no information can be obtained about the nature of the cells expressing the @-gal(i.e., whether they are unfused myoblasts, host fibers that have incorporated donor cells, etc.), this is a rapid way of comparing transplantation efficiencies under different conditions and over time. There are a variety of quantitative assays of &gal, but the following chemiluminescence assay using the P-gal substrate AMPGD (3-(4-methoxyspiro(l,2-dioxetane-3,2’-tricyclo[3..3.1.1 3~7]decan)4-yl)phenyl-@-~-galactopyranoside)is particularly sensitive (Jain and Magrath, 1991).
1. Reagents Extraction buffer: 100mMP-mercaptoethanol,0.1% Triton, 90 pg/ml phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory units/ml aprotinin, 100p M leupeptin in 100 m M Napi, pH 7.0 Assay buffer: 1 m M MgC12, 100 mM NaPi, pH 8.0 AMPGD stock solution: 10 mg/ml in a 1:1 mixture of H 2 0 and methanol (Tropix Inc., Bedford, Massachusetts) Emerald luminescence enhancer stock (Tropix Inc.), diluted 1 : l O in 0.2 N NaOH PETG solution: 26 mM PETG in H20
2. Protocol Sacrifice the animals and dissect out the injected muscles; mince each muscle briefly with a razor blade. Place minced muscles in tubes for homogenization and add 1 ml of ice-cold extraction buffer to each. Homogenize the tissue and then centrifuge at 4OOOg to pellet insoluble material. Transfer a determined volume (e.g., 10 pl) of the supernatant from each tube to a luminometer tube, and bring each sample up to 100pl with assay buffer. Dilute AMPGD stock solution into assay buffer 1:625. Add 200 p1 of diluted AMPGD solution to each tube and begin timing the reaction. After 15 min, add 25 p1 of PETG solution to each tube. Immediately before placing a tube in luminometer to read, add 300 p1 of diluted Emerald luminescence enhancer solution to the tube. Place tube in luminometer and integrate luminescent signal over 10 sec.
12. Methods for Myoblast Transplantation
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As a standard, an extract of the cells used for transplantation can be used to relate the &gal activity of the muscle to the implanted cells. Once the muscle is minced, it can be snap-frozen and stored at -80°C for more than a year with no significant loss of @gal activity, and the &gal activity in the tissue homogenate is stable at 4°C for at least several days, probably longer. The results of this assay give a measure of total &gal activity in the muscle as the chemiluminescent signal is proportional to the amount of &gal enzyme. By extrapolation Prom a cell standard, one obtains an estimate of the number of persisting transplanted cells. Such an estimate is useful for comparing techniques to optimize survival of transplanted cells and as a baseline to compare transplants under different experimental conditions. This estimate, however, assumes a constant relationship between a donor cell nucleus and @gal activity, a relationship that may change with time, especially after differentiation and fusion. Changes in cell biochemical activity that affect transcription, translation, mRNA stability, protein stability, and enzymatic efficiency could all alter the relationship between a donor-cell nucleus and its resulting P-gal activity. An independent measure of implanted cell nuclei, one that is conserved during replication and not dependent on cell biochemical activity, is needed to determine the accuracy to the estimate of implanted cell number by &gal activity.
VI. Concluding Remarks Myoblast transplantation provides a unique means to effect in vivo gene transfer because of the ability of implanted myoblasts to fuse with host myofibers. Among the limitations to more widespread use of myoblast transplantation is the time required for growth, purification, and labeling of cells and for detailed transplant analysis. Future advances will occur through improvement of the techniques described here, in particular more rapid methods of purifymg and labeling donor cells, more efficient methods of delivering myoblasts to the tissue, and, ideally, noninvasive methods of determining the fate of the implanted cells. References Alam, J., and Cook, J. L. (1990). Reporter genes: Application to the study of mammalian gene transcription. Anal. Biochem. 188, 245-254. Barr, E., and Leiden, J. M. (1991). Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 254, 1507-1509. Carlson, B. M. (1973). The regeneration of skeletal muscle. A review. Am. J. Anat. 137, 119-150. Dhawan, J., Pan, L. C., Pavlath, G. K., Travis, M. T., Lanctot, A. M., and Blau, H. M. (1991). Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 254, 1509-1512. Jain, V. K., and Magrath, I. T. (1991). A chemiluminescentassay for quantitationof beta-galactosidase in the femtogram range:Application to quantitationof beta-galactosidase in lacZ-transfected cells. Anal. Biochem. 199,119-124.
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Nolan, G. P., Fiering, S.,Nicolas, J. F., and Henenberg, L. A. (1988).Fluorescence-activated cell analysisand sorting of viable mammalian cellsbased on beta-D-galactosidaseactivity after transduction of Escherichia coli lacZ. Proc. Natl. Acad. Sci. USA 85, 2603-2607. Partridge, T. A. (1991). Myoblast transfer: A possible therapy for inherited myopathies? Muscle Nerve 14, 197-212. Partridge, T. A., Grounds, M., and Sloper, J. C. (1978).Evidence of fusion between host and donor myoblasts in skeletal muscle grafts. Narure 273, 306-308. Pearson, B., Wolf,P. L., and Vazquez, B. S. (1963). Lab. Invest. 12, 1249-1259. Rando, T. A., and Blau, H. M. (1994).Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J. Cell Biol. 125, 1275-1287. Richler, C.,and Yaffe, D. (1970).The in vitro cultivation and differentiation capacities of myogenic cell lines. Dev. Biol. 23, 1-22. Temin, H. M. (1986).Retrovirus vectors for gene transfer: Efficient integration into and expression of exogenous DNA in vertebrate cell genomes. In “Gene Transfer” (R. Kucherlapat, ed.), pp. 149-187. New York Plenum Press.