Experimental Cell Research 174 (1988) 252-265

Isolation of Human Myoblasts with the Fluorescence-Activated Cell Sorter

CECELIA WEBSTER,**’ GRACE K. PAVLATH,*,’ DAVID R. PARKS,? FRANK S. WALSH,3 and HELEN M. BLAU*33

Departments of *Pharmacology and tGenetics, Stanford Uniuersity School of Medicine, Stanford, California 94305, and #Institute of Neurology, Queen Square,

London WCI, United Kingdom

We have established procedures for the rapid and efficient purification of human myoblasts using the fluorescence-activated cell sorter. Our approach capitalizes on the specific reaction of monoclonal antibody 5.1H 11 with a human muscle cell surface antigen. For each of the five samples analyzed, an enrichment of myoblasts to greater than 99% of the cell population was immediately achieved. Following 3 to 4 weeks of additional growth in vitro, sorted myoblast cultures remained 97% pure. Differentiation of the sorted myoblast cultures, assessed by creatine kinase activity and isozyme content, was compara- ble to that of pure myoblast cultures obtained by cloning, and was significantly greater than that of mixed tibroblast and myoblast cultures. An average of lo4 viable myoblasts can be obtained per 0.1 g tissue, each with the potential to undergo approximately 40 cell divisions. Accordingly, if only two-thirds of this proliferative capacity is utilized, the potential yield approximates 10” myoblasts, equivalent to 1 kg of cells. Human myogene- sis in vitro is no longer limited by cell number and is now amenable to molecular and biochemical analysis on a large SCak. @ 1988 Academic Press, Inc.

The optimal in vitro system for the study of differentiation is one that most closely approximates in uiuo development. Primary cultures derived directly from muscle tissues have been widely used to study myogenesis, but the presence of a variable proportion of nonmyogenic cells is inevitable and often complicates the interpretation of results (for discussion see [l]). An alternative is provided by permanent cell lines that are entirely muscle. However, no human muscle cell lines have been isolated.

To overcome these problems we previously defined procedures for isolating and storing pure populations of myoblasts, or clones, derived from human postnatal tissues [2] and demonstrated their use in studies of human muscle differentiation [l-9]. Although our cloning method routinely yields ample num- bers of myoblasts per cell from normal human postnatal muscle biopsies, the yield of myoblasts obtained from muscle at other stages of development or individuals with certain neuromuscular diseases is often inadequate. The reason is that cells isolated from biopsies of older individuals [5, lo] or from patients with Duchenne muscular dystrophy (DMD) [4, 91 have reduced proliferative capacity which is often expended in the process of cloning. Cloning is further

’ Current address: Department of Biochemistry, University of California, Riverside, CA 92521. * Current address: Department of Biology, University of Arizona, Tucson, AZ 85721. ’ To whom reprint requests should be addressed.

Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/88 $03.00

252

FACS isolation of human myoblasts 253

limited by the number of clones that can be monitored and nurtured at any given time. Accordingly, cloning is relatively efficient when myoblasts comprise 95 % of the total cells plated as is the case for normal postnatal biopsies, but is not practical for tissues obtained at embryonic stages of development or from DMD patients in which the proportion of myoblasts is often less than 30% of the cells 141. Finally, cloning limits an analysis of myogenesis to the progeny of only a few myoblasts, which may not be representative of the majority.

Here we report a method for purifying large numbers of primary myoblasts rapidly and efficiently with the fluorescence-activated cell sorter (FACS). Immu- nofluorescence flow cytometry has previously proved useful in the analysis or isolation of different hormone-producing cells from the anterior pituitary [l 11, subpopulations of lymphocytes [ 12, 131, neuronal cells in rat brain [ 141, and pleural cell populations [15]. In some cases, intrinsic cell properties alone were sufficient to distinguish among the cell types of interest [ 13, 151. Our procedures for distinguishing myoblasts from fibroblasts employ a monoclonal antibody, 5.1H11, which specifically recognizes a human muscle cell surface antigen [ 161. In preliminary reports using this approach, different immunolabeling methods were described by us [17] and by others [18]. Here we describe the two different immunofluorescent staining procedures of the many we tested which, in conjunc- tion with intrinsic cell properties, adequately distinguish myoblasts from fibro- blasts with 95 % viability and an immediate increase in purity to -99 %. The yield of myoblasts is several orders of magnitude greater than that obtained by cloning [2] and is primarily limited by the origin and size of the muscle biopsy, since with the FACS approximately 6000 cells per second can be screened [13]. We demon- strate the need for such a method with well-defined mixtures of cloned human myoblasts and fibroblasts or unsorted cell mixtures. Muscle differentiation, assessed by creatine kinase specific activity and isozyme composition, is signifi- cantly inhibited in the presence of fibroblasts.

The potential to purify myoblasts efficiently from human tissues with the FACS should prove invaluable to the in vitro analysis of molecular and biochemi- cal properties of myogenesis. Furthermore, with these methods it is now possible to analyze myoblasts from muscle tissues at all stages of human development derived from normal individuals or those with one of the heritable human muscular dystrophies, such as Becker, Duchenne, or myotonic dystrophy that result from single-gene mutations inherited in a Mendelian manner.

MATERIALS AND METHODS Source of muscle. Muscle samples were obtained coincident with therapeutic abortion or corrective

surgical procedures, in accordance with the guidelines of the Stanford Human Subjects Committee. Parameters for cell sorting were optimized with a total of 20 muscle samples; a summary of the salient findings is presented here using the following 5 samples. Muscle from two fetuses (17 weeks gestation) was taken from the quadriceps (FI) or gastrocnemius (FII). Postnatal muscle was obtained from either the Vastus lateralis (NI, and sample XXIII from which pure populations of myoblasts and tibroblasts were cloned) or the spinalis (NII). In addition, FII was divided into two parts; half was immediately dissociated, and half was retained on ice, in Ham’s FlO, for 3 days prior to dissociation (FIII). XXIII, NI also known as XXXVI, and NII were 1.3,3, and 14 year of age, respectively.

254 Webster et al.

Muscle cultures. Muscle tissues were dissected free of skin, fat, and connective tissue, and then minced and dissociated in trypsin-EDTA. Methods for dissociation of postnatal tissue were as previously described [2]. Conditions for fetal muscle were similar except for the following modifica- tions: trypsinization was carried out in 50.ml conical centrifuge tubes (50 ml trypsin-EDTA/g tissue) without agitation, and cells were dispersed by trituration with a 25-ml pipette followed by filtration through Nytex mesh (85 urn, Tekto Inc.). For each sample, aliquots of dissociated cells were immediately frozen in liquid nitrogen for use at later times.

Cell culture methods for the isolation of myoblast and fibroblast clones, and for the long-term propagation of high-density cultures, have been described [2, 51. Primary cultures of fetal cells were plated at an initial cell density of 2-7x 10’ cells per lOO-mm dish, and sorted by FACS 2 to 5 days later. From fetal samples lo6 viable myoblasts per gram of tissue were obtained, a IO-fold increase over the average cell yield per gram from postnatal muscle plated at the same density. In addition, more cells were obtained from fetal than postnatal samples due to the larger size of muscle biopsies available from abortuses, approximately 2 g versus 0.2 g, respectively. Postnatal cultures were established from frozen dissociated cell stocks which had been stored in small aliquots. Accordingly, ampules con- tained as few as 10’ cells and had to be grown for 12-20 days in vitro to obtain sufftcient numbers for sequential analyses in FACS experiments designed to examine parameters of sorting. We have found that freshly dissociated cells that have not been plated in culture previously and lower cell numbers of 10’ can also be sorted (data not shown). All cultures were initially established in conditioned medium, which was replaced by growth medium when the cells reached 10% confluence as previously described [5]. Cultures were grown on collagen-coated dishes, fed every 2-3 days, and passaged prior to reaching confluence. Special care was required to prevent the precocious fusion of fetal myoblasts into myotubes, because these cells, unlike postnatal myoblasts, fused rapidly at low cell density despite growth in mitogen-rich medium and a greater proportion of fibroblasts. Fetal cultures were therefore fed daily and passaged upon reaching 40% confluence. Differentiation medium contained 2% horse serum, insulin, and dexamethasone [5].

Primary antibody for immunojluorecent labeling. Monoclonal antibody 5.1Hll was produced following immunization of a BALB/c mouse with human myotubes [19]. The specificity of 5.1Hll for a surface antigen present on human myogenic cells has been previously described [16, 19, 201. Primary antibody was either used directly as an undiluted hybridoma supematant or conjugated to Pan F Hapten (4-fluoro-3-nitrotrimethylphenylammonium iodide, PanAb Laboratories, San Jose, CA). For conjugation, 5.1Hll was purified from supematant by precipitation with 50% ammonium sulfate, dialyzed for 2 days against Dulbecco’s phosphate-buffered saline (PBS), and fractionated on an ABi-Gel protein A column using the MAP system for mouse IgG, puritication (Bio-Rad). Fractions containing greater than 1 mg IgG/ml were pooled and dialyzed overnight against PBS. Conjugation of hapten to purified 5.1Hll was carried out at 37°C for 3 h in a reaction mixture containing 2.5 mg antibody, 1.5 mg Pan F hapten, and 80 m&f sodium bicarbonate. Haptenated 5.lHll (H-5.1Hll) was dialyzed, filtered to sterilize, and stored at -20°C.

Immunojluorescent staining procedures. Cultures were harvested in trypsin-EDTA, washed twice by centrifugation for 2 min at 1500 rpm in calcium- and magnesium-free PBS containing 0.5 % bovine serum albumin (used for all washes and antibody dilutions), counted using a hemacytometer, and transferred to Eppendorf tubes. For Texas red staining, single-cell suspensions were incubated for 20 min consecutively with 5.1Hll supematant, biotinylated antimouse IgG antibody (7 &ml, Vector Labs), and Texas red-avidin (10 &ml) conjugated by the method of Hayakawa et al. [21]. For FITC staining, cells were labeled for 15 min each with haptenated 5.1Hll (20 ug/ml) and with FITC- conjugated anti-Pan F hapten antibody (2.5 ug/ml, PanAb Laboratories). Cells were stained at room temperature, and washed three times after each incubation by centrifugation for 15 s at 15,000 rpm in a microfuge. Propidium iodide (1 &ml final concentration, Sigma) was added during the last 5 min of the final labeling step, and was present thereafter [22]. For all reagents, aggregates were eliminated by centrifugation just prior to use.

FACS analysis. Cells were analyzed using a modified Becton-Dickinson cell sorter with a FACS IV optical bench and FACS II electronics [13, 231. FITC and propidium iodide were excited at 488 nm using an argon-ion laser. Texas red was excited at 590 nm by a tunable dye laser charged with rhodamine 6G, which was in turn excited by an argon-ion laser operating in all-lines mode. The light accepted on each fluorescence channel was defined by a six-cavity bandpass filter: 515-545 nm for FITC, 610-640 nm for propidium iodide, and 619-641 nm for Texas red (Becton-Dickinson Immuno- cytometry Systems). In addition to specific red or green immunofluorescence, green and red auto- fluorescence, forward and obtuse angle light scatter, and propidium iodide fluorescence were meas- ured. The fluorescence and light scatter channels were calibrated to standard sensitivity using fluorescent polystyrene microspheres (Polysciences, Inc., or Pandex Laboratories, Inc.). A flow rate

FACS isolation of human myoblasts 255

of 1000 cells per second was optimal for our purposes. For each sample, data from 5000 to 30,000 cells were collected in list mode on a VAXll/780 computer and processed as previously described [13,231. The proportion of each cell type present was determined by integration over selected regions of the multiparameter data space. Cells were collected into growth medium, either in %-well tissue culture plates at a frequency of one cell per well, or in 6-ml tubes for replating in polyclonal mass cultures.

Assays for creatine kinase (CK). The differentiation of myoblasts in pure cultures was compared to that in mixed cultures. Cultures were harvested and assayed for CK specific activity (milliunits/milli- gram of protein) and isozyme composition as previously described [24, 251, using a reagent kit from Boehringer Mannheim. Total protein was determined by the method of Bradford [26] with bovine serum albumin (Sigma) as a standard. Cell lysates were electrophoresed on nondenaturing 7.5% polyacrylamide slab gels (2000 V. h at 4”C), overlayed with 0.5% agarose containing the same reagents used in the activity assay, and incubated for 30 min at 37°C. Isozyme bands were visualized under uv illumination and photographed by exposure of Polaroid 665 film with a Wratten No. 21 filter for 20 min. Negatives were scanned on an EC densitometer, and the proportion of each isozyme present was determined by cutting out and weighing the peaks.

RESULTS

Criteria for Distinguishing Myoblasts from Fibroblasts with the FACS

To optimize the conditions for cell separation with the FACS, we used pure populations of myoblasts and fibroblasts derived from clones of individual disso- ciated cells obtained from postnatal muscle tissue. Procedures for cell handling, for immunofluorescent staining with monoclonal antibody 5.1H11, and for assay- ing intrinsic cell properties that could be useful in distinguishing between the two cell types were tested.

A major source of error in sorting arises when an unlabeled and a labeled cell coincide in the same drop and are identified as a single positive cell. Thus, dissociation of monolayers into a true single-cell suspension is essential. Several dissociation methods were evaluated and no difference in staining intensity with 5.1Hll was observed when cells were removed by scraping with a rubber policeman or by treatment with collagenase, dispase, trypsin-EDTA, or EDTA alone. We found that the mild trypsinization procedure routinely used in our laboratory (0.05 % trypsin, 0.02 % EDTA in PBS at 37°C for 2 min) resulted in the highest cell viability with the fewest cell clumps. Cell clumping was observed when 2 % horse serum in Ham’s FlO was used as the incubation medium for cell staining. Replacement of this medium with 0.5% BSA in calcium-magnesium- free PBS substantially reduced cell clumping without adversely affecting viabil- ity.

The low density of the 5.1Hll antigen on proliferating myoblasts made amplifi- cation of the specific immunofluorescence signal essential. Only two of the six staining procedures tested resulted in a suffkiently intense signal on myoblasts to permit a clear separation from fibroblasts. Figure 1 shows the staining patterns obtained from myoblasts, fibroblasts, and a mixed population following a three- step labeling procedure with S.lHll, biotinylated anti-mouse IgG antibody, and Texas red-avidin. Staining is clearly specific for myoblasts, and fewer than 10% of each cell type is within the area of signal overlap. By collecting only the 80% of myoblasts which stain at high intensity, the proportion of fibroblasts in the myoblast sample can be reduced to less than 0.02 %. An equally good discrimina-

17-888331

256 Webster et al.

Fig. 1. Myoblasts and tibroblasts are distinguished by the FACS following immunofluorescent labeling with 5.1Hll. Histograms of cell number (ordinate) and log Texas red immunofluorescence (abscissa) are shown for myoblasts, fibroblasts, and a mixed-cell population containing 40% tibro- blasts and 60% myoblasts. Broken lines: control staining in which 5.1Hll was omitted from the labeling procedure and the cells were exposed to second and third step reagents only. Solid lines: the cells were labeled with the 5.1Hll primary antibody, biotinylated anti-mouse IgG antibody, and Texas red-avidin.

Myoblasts

Fibroblasts

5.1Hll

tion between cell types can be achieved using fluorescein (FITC), despite a high background signal in the green due to cellular autofluorescence. A comparison of the two methods is shown in Fig. 2. Integration by computer revealed that the proportion of positive cells differed by only 0.1% when cells from the same sample were stained either with haptenated-5.1H11 (H-5.1Hll) followed by FITC anti-hapten antibody or by the Texas red procedure described above. The two- step method is rapid, requires less cell handling, and most importantly utilizes the more commonly available argon-ion laser for dye excitation. Nonspecific staining by second or third step reagents alone was negligible with both methods shown in Fig. 2. In contrast, the substantial overlap in the signals generated by myoblasts and fibroblasts precluded cell sorting with other reagents tested, including 5.1Hll directly conjugated to phycocyanine or phycoerythrin [13], or anti-mouse IgG antibodies conjugated to fluorescein or rhodamine. The last observation contrasts with that presented in a preliminary report by others [ 181 in which rhodamine was used. All of the experiments described below were performed using the three- step Texas red labeling procedure.

Several parameters that measure intrinsic cell properties were found to en-

FACS isolation of human myoblasts 257

0. II II I II II I .I 1 IO 100 .l 1 10 100

Texas Red Fluorescein

Fig. 2. Efficient separation of myoblasts from tibroblasts can be achieved by labeling with either Texas red or fluorescein. The same mixed-cell population is shown labeled with Texas red (A) or fluorescein (B) in two-dimensional contour plots of red or green immunofluorescence (abscissa) and intrinsic obtuse angle light scatter (ordinate), where 10% of the cells lie between each contour line (13). In this case, 87% of the cells were myoblasts (right-hand plots).

A I 200

100

0

200

100

tl

2

s 0

2 200 I

8

100

0

200

100

0

I a

B

.l 1 10 100 .l 1 10 100

5.1Hll

FI NI

Fig. 3. Enrichment of myoblasts from mixed primary cultures. Fibroblasts and myoblasts from Fetal I (F I) and Postnatal I (N I) samples are separated in two-dimensional contour plots of Texas red immunofluotescence (abscissa) and intrinsic obtuse angle light scatter (ordinate), gated for propidium iodide, forward scatter, and green autofluorescence. (A) The total initial population, containing both cell types. (B) The same population after setting a window for Texas red that allowed collection of nonoverlapping populations of myoblasts and tibroblasts. (C) An immediate reanalysis of the sorted myoblasts by the FACS. (D) Reanalysis of sorted myoblasts after growth for 17 (FI) or 18 (NI) days in culture.

258 Webster et al.

TABLE 1

Percentage of myoblasts in cell cultures before and after cell sorting

Sample (age) Initial Immediately Following growth

after sort in culture (days)

A. Fetal I(17 weeks)

II (17 weeks)

III (17 weeks)

B. Postnatal I (3 years)

II (14 years)

80 99.8 100.0 (17) 100.0 (27)

24 99.7 100.0 (17) 100.0 (20) 100.0 (27)

56 99.2 99.4 (14) 99.9 (21)

81 99.7 97.5 (17) 79 99.4 97.8 (18)

hance the yield and purity of myoblasts obtained with the FACS, in addition to the specific fluorescence due to staining with 5.1H11. With the FACS, informa- tion on multiple parameters can be gathered at a rate of several thousand cells per second. This information is then used for setting “gates” for each parameter, so that only those cells which fall within a defined window are collected. For example, light scatter measurements alone have previously been used to distin- guish among four cell populations in blood [ 131. We found that obtuse angle light scatter, which is influenced by internal cell structure, was consistently higher for fibroblasts than for myoblasts (Figs. 2 and 3). Forward angle light scatter, which is indicative of cell size, was used to gate-out cell clumps and noncellular debris. Cells with high autofluorescence, which are a source of false positives in immu- nofluorescent staining, were eliminated. Finally, propidium iodide, which is effkiently excluded from living cells but which stains dead cells intensely 1271, was used to measure cell viability and to eliminate dead cells from FACS analyses [22]. Each of these parameters contributed to the optimal discrimination of viable myoblasts, beyond that possible by immunofluorescence alone.

Separation of Myoblasts from Mixed Cultures

We determined that sorting conditions established for pure cultures of myo- blasts and fibroblasts derived from individual clones were directly applicable to mixed cultures obtained from dissociated fetal or postnatal muscle. In theory, cloned cells that had been in culture for 1 to 2 months might differ from freshly dissociated cells. Similarly, cells derived from muscle tissue at different stages of development, fetal and postnatal, might differ. However, the amount of antigen expressed per cell and intrinsic cell properties, such as autofluorescence and granularity that are frequently altered by long-term cultivation [ 131, did not differ significantly between clones that had undergone many doublings and early pas- sage cells, or between fetal and postnatal cells.

FACS isolation of human myoblasts 259

4,000, r

0 20 40 60 80 100

Fibroblasts in culture (%I

Fig. 4. CK activity in mixed cultures is disproportionately influenced by the presence of fibroblasts. The specific activity of CK was determined spectrophotometrically using a coupled enzyme reaction in triplicate assays of duplicate dishes, and the mean +SEM was graphed relative to the proportion of tibroblasts in the culture at the onset of fusion. Myoblasts and fibroblasts derived by cloning single cells from the muscle tissue of the same individual had been in culture for 30 or 32 days, respectively, prior to the experiment. Cells were harvested, counted, combined in ratios as indicated above, and plated at a confluent density of 8x 10’ cells per 60-mm dish. Cells were allowed to attach overnight in growth medium, fed with differentiation medium (5) the following day, and harvested for CK assays 4 days later. Under these conditions, contact inhibition and mitogen depletion combine to inhibit proliferation and the ratio of cell types in the cultures changes minimally overtime.

The effective use of multiple parameters in the isolation of myoblasts from mixed primary cultures derived from the muscle of two different individuals is shown in contour plots in Fig. 3, and the data for all of the samples analyzed are summarized in Table 1. In Fig. 3a, the samples were labeled with 5.1Hll antibody and gated on the FACS for propidium iodide, green autofluorescence, and forward angle light scatter. As a result, only viable single cells are plotted as a function of obtuse scatter and of specific immunofluorescence. Less than 5 % of the cells were labeled by propidium iodide, indicating that greater than 95% of cells presented to the FACS were viable. For collection, gates for Texas red intensity were then selected that eliminated the area of signal overlap and optimized separation of the two cell types (Fig. 3 b). The proportion of myoblasts within a window that eliminated all but 0.01% of the fibroblasts relative to the total myoblasts detected by the FACS reached as much as 80% (Fig. 2), but ranged from 35 to 86% in the different samples analyzed.

To determine the validity of the FACS separation, myoblast, fibroblast, and ungated control populations that were cell mixtures were collected separately. The degree of cross contamination of one cell type by the other was determined by an immediate reanalysis of aliquots of the sorted cells by the FACS. We found that the fidelity of the FACS separation was greater than 99% in each case and was not influenced by the proportion of fibroblasts initially present in the cultures (Figs. 3 a, c, and Table 1). Although fibroblasts comprised between 19 and 76% of the cells in the five samples analyzed, in each case this proportion was reduced to less than 1% by the FACS. Furthermore, the high level of purity of sorted muscle cultures was maintained following extended growth in vitro (Fig.

260 Webster et al.

3 d and Table 1). Cells collected by the FACS were replated in mass cultures in growth medium and cultured for an additional 14-27 days, at which time the purity of the resulting population was reassessed by the FACS. Fewer than 3% fibroblasts were present in any sample, and in two of the three fetal samples no fibroblasts were detectable.

We found that maintenance of muscle biopsies at 4°C for a period of days can serve as an effective initial enrichment for myoblasts prior to isolation by the FACS. We previously showed that neither the yield nor the viability of myoblasts was affected by storage of muscle biopsies at 4°C for up to 1 week prior to tissue dissociation, but that the majority of postnatal fibroblasts did not survive (5). This temperature effect is also apparent in the present study. The yield of myoblasts was the same, 1.7~ lo6 cells per gram tissue, for FII and for its derivative FIII, which was kept on ice for 3 days prior to dissociation. However, the number of fibroblasts obtained was reduced by two-thirds, from 5.3~ lo6 for FIT to 1.4~ lo6 for FIII, and is the basis for the relative increase in the proportion of myoblasts present in FIII (Table 1).

Differentiation of Sorted Myoblasts Is Increased Relative to Mixed Cultures

We assessed the capacity of myoblasts purified by the FACS to differentiate in vitro by assaying the specific activity and isozyme composition of creatine kinase (CK). Synthesis of the muscle-specific M-CK subunit is induced upon fusion of myoblasts into myotubes, and its expression correlates with an increase in CK enzyme activity. The nonmuscle isozyme is a BB dimer, and the muscle isozymes are MB and MM dimers. We compared CK activity and isozyme composition in cultures of myoblasts alone that were isolated either by cloning or by the FACS, and in mixed-cell cultures that contained myoblasts and fibroblasts. Cell mixtures were either predetermined, well-defined proportions of previously cloned and characterized fibroblasts and myoblasts (Fig. 4 and Table 2A) or derived directly from trypsin-dissociated muscle tissues (Fig. 5 and Table 28).

Following differentiation, CK-specific activity was significantly lower in the mixed-cell cultures of defined composition than in the pure myoblast cultures (Fig. 4). A 66% reduction in the specific activity of CK occurred when only 20% of the cells were fibroblasts. This result suggests that the presence of fibroblasts can adversely affect differentiation of mixed cultures to a degree which exceeds their proportion in the population. Similar results were obtained when myoblasts collected by the sorter were compared with unsorted mixed cultures from the same samples (Fig. 5). Although the absolute values differed between the two samples, in both cases the CK activity obtained from the sorted myoblast cultures was increased twofold relative to unsorted mixed cultures. The differ- ence in the magnitude of the inhibitory effect of fibroblasts on CK activity shown in Figs. 4 and 5 is likely to be due to the developmental stage from which the cells were derived, neonatal and fetal stages, rather than the method of isolation, cloning and cell sorting, respectively. We have noted that fetal cells have a greater tendency to fuse than neonatal myoblasts, irrespective of density, the

FACS isolation of human myoblasts 261

TABLE 2

CK isozyme expression in mixed and sorted cultures

Myoblasts M-CK (% of (% control

total cells) M-CK)”

M-CK (% of total)

subunits)

CK isozymes (% of total isozymes)

MM MB BB

A. Postnatalb 0 0 0 0 0 100

20 4 2 0 3 97 40 18 7 0 14 86 60 48 19 0 37 63 80 63 25 4 42 54

100 100 39 16 46 38

Il. Fetal' F I mixed 81 67 25 3 44 53 FI sorted 100 100 38 15 46 39 FII mixed 39 83 33 11 45 44 FII sorted 100 100 40 17 46 37

a M-CK content in cell mixtures was normalized in each case to the absolute value obtained in the corresponding control myoblast culture (100% myoblasts) to show correlation with the myoblast content of the culture. Absolute values for M-CK subunits relative to total M- and B-CK subunits in control myoblast cultures were 39 % for postnatal cells, 38 % for FI, and 40 % for FII. The proportion of CK isozymes in fetal cultures was determined on the day of peak CK activity (see Fig. 5).

b Postnatal cells were purified by cloning, and well-defined mixtures of cloned tibroblasts and myoblasts were analyzed 4 days after plating in differentiation medium (see Fig. 4).

’ Fetal cells were purified from mixtures with the FACS (sorted) and the proportion of myobasts in mixtures was determined by analysis of replicate cultures. The values shown here differ slightly from those given in Table 1 for fetal samples because the cultures were established at and propagated for different times. Mixtures shown here were from passage 4.

presence of increased proportions of fibroblasts, or mitogen-rich media (unpub- lished observations). In addition, the induction of CK activity upon differenti- ation of pure fetal muscle cultures (Fig. 5) was higher than that seen in pure neonatal muscle cultures (Fig. 4) [l, 21.

A measure of muscle differentiation that is frequently used is the proportion of CK isozymes that is MM. A two- to fivefold decrease in the proportion of MM- CK homodimer was observed in mixtures of purified cells (Table 2A), and in unsorted mixed cultures (Table 2B) relative to the levels obtained with the corresponding cultures of myoblasts alone. MM-CK was not detected in cultures containing less than 20% M-CK subunits, but increased linearly with M-CK content beyond this level in all samples analyzed. Thus, accumulation of the MM- CK isozyme was a function of M-CK subunit content. These data show that a comparison of the proportion of the MM-CK homodimer in different cultures is a less sensitive measure of the relative degree of differentiation than the proportion of M-CK subunits.

We determined the proportion of M-CK subunits relative to total M-CK and B- CK subunits. In pure myoblast cultures isolated from postnatal tissue by cloning, M-CK comprised 39 % of the total CK subunits (Table 2 A). Similar values were

262 Webster et al.

z FI F 11

6.000 al z h 6,000 - F 3 .5 4.000-

c’ 4

Days in fusion medium

Fig. 5. CK activity in mixed and sorted fetal muscle cell cultures. Mixed cultures from dissociated muscle tissues of F I and F II at passage 4 (0) and either passage 7 (F II) or 8 (F I) (A), were compared with myoblasts isolated from the same samples using the FACS (0). Duplicate dishes were harvested over a 3-day time course when myoblast fusion was at its peak (Days 2, 3, and 4 after transfer to differentiation medium) and assayed in triplicate at each time point. Values for CK activity are the means +SEM.

obtained with sorted myoblast cultures from fetal samples F I and F II (Table 2B), in which 38 and 40% of CK subunits were M-CK, respectively. Thus, the extent of myotube differentiation attained in enriched myoblast cultures isolated by the FACS is comparable to that in pure myoblast cultures isolated by cloning. In contrast, M subunit accumulation in mixed cultures was reduced, often to levels below that expected from the proportion of myoblasts present. This is apparent in Table 2, where we compare the proportion of myoblasts in mixed cultures (second column) with the proportion of M-CK subunits in the same cultures expressed relative to the corresponding (100%) pure control myoblast culture (third column). For example, in postnatal cultures containing 80% myoblasts, M- CK reached only 63% of the control value. A similar result was obtained for mixed cultures from the fetal sample F I. Although to a lesser degree, M-CK was also reduced in mixed cultures from the fetal sample FII.

DISCUSSION

The number of cells obtained with the FACS is sufficient for most biochemical and molecular analyses of human muscle gene expression in tissue culture. In fact, the number of human myoblasts that can be studied is now primarily limited by incubator capacity and the need to grow myoblasts as monolayers, rather than in suspension. The substantial number of myoblasts that can be obtained by cell sorting is illustrated by the following example. As we have shown previously, a typical size 0.2-g muscle biopsy from a normal postnatal donor will yield 2x lo4 myoblasts [2], a number two orders of magnitude greater than that which can be handled by cloning but which is readily handled by the FACS. The setting of gates in order to obtain myoblasts that are 99 % pure results in an average loss of only 50% of the myoblasts. Once plated in culture, each cell is capable of 40 doublings, or giving rise to a total of IO’* cells, a value which we determined by

FACS isolation of human myoblasts 263

serially passaging and counting the cells of single clones until they senesced [9]. In practice, only two-thirds of this in vitro lifespan, or lo8 cells, can be expended before the capacity to differentiate declines. Taken together, these data demon- strate that the potential yield from such a biopsy is lOI myoblasts, equivalent to 1 kg of cells from which approximately 10 g RNA, 8 g DNA, or 200 g protein can be isolated. This cell yield can be even higher, if the biopsy size is greater or the proliferative capacity is increased, as is the case for fetal myoblasts which are capable of at least 60 cell divisions [IO]. Thus, the study of human myogenesis in vitro is no longer limited by inadequate cell numbers.

The unique ability of the FACS to maximize cell yield while at the same time minimizing time in culture should allow us to analyze myoblasts which have extremely limited in vitro lifetimes. This is a common problem in the cultivation of cells from older individuals and from young patients suffering from neuromus- cular disorders. On average, myoblasts isolated from normal human donors approximately 50 years of age are capable of only 20 cell divisions [5,9], whereas those from a S-year-old boy with Duchenne muscular dystrophy can undergo only 15 divisions [9]. Thus, our sorting methods, which can be used with as few as lo5 freshly dissociated cells which were not previously plated in culture (data not shown) should facilitate the study of muscle differentiation with cells isolated from naturally occurring muscle mutants, such as Duchenne, Becker, or Myoton- ic dystrophies, which are inherited as Mendelian single-gene defects. In addition, cell sorting can be used to facilitate cloning when pure myoblast populations derived from single cells are desired, as in studies designed to examine potential myoblast heterogeneity at different stages of development or in disease states. When individual myoblasts were sorted by the FACS at a frequency of one cell per well directly into 96-well plates, between 30 and 60% gave rise to viable colonies (data not shown), a remarkably high viability and plating efficiency at clonal density. Finally, whereas previous methods for purifying myoblasts by cloning required a minimum of 2 weeks 121, cell sorting requires less than 5 h to perform.

The importance of using pure populations for assays of differentiation is apparent from our results with well-defined cell mixtures. Both the specific activity of CK and the relative content of the muscle-specific M-CK subunit were significantly reduced in mixed cultures in the presence of only 20 % fibroblasts, a proportion frequently obtained in freshly dissociated cells isolated from normal muscle tissue (Table 1). Moreover, the decrease in CK activity was not directly proportional to the percentage of fibroblasts in the cultures, illustrating that even when the cell composition is known, biochemical assays of differentiation may give misleading results. With biopsies from individuals with heritable myopath- ies, in which the proportion of tibroblasts may be 70 %, the problem is exacerbat- ed. This may in part explain previous reports of decreased CK activity and reduced proportions of MM-CK isozymes in myopathic relative to normal muscle cultures [28-301. In fact, when we analyzed pure muscle cultures, these differ- ences were not apparent [ 11.

The reduced differentiation of mixed cultures relative to controls may, in part,

264 Webster et al.

result from an inhibitory influence of tibroblasts on myogenesis. Possibly non- myogenic cells physically hinder myoblast contact and fusion. It is unlikely that tibroblasts secrete an inhibitory factor into the medium, since the conditioned medium in which we and others routinely culture myoblasts is frequently derived from fibroblast cultures and has no detectable deleterious effects [2, 311. Possi- bly the extracellular matrix produced by the fibroblasts is inhibitory to myoblast differentiation. Support for this possibility is provided by studies which demon- strate that myoblasts will not differentiate when plated on a matrix produced by fibroblasts, even after removal of the cells that produced it [32].

The need for primary myogenic cells to study normal differentiation is further underscored by recent evidence that some established myogenic cell lines may be regulatory mutants that do not precisely recapitulate normal muscle development [33]. The methods described here for human muscle cells should now also be applicable to muscle cells from other species, since antibodies have been de- scribed that are specific to antigens on rat [34] myoblasts. Finally, the ability to separate cell types directly from dissociated tissue with minimal or no prior exposure to cell culture conditions should facilitate the in vitro analysis of the impact of physiological and pharmacological alterations of cell function in vivo.

We thank Dr. Leonard Herzenberg for advice and encouragement, Ms. Lisa Peterson and Dr. Kimber Lee Poffenberger for providing Texas red-avidin, Dr. James Dasch for advice and assistance on antibody purification and conjugation, and Dr. Kimber Lee Poffenberger for a critical reading of the manuscript. Flow cytometry for this study was performed in the Shared Cell Sorter Facility at Stanford University (D. R. Parks, Director). C. W. and G. K. P. were supported by predoctoral fellowships from the NIH (CA 09302 and GM 07149, respectively). This work was supported by grants to H. M. B. from the Task Force on Genetics of the Muscular Dystrophy Association of America and thg NIH (HD18179), and NIH Research Career Development Award HD00850.

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Received June 22, 1987

Printed in Sweden