Proliferation and differentiation of human fetal myoblasts is regulated by PDGF-BB

Differentiation (1993) 54:47-54

Differentiation Ontogeny, Neoplasia and Differentiation Therapy

0 Springer-Verlag 1993

Proliferation and differentiation of human fetal myoblasts is regulated by PDGF-BB Pei Jin', Karen Farmer 2 , Nils R. Ringertzl, Thomas Sejersen' ' Department of Medical Cell Genetics, Medical Nobel Institute, Karolinska Institutet, Box 60 400, S-104 01 Stockholm, Sweden ' Department of Cell Biology and Neuroscience, University of Texas, Dallas. USA

Accepted in revised form February 10, 1993

Abstract. A myoblast clone, G6, was obtained from thigh muscle of an 11 week old human fetus, and used to examine the effect of platelet-derived growth factor (PDGF) on cell multiplication and differentiation. G6 myoblasts showed extensive fusion, and expressed creatine phosphokinase activity and muscle specific gene mRNA (myosin heavy chain, a-actin) when switched to a differentiation medium. The cells expressed PDGF P-receptor mRNA, and bound 251-PDGF-BB specifically. Expression of PDGF P-receptors declined during in vitro differentiation. Relative levels of transcripts for the myogenic regulatory factors Myf4 (myogenin), Myf5, and Myf6 (MRF4) increased during the differentiation process, whereas Myf3 (MyoDl) was preferentially expressed in undifferentiated myoblasts. Treatment of the myoblasts with PDGF-BB increased DNA synthesis and cell density. Myogenic differentiation, analyzed as number of nuclei present in myotubes and expression of creatine phosphokinase and myosin heavy chain, was partly inhibited by the presence of PDGF-BB in the differentiation medium. PDGF-BB may, therefore, have the potential of regulating human muscle development and muscle regeneration.

Introduction

Growth factors play important roles in controlling myogenic differentiation [14,35]. Both fibroblast growth factor (FGF) and transforming growth factor type p (TGF- p) are potent inhibitors of muscle differentiation [30, 361. By contrast, the insulin-like growth factors (IGFs) are active in stimulating myoblast differentiation [l 1- 131.

Another potent growth factor for mesodermal cells, platelet-derived growth factor (PDGF), was originally discovered as a constituent of platelet a-granules with growth-promoting activity for smooth muscle cells and

fibroblasts. Subsequent studies have shown that this growth factor is synthesized by a number of normal and transformed cell types [23, 38, 391. PDGF is a dimer of two polypeptides, denoted A chain and B chain, linked by disulfide bonds. All three possible isoforms, AA, AB and BB, have been identified, and they bind with different activities to two distinct types of cell surface receptors known as PDGF c1- and P-receptors [17, 18, 19, 24, 411. The PDGF a-receptor binds all three forms of PDGF with high affinity. The PDGF P-receptor binds PDGF-BB with high affinity, and PDGF-AB with low affinity, but does not bind PDGF-AA [9, 10, 15, 19, 25, 31, 371.

Recent studies on rat and mouse myoblast lines suggest that PDGF is capable of regulating myoblast differentiation. Rat L6 myoblasts express the gene encoding PDGF P-receptors, and bind PDGF-BB specifically [27]. PDGF-BB stimulates proliferation and inhibits differentiation of L6 myoblasts [28]. Similar results were obtained using mouse C2 myoblasts [46]. However, nothing has hitherto been known about the role of PDGF in human myogenesis.

The aim of the present investigation was to examine if PDGF regulates human myoblast differentiation. The optimal in vitro system to study this is one that most closely approximates in vivo development. Primary cultures derived directly from muscle tissues have been widely used to study myogenesis. With this technique, muscle cells are inevitably contaminated by diverse cell types including nerve, adipocytes, and fibroblasts. These non-myogenic cells may affect results intended to reflect the situation in differentiating myoblasts. Furthermore, the ratio of myoblasts to non-myogenic cells influences the behavior of the myoblasts present, and muscle differentiation has been shown to be significantly inhibited by the presence of fibroblasts [44]. Therefore, we have used single-cell derived, cloned myoblasts from an aborted human fetus for analysis of myogenic markers, PDGF receptor expression, and effects of PDGF on proliferation and differentiation.

Correspondence to: T. Sejersen

4s

Methods

Preparation qf human fetal myoblasts. Muscle samples were obtained from an aborted human 73-day-old fetus with approval of the Ethical Committee of the Karolinska Hospital. Thigh muscle was dissected free of skin and bones under a dissecting microscope. The muscle was minced by cutting with scissors, and dissociated in 0.25% trypsin at 37" C with agitation. Cells in suspension were filtered, and collected by centrifugation. Myoblasts were enriched by preincubating the cells in cell culture dishes at 37" C in growth medium. Cells remaining floating in the medium after 30 rnin incubation were collected, seeded in 40-cm dishes, and cultured for 3 days in growth medium. To make a single cell suspension, the culture was rinsed twice and then trypsinized at 37" C for 15 rnin with 0.25% trypsin in calcium-, magnesium-free phosphate buf- fcred saline (PBS). Following trypsinisation, cells were seeded into a 96-well plate at a statistical density of 0.5 cell/well.

Cell culture media and PDGF. Growth medium used for myoblast proliferation was Ham's nutritent mixture F-10 with 20% fetal calf serum (Gibco/BRL AG. Basel, Switzerland), and 0.5% chicken embryo extract made in this laboratory [4]. Differentiation medium was Dulbecco's modified Eagle's medium (DMEM) with 5% horse serum (HS) (Gibco/BRL AG, Basel, Switzerland).

PDGF and its 'Z51-labeled product used in the present experiments werc provided by Dr. C.-H. Heldin (Ludwig Institute, Upp- sala, Sweden) and have been described previously [27, 281. Briefly, recombinant human forms of PDGF-AA and BB homodimers were purified from. the Saccharomyces cerevisiae expression system as described by Ostman et al. [37]. PDGF isoforms were lz5I-

labeled using the Bolton and Hunter protocol [5] to a specific activity of 160000 cpm/ng for PDGF-AA, and 66000 cpm/ng for PDGF-BB, respectively.

R N A analysis and D N A probes. Preparation of poly A+ RNA from cultured cells was performed as described previously [28]. Crude thigh muscle from the same fetus used to establish cloned myoblasts was freed of skin and bones, minced by cutting with scissors, and dissolved in 4 M guanidinium thiocyanate, 25 mM Na-citrate, 100 mM P-mercaptoethanol, 0.5% lauroylsarcosine, 0.1 % Anti- foam A (Sigma Chemical, St. Louis, Mo., USA). The sample was homogenized and extracted once with phenol/chloroform (1 : I), and precipitated in isopropanol. The pellet was dissolved in ice-cold sodium chloride-TRIS-EDT A buffer (STE), followed by the proce- dures used for preparation of RNA from cultured cells. Samples of 5 pg of poly(A)+ RNA were fractionated by electrophoresis through 2.2 M formaldehyde/l.l% agarose gels. Probes used for hybridization were as follows: the 1.5-kb cDNA fragment of human PDGF a-receptor clone 15.3 pUC [lo], the 1.0 kb PstI cDNA fragment of human PDGF p-receptor [9], the 1.3-kb cDNA fragment of human PDGF A-chain clone D1 [I], the mouse a-actin pAM 91 [32], the mouse fast myosin heavy chain MHC 32 1451, the recombinant plasmids MyD (MyoDI), Myf4 (myogenin), Myf5, and Myf6 (MRF4) [6, 71. Probes were labelled with 32P- dCTP (Amersham, Aylesbury, England) by random oligonucleo- tide priming or nick translation to a specific activity of 1-7x 10' cpm/pg DNA. X-ray films (Amersham) were exposed to hybridized blots for 16-72 hours.

Receptor binding assays. For ligand-receptor down-regulation assays, myoblast cultures in growth medium were treated with increasing amounts of cold PDGF-AA or -BB, at 37" C for 2 h. Cells were then washed once with ice-cold binding buffer (PBS containing 0.1 % bovine serum albumin, 0.9 niM CaC1, and 0.8 mM MgSO,), and incubated for 2 h at 4" C in 0.5 ml binding buffer containing 2 ng/ml of '251-labeled PDGF-AA or -BB, corresponding to their pretreatment

For receptor competition binding, cultures in growth medium or in differentiation medium were rinsed once with binding buffer and incubated for 2 h at 4" C in 0.5 ml binding buffer containing 2 ng/ml of 1251-labeled PDGF-AA or -BB in the absence or pres-

ence of 100-fold excess of the corresponding cold ligands. Cultures were then washed five times in ice-cold binding buffer and lysed by incubation in 0.5 nil lysis buffer (1% Triton/lO% glycerol/ 20 mM TRIS, pH 7.4) for 60 rnin at room temperature. Radioactiv- ity was determined in a gamma spectrometer (1282 CompuGamma, LKB Wallac, Stockholm, Sweden).

Assay of creatine kinase activity and D N A content. Cultures were washed twice with cold PBS and stored at -70" C after cell sur- faces were covered by the addition of 0.25 ml of 0.05 M glycylgly- cine, pH 6.75. Just before enzyme determinations, the cultures were thawed, scraped from the dishes and sonicated. Aliquots were taken for determination of creatine phosphokinase (CPK) activity as well as DNA content. CPK was measured using a Sigma kit 45-5 (Sigma Chemical, St. Louis, Mo., USA), and the activity was expressed in Sigma units at 25" C per min per pg DNA. DNA content was measured by a modification [I31 of the fluorometric method [29], using a Fluor-spectrophotometer (Shimadzu RS-SlOLC, Kyo- to, Japan) with the excitation wave length at 400 nm and the emis- sion wave length at 520 nm.

3H-thymidine incorporation. Human fetal myoblasts were cultured in growth medium in 16-mm wells for 48 h. The cells were then switched to DMEM containing either 2% or 5% horse serum as indicated in the text. Control wells received no further additives, while experimental wells received PDGF-AA or -BB at 20 ng/ml. Media were replaced at 24-hour intervals to ensure continuous supply of PDGF. Cells exposed to PDGF for different time peri- ods, were pulsed for 2 h with l pCi/ml [3H]-thymidine (Amersham, Aylesbury, England). The cells were thereafter rinsed three times with ice-cold DMEM. This was followed by washing 3 x 15 rnin in 1 ml of 5% ice-cold trichloroacetic acid (TCA). Cells were lysed for 30 rnin at 37" C in 0.5 ml of 0.5 N NaOH on a rocking plate. The NaOH suspensions were transferred into scintillation vials containing 3 ml of SAFE-E'LUOR-S (Lumak, The Netherlands). Ra- dioactivity was determined in triplicate wells by a liquid Scintilla- tion counter (1214 RACKBETA, LKB Wallac, Sweden).

Immunoblotting. Cells were scraped in PBS, lysed in sodium dode- cyl sulphate (SDS) sample buffer, and briefly boiled. Aliquots of cell lysates were compared for total protein content in dilution series in a dot blot assay (Amido black staining), and normalized amounts of protein samples were controlled by Coomassie blue staining following SDS polyacrylamide gel electrophoresis. Pro- teins were separated according to size by SDS polyacrylamide (12%) gel electrophoresis and electroblotted onto a nitrocellulose filter. After preadsorption with 5% non-fat drymilk powder in PBS overnight at 4" C, the filter was incubated for 2 h at room temperature with monoclonal anti-myogenin antibodies IF5-D7 or monoclonal anti-myosin heavy chain antibodies MF20, diluted 1.10 and 1:20, respectively, in PBS/O.lX Tween 20. The IF5-D7 antibodies were raised against amino acids 144170 of rat myogenin. The MF20 was obtained from the Developmental Studies Hy- bridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medi- cine, Md., USA, and the Department of Biology, University of Iowa, Iowa City, Iowa, USA, under contract N01-HD-6-2915 from the NICHD. Alkaline phosphate-conjugated anti-mouse IgG, diluted 1 : 1000 in PBS, were used as secondary antibodies. The filter was stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Rainbow protein molecular weight markers (Amer- sham, Aylesbury, England) were used.

Results

Generation of human muscle cell clones

Thigh muscle, dissected from a 73-day-old aborted fetus, was cut and dissociated by trypsinization. Myoblasts,

49

p-rec.

A-chain

MHC

p-actin

- m = )I Y

f 200 y. '1 1. i! l o O D 1 T

2 Days in differentiation medium

6

a-actin Fig. 2. Fusion of human fetal myoblasts in culture. G6 human fetal myoblasts were plated at 100 x lo3 cells per well in differentiation medium, with or without addition of 20ng/ml of platelet- derived growth factor-BB (PDGF-BB), for up to 6 days. Samples were removed and fixed every 24 h for Giemsa-staining and micro- scopic examination. Cells with three or more nuclei were counted as myotubes. The figure illustrates calculated total number of nuclei present in the cultures (open bars), without (lejt) or with addition of PDGF-BB, and the number of nuclei present in myotubes (striped bars). More than 1000 nuclei were scored in 15 random fields for each timepoint. Diagonal striping indicates absence of PDGF-BB, vertical striping indicates presence of PDGF-BB. Re- sults are the mean of triplicate wells from a single experiment. Standard error was less than 10%. The experiment was repeated twice with similar result

MYf3

Myf4

Myf5

Myf6

-1 0 1 2 3 4 5 6 days Fig. 1. Kinetics of messenger RNA levels during myoblast differentiation. G6 human fetal myoblasts were plated at low density and cultured to subconfluence in growth medium. The cells were then shifted to differentiation medium (day 0) and maintained in this medium for up to 6 days. Cultures were removed daily for RNA preparation. Five micrograms of poly A + RNA from each sample was fractionated through a 2.2 M formaldehydeil .I YO agarose gel, transferred to a nylon filter and hybridized with gene specific probes as detailed in Methods. MHC, myosin heavy chain

enriched by pre-adhesion, were seeded into a 96-well plate at a statistical density of 0.5 cell/well. The dissocia- tion of monolayer into a true single-cell suspension is essential in this cloning procedure. Following digestion of the cells with 0.25% trypsin in calcium-, magnesium- free PBS at 37" C for 15 min, no cell clumping was observed under an inverted phase contrast microscope. Clones arising from individual dissociated cells in the 96-well plate were trypsinized, subcultured and frozen in liquid nitrogen for long term storage. Simultaneously, individual clones were tested separately for their myogenic potential by plating the cells into 35-mm dishes, and analysing fusion capacity and CPK activities. A total of 15 clones were obtained in this experiment. Seven of these showed some initial ability to form myotubes, but most clones lost this ability upon sub-cultivation. Two clones, E6 and G6, were stably myogenic, forming

multinucleated myotubes and expressing creatine phosphokinase (CPK) (data not shown).

Proliferation and differentiation capacity

The limitation of longevity and the stability of differentiation potential are two major concerns when primary myoblasts are used to study myogenic differentiation. To minimize these problems, all experiments were performed using fresh, frozen-thawed myoblast cultures, and cells maintained in culture for more than 3 weeks were no longer used.

The two myogenic clones, E6 and G6, were similar in proliferation and differentiation capacity and in responsiveness to PDGF treatment. Therefore, we ran- domly picked clone G6 for more detailed studies. Results presented below are all obtained with this clone.

The cell doubling time for G6 myoblasts was approximately 34 h when cultured in 20% fetal calf serum (FCS). This could be reduced to approximately 25 h if the cells were fed fresh growth medium daily. Occasional myotubes were observed ( < 1 YO of nuclei), and accumu- lation of muscle specific a-actin transcripts already started in subconfluent myoblast cultures before they were exposed to differentiation medium (Fig. 1, compare day - 1 and day 0).

Fusion was rapid after shifting to differentiation medium (DMEM/5% HS). The fusion index (% nuclei in myotubes) increased from 3 to 64% between days 1 and 2 (Fig. 2). Formation of myotubes was parallelled by

50

a-rec.

p-rec.

A-chain

Myf5

Myf3

Fig. 3. Northern blot illustrating relative abundances of mRNA for PDGF receptors, PDGF A-chain, Myf3, and Myf5 in crude thigh muscle and myogenic and fibroblast-like cells derived from the same 11 week old fetus. Five micrograms each of poly A + RNA from G11 fibroblast-like cells cultured in growth medium ( G f l Fb), G6 myoblasts in growth medium (C6 Mh), G6 myotubes 6 days in differentiation medium (G6 Aft), and crude thigh muscle was analyzed

increased levels of myosin heavy chain (MHC) and cc- actin transcripts (Fig. 1) and CPK activity (Fig. 6). Fol- lowing day 3-4 in differentiation medium, a slight decrease was observed in fusion index (Fig. 2), and in expression of MHC and cc-actin (Fig. 1).

Expression of myogenic regulatory genes

Four human myogenic regulatory genes have been identified and isolated [6, 71. Transcripts of two of them, Myf3 and Myf4, were abundantly expressed in G6 cells. The expression of Myf3 (the human homolog of M y o D f ) reached its maximum when myoblasts became confluent. The relative abundance of Myf3 transcripts was significantly downregulated as the cells were shifted from growth medium to differentiation medium. In contrast, the expression of Myf4 (the human homolog of myogenin) was activated one day after the cultures were shifted to differentiation medium, and remained at a high level throughout the time period studied (Fig. 1). Transcripts of MyjS and Myf6 (the human homolog of MRF4) showed kinetics similar to that of Myf4. Both Myf5 and Myf6 expression was below the detection limit in G6 myoblasts, but became clearly detectable when myoblasts started to differentiate (Fig. 1). The relative levels of Myf3 and Myf5 transcripts in the G6 myoblasts diff- fered from the situation in total crude muscle from the same fetus (Fig. 3) . A high abundance of Myf3 transcripts was observed in G6 myoblasts, while Myf3 mRNA was barely detectable in the crude muscle. Myf5 transcripts, on the other hand, were below the detection

limit in G6 myoblasts but abundantly expressed in the crude muscle. G6 myotubes showed an expression pattern intermediate between that of G6 myoblasts and the crude muscle tissue. No cross-hybridization was observed between any of the different Myf members at the stringent hybridization conditions used.

E,xpression of PDGF A-chain and P-receptor mRNA

The PDGF A-chain gene was expressed in crude human fetal muscle tissue (Fig. 3), and in cloned G6 myogenic cells, proliferating as well as differentiated (Figs. 1 and 3) . Expression of this gene was, however, not detected in a non-myogenic clone G11. Clone G1 I is one of the 15 clones obtained from the 11-week-old fetus, and is most likely derived from muscle fibroblasts since these cells neither underwent fusion nor expressed any detectable muscle specific markers, including CPK and transcripts of the Myf family genes (Fig. 3 and data not shown).

PDGF P-receptor transcripts were expressed in the 1 1-week-old fetal muscle tissue, G6 myogenic cells as well as in GI1 non-myogenic cells (Figs. 1 and 3). The transcripts were most abundant in undifferentiated G6 myoblasts (Figs. 1 and 3) and were barely detectable in differentiated G6 inyotubes and in non-myogenic GI 1 cells (Fig. 3 ) . The expression of PDGF P-receptor mRNA in G6 myoblasts declined when differentiation occurred, and was inversely correlated to the appearance of the differentiation marker MHC and a-actin transcripts (Fig. 1). The expression of PDGF a-receptor mRNA was detected in crude muscle tissue of the 11- week-old fetus as well as in the non-myogenic G I 1 cells, but not in the G6 myogenic cells (Fig. 3).

Binding of 12'I-PDGF to hurnan,fetal muscle cells

In agreement with the results from Northern hybridization, G6 human fetal muscle cells showed specific binding of PDGF-BB, but not PDGF-AA. Pre-incubation of the myoblasts with cold PDGF-BB for 2 h at 37" C depleted cell-surface binding sites for PDGF-BB. The down-regulation of the cell surface binding sites by cold PDGF-BB was significant, dose-dependent, and satur- able (Fig. 4a). PDGF-AA, by contrast, showed no such effect a t any of the concentrations tested. The myoblast cultures bound more '"1-PDGF-BB than did the differentiated myotubes (Fig. 4b).

Increase in number of D N A synthesizing cells by PDGF-BB is dependent on serum concentration

Addition of PDGF-BB to mitogen-poor medium increased 3H-thymidine incorporation in the human fetal G6 myoblasts, while PDGF-AA showed no such effect (Fig. 5) . The increase in number of DNA synthesizing cells by PDGF-BB was affected by the serum concentration in the tissue cultures. Figure 5 b shows that PDGF-

51

a

40

I F : I l 20 0 0 1 0 2 0 30 4 0

Concentration of Cold PDGF (ng/ml)

b

Fa lZSI-BB+Cold BB

Mb Mt

Fig. 4. Receptor down-regulation assay. a G6 human fetal myoblasts were seeded at 4 x 104/16-mm well and cultured for 3 days in F10 medium containing 20% fetal calf serum (FCS) and 0.5% chicken embryo extract (CEE). This was followed by addition of increasing concentrations of cold PGFF-AA (0-0) or -BB (A-A) to the culture medium. Cells were then incubated for 2 h at 37" C to allow receptor-ligand internalization to occur. The cells were washed to remove unbound ligand, and further incubated at 4" C for 2 h with '251-PDGF-AA or -BB (2 ng/ml) corresponding to their pre-treatment. The cells were washed, lysed and the total cell-associated '''1 was determined. Data were plotted as mean binding as percentage of control binding (no preincubation with cold PDGF) for determinations made from triplicate wells. Variation in triplicate wells was less than 10%. b G6 human fetal myoblasts were seeded into 16-mm wells. Subconfluent myoblast cultures (Mb) in growth medium, or cultures maintained in differentiation medium for 4 days (Mt) were rinsed with binding buffer, and then incubated for 2 h at 4" C in binding buffer containing 2 ng/ml of 12sI-labeled PDGF-BB with or without 100 times excess of cold PDGF-BB. Results are means of triplicate wells, and variation was less than 10%. Cell densities were determined in parallel cultures by counting the trypsinized cells using an automatic parti- cle counter, or by counting Giemsa-stained cell nuclei using a light microscope

BB increased 'H-thymidine incorporation by four to five times in G6 inyoblasts maintained in DMEM containing 5% horse serum. PDGF-BB was, however, unable to increase DNA synthesis significantly in DMEM containing 2% horse serum (Fig. 5 a).

PDGF-BB inhibits differentiation of human fetal myoblasts

PDGF-BB partially inhibits differentiation of the human fetal myoblasts. As shown in Fig. 2, the number of nuclei present in myotubes was reduced by 50% in cultures treated with PDGF-BB for 2-4 days. At later timepoints the number of nuclei present in myotubes decreased in all cultures, with or without PDGF-BB treatment, due to detachment of myotubes from the plastic dish. Crea- tine phosphokinase activity was also repressed by PDGF-BB (Fig. 6a), and the cell density, measured as DNA content per dish, was increased by the presence of PDGF-BB in the differentiation medium (Fig. 6 b).

Because of the drastic increase in Myf4 expression in G6 cells after switch to differentiation medium (Fig. l), we analyzed if PDGF-BB down-regulates expression of Myf4 protein. As shown in Fig. 7, Myf4 and MHC proteins, migrating with apparent molecular

Control a H PDGF-AA

& 800

600

400

8 c c1

3

2 200

x J=

m 0

24 48 72

Time (hours) after PDGF Addition Fig. 5. Serum concentration dependent induction of 3H-thymidine incorporation by PDGF-BB. G6 human fetal myoblasts were cultured at 2 x 104/16-mm well in growth medium for 48 h. This was followed by a shift to 2% (a), or 5% (b) horse serum in Dulbecco's modified Eagle's medium (DMEM) for the indicated length of time in the absence or presence of 20 ng/ml of PDGF-AA or -BB, respectively. The media with or without PDGF were replaced every 24 h The cultures were exposed to 3H-thymidine at 1 pCi/ml 2 h before cell harvesting, and the extent of 3H-thym~dinc incorporation into trichloroacetic acid (TCA) precipitable material was ana- lysed. Results are shown as means of triplicate wells

18

15 ?

0 1 2 3 4 0 1 2 3 4

Days in Differentiation Medium Fig. 6. Effect of PDGF-BB on creatine phosphokinase activity and DNA content. G6 human fetal myoblasts were plated at low density and cultured to sub-confluence in growth medium. The cells were shifted to differentiation medium and maintained in thc medium for up to 4 days in presence (0-o), or absence (0. . . . '0) of PDGF-BB. Cultures were removed at 24-hour intervals for determination of creatine phosphokinase activity (a), and DNA content (b)

weights of 35 and 200 kDa, respectively, were both in- duced in G6 cells cultured in differentiation medium. However, PDGF-BB treatment did not cause any decrease in Myf4 protein as determined by Western blotting, although there was a decrease in MHC expression in G6 cells treated with PDGF-BB for 3 days. Similar results were obtained with immunofluorescence. In proliferating G6 myoblasts 6% of the nuclei were strongly positive for Myf4 immunoreactivity. After 3 days in differentiation medium, the percentage of strongly immu- noreactive nuclei was 35% in the absence of PDGF-BB and 29% in the presence of PDGF-BB. This difference

52

0 hl M w w w u - c M M

u u - c l c l c y . Fig. 7. Immunoblot illustrating: g z z r . -

Z Z M M lc

a specificity of anti-myogenin antibodies, b expression of Myf4 (human homolog of myogenin) in differentiating G6 myoblasts in the absence or presence of PDGF-BB, and c expression of MHC protein in differentiating G6 myoblasts in the absence or presence of 20 ng/ml of PDGF- BB. A minor 70 kDa band was detected by the anti-myogenin antibodies, except for the major 35 kDa Myf4 band (a and b)

I I P ~ P O

M H C

was not statistically significant. At the same timepoint, the number of nuclei present in myotubes in the PDGF- BB treated culture was reduced by 50%.

Discussion

PDGF has recently been shown to regulate myogenesis in muscle cell lines derived from rat [27, 281, and mouse [46]. However, the effect on human myoblasts has hitherto been unknown. In the present study we employed human fetal myoblasts to study the regulation of differentiation by PDGF. These myoblasts are single-cell derived to avoid contamination by non-muscle cells, and have received a minimum exposure to tissue culture conditions. Myoblasts capable of differentiation in vitro ap- pear in limb buds of human embryos before day 36 of development [20]. Muscle cells obtained from fetal stages of development often contain less than 30% myoblasts [4], and not all myoblasts obtained are capable of differentiation [3]. The majority of the clones obtained in our experiments were differentiation-incapable (13 out of 15 clones). The non-differentiating clones may be non-muscle cells, or they may include determined muscle cells which have not progressed to the point where they are capable of undergoing in vitro myogenic differentiation [20-221.

The two myogenic clones obtained require high concentrations of mitogens to proliferate. Similar cell culture conditions have previously been used to analyze proliferation of human muscle satellite cells [2, 161. Max- imal myoblast fusion occurred in mitogen-poor culture condition (5% HS). The differentiation of the human fetal myoblasts was prompt. A fraction of cells, however, underwent fusion gradually in one or two weeks. This heterogeneity in myoblast differentiation has also been observed in cultures of human muscle satellite cells [ 161. Initially formed myotubes seem less stable and undergo degradation soon after they were formed. Addition of insulin or dexaniethasone to the cultures did not change the differentiation potential significantly, nor did these agents stabilize the myotubes formed (data not shown).

The kinetics of expression of the four Myf family genes were examined in the human fetal muscle cells for two purposes. First, the expression of Myf genes are muscle lineage specific and also to some extent differentiation-stage specific. Therefore, patterns and kinetics of the Myf gene transcripts should be useful both as a muscle lineage marker and as a differentiation marker. Second, the expression pattern of the four Myf genes has not previously been analyzed in cloned human fetal myoblast cultures undergoing in vitro differentiation. Results obtained with the G6 myogenic cells showed that Myf3 was preferentially expressed in undifferentiated myoblasts, and that the expression of this gene was down-regulated as the cells started to differentiate. In contrast, expression of the other Myf genes was either extremely low or below the detectable level in undifferentiated myoblasts. In mitogen-poor medium, however, expression could be demonstrated as the cells began to differentiate. The fact that the expression pattern of Myf3 was the inverse of that of the other Myfgenes in human fetal myoblasts is consistent with the notion that Myf3 is controlled differently than the other Myf genes, and that different Myfgenes are used at different differentiation stages [34]. In agreement with previous observations [6,7], our results showed that the ll-week- old human fetal thigh muscle expressed an easily detectable level of Myf5, and a low level of Myf3. In G6 myoblasts obtained from the same fetus, the relative expression of Myf5 and Myf3 was opposite to that in the crude thigh muscle. This may be explained by differences in differentiation stage, the G6 cells representing a more immature phenotype than the majority of cells in the crude muscle tissue. However, it is also possible that the fetal muscle contains subclasses of myoblasts differ- ing in their Myfexpression pattern. In this context it is worth to note that, in contrast to primary muscle cultures derived from 20-22 week old human fetuses [6, 71, G6 myoblasts established from an 11 week old fetus expressed Myf4, Myf5, and Myf6 only after induction of differentiation, possibly reflecting the different ages studied. However, because of the low sensitivity of the RNA blotting method, we cannot rule out that low levels of one or more of these Myf transcripts are

53

present in G6 myoblasts. This was the case in cultured mouse C2 and So18 myoblasts, where cDNA-PCR procedure demonstrated Myf5 transcripts, previously unde- tected by Northern blot analyses [33].

The expression pattern of PDGF and PDGF receptor genes in the cloned G6 myoblasts and crude human fetal thigh muscle tissue correlate well with previous results on rat and mouse muscle cell lines. Skeletal muscle cells in all three species express the PDGF p-receptor, but no or very few PDGF a-receptors. The inverse correla- tion between down-regulation of the PDGF P-receptor expression and the appearance of muscle specific markers is similar to results previously obtained with rat L6J1 myoblasts [27, 281. The down-regulation of PDGF-P receptors is also analogous to the down-regulation of receptors for FGF and TGF-P in differentiating mouse or rat myoblasts [14, 361. Selective down-regulation of growth factor receptors may, thus, be important for myoblasts to transit from proliferation to differentiation. Interestingly, although both PDGF a- and b-receptor transcripts were present in crude thigh muscle, G6 myoblasts expressed exclusively the P-type, whereas muscle-derived G11 fibroblast-like cells expressed al- most exclusively the a-type PDGF receptor. The different distribution of PDGF receptor subtypes between G6 and GI 1 clones suggest that different isoforms of PDGF may affect different populations of cells present in muscle. The expression of the PDGF a-receptor mRNA in GI1 non-myogenic cells also suggests that PDGF A- chain expressed and secreted by the myogenic cells [40] may target muscle supporting cells, e.g. muscle fibroblasts, in a paracrine manner. Such effects have been speculated to be important in forming the myotendinous junctions [26].

Activation of the PDGF P-receptors by addition of PDGF-BB to the cell culture medium stimulates DNA synthesis and inhibits differentiation in human myoblasts. This observation is in agreement with the results obtained previously with the rat L6J1 line and the mouse C2 line. The kinetics of Myf4 expression in differentiating G6 cells suggested that it could be a likely target gene for the inhibitory myogenic effect of PDGF-BB, similar to the inhibitory effects of FGF and TGF-P on MyoDl expression [43]. However, our results from immunoblotting and immunofluorescence indicate that PDGF-BB does not repress the expression of Myf4 protein. Thus, the inhibitory effect of PDGF-BB on myogenic differentiation is likely to be independent of Myf4 content. Interestingly, also T G F - j appears to uncouple Myf4 (myogenin) expression from induction of other muscle-specific genes [8]. Furthermore, TGF-fi was re- ported to block myogenic differentiation in the presence of constitutively expressed myogenin [ 81.

It is intriguing to note that serum-concentration af- fects the effect of PDGF-BB on DNA synthesis in fetal myoblasts. It suggests that factor(s), present in the horse serum is required for a significant induction of DNA synthesis by PDGF-BB. We have also observed that the mitogenic response of PDGF-BB on G6 myoblasts is affected by cell density (data not shown). These findings require further analyses on the interaction of PDGF-BB

with other mitogens in stimulating proliferation of human myoblasts.

Taken together, our results demonstrate that cloned human myogenic cells in culture express PDGF p-receptors, which undergo down-regulation during in vitro differentiation. These cells also express the PDGF A-chain gene and respond to PDGF-BB treatment by increased DNA synthesis and decreased myogenic differentiation. This suggests that PDGF is involved in regulating normal human muscle development and regeneration, supported also by the recent finding that PDGF-receptor concentration is elevated in regenerating mouse muscle fibers [42]. Further work will attempt to determine if possible differences in growth factor or growth factor receptor setup may account for the markedly higher capacity of muscle from mice than humans to regenerate in dystrophic conditions.

Acknowledgments. We thank Mrs. Gabriella Dombos for excellent technical assistance. We are indebted to the following colleagues for providing us with material: Dr. C.-H. Heldin (recombinant and '25T-labeled PDGF), Dr. C. Betsholtz (PDGF A-chain gene), L. Claesson-Welsh (PDGF a- and P-receptor genes), Dr. H.H. Ar- nold (Myfgenes distributed by American Type Culture Collection).

This work was supported by the Swedish Medical Research Council, the Swedish Cancer Society, and funds of Karolinska In- stitutet.

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Proliferation and differentiation of human fetal myoblasts is regulated by PDGF-BB