Embryonal carcinoma (EC) cells are developmentally pluripotential cells which can be induced to differentiate in cell culture to form a wide variety of cell types. To investigate the lineage relationships between cells of different types, we set out to isolate cell lines with multiple but restricted developmental potentials from differentiating cultures of P19 cells, a line of EC. By selecting for differentiated cells capable of anchorage independent growth, we isolated cell lines which differentiated in high density cultures to form at least two cell types; myocytes that resembled fetal skeletal muscle cells and loose connective tissue cells that secreted large amounts of type I collagen. These results suggest that skeletal myocytes and connective tissue share a common precursor and that stem cells with limited but multiple developmental potentials can be isolated from differentiating cultures of P19 cells.

Embryonal carcinoma (EC) cells are the stem cells of mouse teratocarcinomas. Some EC cell lines have developmental potential similar to the cells of the early embryo (Graham, 1977). Because EC cells can grow and differentiate in culture, they are an attractive system with which to investigate some of the processes of early mammalian development.

P19 cells are a pluripotential line of EC cells that develop into many differentiated cell types both in cell culture and in vivo (Rossant and McBurney, 1982). In culture, P19 cells differentiate into neurons, glial and fibroblast-like cells when aggregated and exposed to retinoic acid (RA) (Jones-Villeneuve et al. 1982; 1983). After aggregation and exposure to dimethyl sulfoxide (DMSO), P19 cells differentiate into various other cell types including cardiac and skeletal myocytes (McBurney et al. 1982; Edwards et al. 1983; Smith et al. 1987; Rudnicki et al. 1988). Although the physiological significance of RA and DMSO action remains unclear, the differentiated cells that appear in RA- or DMSO-treated cultures are similar to those found in the embryo and they appear with similar kinetics, suggesting that the developmental processes occurring in the differentiating P19 cultures closely parallel those that occur in embryos.

The cells of the early mammalian embryo are initially developmentally totipotent and become progressively more restricted in their developmental potential until fully differentiated cells with specialized characteristics are ultimately formed (Rossant, 1984). If the differentiation of P19 cells in culture occurs by progressive developmental restriction, one would expect that differentiating cultures should transiently contain cells with restricted potential. To isolate lines of cells with restricted developmental potential, we assumed that the properties of such cells might be intermediate between those of the parental EC cells and their fully differentiated progeny. Since EC cells are anchorage-independent and immortal while their fully differentiated derivatives are anchorage-dependent and senescent (Rodrigues et al. 1985; Bell et al. 1986), we selected for cells in DMSO-treated P19 cultures capable of forming colonies in suspension cultures. Cell lines recovered in this way were not pluripotential EC cells but appeared to be committed to develop into a subset of mesodermal lineages including embryonic skeletal muscle and loose connective tissue.

Cell culture

A subclone of P19 cells (McBurney and Rogers, 1982) transfected with pSV2neo (Southern and Berg, 1982) was used for these experiments. These P19(neo) cells were cultured and induced to differentiate with DMSO as described elsewhere (Rudnicki and McBurney, 1987). The cell lines derived from these differentiated cultures were maintained in exponential growth by subculturing before cultures became confluent. Differentiation was induced by allowing the cells to grow to confluence. These cultures were subsequently maintained undisturbed with changes of medium every two days.

Histological examination

To prepare MR16 and MR322 cultures for light and electron microscopy, the cultures were washed with buffered saline and the sheet of cells carefully removed from the culture dish with the aid of a rubber policeman. For light microscopy, the cell sheet was fixed with 3 % formaldehyde in buffered saline, dehydrated in ascending concentrations of ethanol, cleared in xylene, and embedded in Paraplast. 6 μm sections were cut and stained with hematoxylin-eosin-phloxine, alcian blue (pH 1.0 and 2.5), periodic acid-Schiff reagent and Masson’s trichrome.

Samples for electron microscopy were fixed in 3 % glutaraldehyde in 0.1 M-phosphate buffer, pH7.35, for at least 48h. Samples were postfixed in 1.0% osmium tetroxide, dehydrated in ethanol, and embedded in Epon-Araldite. Semithin sections were stained with toluidine blue. Thin sections (60nm) were cut on a Reichert Ultracut microtome, stained with uranyl acetate and lead citrate and examined with a Zeiss 10CA electron microscope.

Immunofluorescence experiments

Immunofluorescence experiments were performed as described previously (Rudnicki and McBurney, 1987) using the antibodies listed in Table 1.

Table 1.

Summary of muscle antigens detected by indirect immunofluorescence experiments

Summary of muscle antigens detected by indirect immunofluorescence experiments
Summary of muscle antigens detected by indirect immunofluorescence experiments

RNA analysis

Total RNA was isolated by the procedure of Auffray and Rougeon (1980). Northern blot analysis was as described (Rudnicki et al. 1988). The cardiac-actin isotype specific probe was the 0.45 kb HaeUI-Pstl fragment containing the 3’-untranslated (UT) region of the human cardiac-actin gene (Hamada et al. 1982; Rudnicki et al. 1988). The skeletal-actin isotype specific probe was the 0.42 kb Psii-BatriHI fragment containing the 3’-UT region of the mouse skeletal-actin gene (Hu et al. 1986). The muscle myosin heavy chain (MHC) mRNA was detected with the rat embryonic MHC cDNA (pMHC-25; Medford et al. 1980). The troponin-T (TNT) mRNA was detected with the rat skeletal-muscle TNT cDNA (pTNT-15; Garfunkel et al. 1982). The myosin light chain 2 (MLC2) mRNA was detected with the rat skeletal-muscle MLC2 cDNA (pLC2-18; Garfunkel et al. 1982). The MLC1&3 mRNAs were detected with the rat skeletal-muscle MLC1&3 cDNA (pLC-84; Garfunkel et al. 1982). The slow-MLC1 mRNA was detected with the human V-MLC1 cDNA 3’-UT region (Hoffmann et al. 1988). Tubulin mRNA was detected with the mouse alpha-tubulin cDNA (Lemishka et al. 1981).

Isolation of cell lines

P19(neo) cells were induced to differentiate by aggregation in the presence of DMSO (McBurney et al. 1982). On day 7, beating cardiac muscle cells but no skeletal muscle cells were present. These day 7 cultures were dispersed into single cells and suspended in growth medium containing 1.2% methyl cellulose to maintain the cells suspended above the solid plastic surface. After 10 days incubation in the methyl-cellu-lose-containing medium, one or two small colonies were found from each 103 cells seeded (Fig. 1, panels A and D). Individual colonies were picked from the methyl-cellulose, dispersed onto plastic surfaces, and expanded into cell lines.

Fig. 1.

Phase-contrast photographs of cell lines derived from DMSO-treated P19 cultures. Two myogenic cell lines, MR322 (A, B and C) and MR16 (D, E and F), were isolated from DMSO-treated P19 cultures. Single cells from both lines formed colonies 8 days after suspension in growth medium containing 1.2% methylcellulose (panels A and D). Both cells have fibroblast-like appearance when cultured on plastic surfaces (panels B and E). Cultures maintained following growth to confluence developed multinucleate myotubes some of which contracted rhythmically (panels C and F). Bar=50 μm.

Fig. 1.

Phase-contrast photographs of cell lines derived from DMSO-treated P19 cultures. Two myogenic cell lines, MR322 (A, B and C) and MR16 (D, E and F), were isolated from DMSO-treated P19 cultures. Single cells from both lines formed colonies 8 days after suspension in growth medium containing 1.2% methylcellulose (panels A and D). Both cells have fibroblast-like appearance when cultured on plastic surfaces (panels B and E). Cultures maintained following growth to confluence developed multinucleate myotubes some of which contracted rhythmically (panels C and F). Bar=50 μm.

Eighteen cell lines were derived in this fashion. In 7 of the 18 cell lines, cells spontaneously developed into multinucleate myotubes on reaching confluence (Fig. 1, panels C and F). These myotubes often contracted rhythmically. Two of these cell lines (MR16 and MR322) were investigated because they formed myocytes with different morphologies. Mononucleate and multinucleate myocytes were present in both cultures but myocytes in MR16 cultures developed into bipolar myotubes whereas, in MR322 cultures, myocytes and myotubes were spherical and seldom elongated.

The MR16 and MR322 cells had fibroblast-like morphology (Fig. 1, panels B and E), did not react with the anti-EC antibody AEC3A1-9 (Harris et al. 1984), and did not respond to RA by developing into neurons. Thus, these cells were not EC and were distinct from the parental P19 cells. However, the cells from these lines formed colonies in suspension with about 20 % the efficiency with which they formed colonies on plastic surfaces. Both of these lines have been cultured continuously for 30 or more passages, suggesting that they are immortal.

Not all cells in confluent cultures of MR16 or MR322 developed into muscle. Those cells that did not become muscle appeared to retain fibroblast-like morphology for at least 2 weeks following confluence.

Cells from the MR16 and MR322 lines have both been rigorously cloned by picking individual cells with capillary tubes (Rudnicki and McBurney, 1987). All nine subclones from MR16 and four of the five subclones from MR322 behaved like the parental cells in that both multinucleate muscle and nonmuscle cells were present in confluent cultures and the proportion of these two cell types appeared to vary only slightly from clone to clone. In addition, both MR16 and MR322 cells were plated out at clonal density and the colonies examined after 10 days of incubation. The plating efficiencies of these cells exceeded 50 % and in virtually all of the colonies both muscle and nonmuscle cells were present.

Microscopic examination

Differentiated cultures of both MR16 and MR322 were fixed and prepared for light and electron microscopy.Although the muscle cells in MR16 and MR322 cultures appeared different when viewed live, the sectioned < material from both cultures was indistinguishable and 1 will be discussed together.

Cultures of MR16 and MR322 cells prepared 7 to 20 days after reaching confluence contained three morpho-logically distinct cell types; muscle, a basal cell and loose connective tissue.

The muscle cells were variable in size with some measuring over 100 urn in diameter (Fig. 2). At least some of these muscle cells were multinucleate and contained extensive arrays of thin and thick filaments which occasionally formed myofibrils with distinct periodicity (Fig. 3C inset) and which were often so dense that they displaced other organelles such as mitochondria to the periphery of the cytoplasm (Fig. 3C).

Fig. 2.

Light microscopic photographs of cross-sections of differentiated MR16 and MR322 cultures. Cultures were fixed 7 to 10 days after confluence and prepared for microscopy. Panels A, B, and C show semithin (1 μm) sections stained with toluidine blue. (A) Low magnification illustrating the multilayered MR322 culture containing large myocytes (M), flattened epithelioid cells forming a basal layer (arrows) which was originally attached to the plastic surface, and irregularly shaped cells enmeshed in a stromal matrix. (B) Higher magnification photomicrograph of the matrix of a differentiated MR16 culture. Irregularly shaped stromal cells (arrows) and long cytoplasmic processes (arrowheads) were surrounded by a faintly staining, somewhat fibrillar matrix. (C) High magnification of the field in panel A showing the multinucleated myocyte (M) and stromal cells separated from the flat basal cells by a zone of matrix material (arrows). (D) A paraffin-embedded section stained with Masson’s trichrome showing the staining of stroma (S) composed of collagen fibrils (this material stained turquoise). Bar=10 μm in each panel.

Fig. 2.

Light microscopic photographs of cross-sections of differentiated MR16 and MR322 cultures. Cultures were fixed 7 to 10 days after confluence and prepared for microscopy. Panels A, B, and C show semithin (1 μm) sections stained with toluidine blue. (A) Low magnification illustrating the multilayered MR322 culture containing large myocytes (M), flattened epithelioid cells forming a basal layer (arrows) which was originally attached to the plastic surface, and irregularly shaped cells enmeshed in a stromal matrix. (B) Higher magnification photomicrograph of the matrix of a differentiated MR16 culture. Irregularly shaped stromal cells (arrows) and long cytoplasmic processes (arrowheads) were surrounded by a faintly staining, somewhat fibrillar matrix. (C) High magnification of the field in panel A showing the multinucleated myocyte (M) and stromal cells separated from the flat basal cells by a zone of matrix material (arrows). (D) A paraffin-embedded section stained with Masson’s trichrome showing the staining of stroma (S) composed of collagen fibrils (this material stained turquoise). Bar=10 μm in each panel.

Fig. 3.

Electron micrographs of differentiated MR16 and MR322 cultures. (A) Low magnification photomicrograph showing epithelioid basal cells (B) possessing intercellular junctions (arrow) and the sparse underlying stromal matrix containing collagen fibers. A mesenchymal cell (M) is also shown. (B) Low magnification showing the complex arrangement of collagen fibers, mesenchymal cells (M), and cellular processes. (C) Low magnification of a myocyte containing extensive arrays of myofibrils that displace mitochondria to the cytoplasmic periphery. Occasionally, the myofibrils were organized and showed distinct Z-bands (inset). (D) High magnification of collagen fibrils, some of which were associated with fine filaments (arrowheads). The collagen fibrils were about 600nm in thickness, but rarely showed structural periodicity. Bar=2 μm in A, B, C and 1 μm in D.

Fig. 3.

Electron micrographs of differentiated MR16 and MR322 cultures. (A) Low magnification photomicrograph showing epithelioid basal cells (B) possessing intercellular junctions (arrow) and the sparse underlying stromal matrix containing collagen fibers. A mesenchymal cell (M) is also shown. (B) Low magnification showing the complex arrangement of collagen fibers, mesenchymal cells (M), and cellular processes. (C) Low magnification of a myocyte containing extensive arrays of myofibrils that displace mitochondria to the cytoplasmic periphery. Occasionally, the myofibrils were organized and showed distinct Z-bands (inset). (D) High magnification of collagen fibrils, some of which were associated with fine filaments (arrowheads). The collagen fibrils were about 600nm in thickness, but rarely showed structural periodicity. Bar=2 μm in A, B, C and 1 μm in D.

The basal cells were those attached directly to the culture dish. They formed a monolayer consisting of a continuous sheet of flattened epitheloid cells (Figs 2A, 3C) upon which the other two cell types rested. These cells did not contain intermediate filaments containing cytokeratins as determined by immunofluorescence staining (data not shown). The basal layer was separated from the cells above by a translucent layer 2–6 μm thick which contained collagen fibrils and amorphous matrix material (Fig. 3A). Adjacent basal cells were connected by junctional complexes.

The majority of cells in these cultures appeared to be loose connective tissue. These cells were of various shapes and were enmeshed in a loose stromal matrix composed of collagen fibrils and a faintly staining ground substance. Many of these cells contained active granular endoplasmic reticulum and formed thin cytoplasmic processes which coursed randomly through the extracellular matrix (Figs 2B, 3A,B).

The extracellular material stained intensely green with Masson’s trichrome stain (Fig. 2D) confirming that collagen was present within this matrix. Variable staining was obtained with alcian blue and periodic acid-Schiff procedures suggesting the presence of some sulfonated proteoglycans. Three different antibody preparations reactive with type II collagen failed to stain the extracellular material indicating that these mesenchymal-like cells are not chondrocytes.

Characteristics of the myocytes

The muscle cells in the differentiated MR16 and MR322 cultures often contracted rhythmically and many were multinucleate. To determine whether they were cardiac or skeletal in nature, we used a number of antibodies and cDNA probes that recognize proteins and mRNAs specific to cardiac or skeletal muscle.

The muscle cells in MR16 and MR322 cultures were labeled in immunofluorescence experiments with the mouse monoclonal antibody, MF20 (Bader et al. 1982), which reacts with all forms of cardiac and skeletal muscle myosin heavy chain (MHC) protein (C/S-MHC). The cardiac-specific proteins, atrial-MHC (A-MHC) and atrial-myosin light chain 2 (A-MLC2), were not detected in any myocytes within differentiating MR16 and MR322 cultures (Fig. 4).

Fig. 4.

MLC proteins in MR322 myotubes. A and D are phase-contrast photographs of differentiated cultures of MR322 cells stained with either rabbit anti-A-MLC2 antiserum (B) or rabbit anti-V-MLCl antiserum (E), and mouse monoclonal antibody MF20 (C and F) reactive with muscle-specific MHC protein. Second antibodies were fluoresceiri-conjugated antirabbit and rhodamine-conjugated anti-mouse IgGs. Bar=50 μm.

Fig. 4.

MLC proteins in MR322 myotubes. A and D are phase-contrast photographs of differentiated cultures of MR322 cells stained with either rabbit anti-A-MLC2 antiserum (B) or rabbit anti-V-MLCl antiserum (E), and mouse monoclonal antibody MF20 (C and F) reactive with muscle-specific MHC protein. Second antibodies were fluoresceiri-conjugated antirabbit and rhodamine-conjugated anti-mouse IgGs. Bar=50 μm.

Antisera directed against the cardiac ventricular-myosin light chain 1 (V-MLC1) also react with skeletal slow MLC1 because these two proteins are encoded by the same gene (Barton and Buckingham, 1985). This antiserum labeled all of the MR16 and MR322 myocytes which also contained C/S-MHC protein (Fig. 4). Low levels of staining with antibodies against the cardiac ventricular-MHC (V-MHC) was observed in a subpopulation of the muscle cells (Fig. 5). This staining is likely a consequence of identity between the V-MHC and the skeletal-muscle slow (type I)-MHC proteins (Mahdavi et al. 1987).

Fig. 5.

MHC proteins in MR16 myotubes. A and D are phase-contrast photographs of differentiated cultures of MR16 cells stained with both rabbit anti-V-MLCl antiserum (B and E) and mouse monoclonal antibodies reactive against either A-MHC (C), or V-MHC proteins (F). Second antibodies were fluorescein-conjugated anti-rabbit and rhodamine-conjugated anti-mouse IgGs. Bar=50 μm.

Fig. 5.

MHC proteins in MR16 myotubes. A and D are phase-contrast photographs of differentiated cultures of MR16 cells stained with both rabbit anti-V-MLCl antiserum (B and E) and mouse monoclonal antibodies reactive against either A-MHC (C), or V-MHC proteins (F). Second antibodies were fluorescein-conjugated anti-rabbit and rhodamine-conjugated anti-mouse IgGs. Bar=50 μm.

The results of immunofluorescence experiments are summarized in Table 1. The multinucleate myocytes in differentiating MR16 and MR322 cultures do not express the cardiac-specific A-MLC2 and A-MHC isoforms suggesting that they are skeletal muscle.

The accumulation of muscle-specific transcripts in differentiating MR16 and MR322 cultures was examined by Northern slot blot analysis with a panel of DNA probes (Fig. 6). The level of alpha-tubulin mRNA remained relatively constant and was used to control for the amount of RNA in each slot.

Fig. 6.

Appearance of musclespecific transcripts in differentiating cultures of MR16 cells. RNA was isolated at daily intervals (days 0 to 10) from cultures which became confluent at day 0. Slot blots of this RNA were probed for a number of muscle-specific transcripts: A, cardiac-actin; B, skeletal-actin; C, embryonic myosin heavy chain (MHC); D, skeletal muscle troponin-T; E, skeletal-muscle myosin light chain 2 (MLC2); F, skeletal-muscle MLC1&3; G, slow-MLCl; H, alphatubulin. The relative mRNA levels were determined by scanning densitometry of the autoradiograms shown on the left and were plotted on the right as the percentage of the maximum level of expression normalized to the level of tubulin mRNA. Slots labeled C and S contained RNA from adult mouse cardiac and skeletal muscle, respectively.

Fig. 6.

Appearance of musclespecific transcripts in differentiating cultures of MR16 cells. RNA was isolated at daily intervals (days 0 to 10) from cultures which became confluent at day 0. Slot blots of this RNA were probed for a number of muscle-specific transcripts: A, cardiac-actin; B, skeletal-actin; C, embryonic myosin heavy chain (MHC); D, skeletal muscle troponin-T; E, skeletal-muscle myosin light chain 2 (MLC2); F, skeletal-muscle MLC1&3; G, slow-MLCl; H, alphatubulin. The relative mRNA levels were determined by scanning densitometry of the autoradiograms shown on the left and were plotted on the right as the percentage of the maximum level of expression normalized to the level of tubulin mRNA. Slots labeled C and S contained RNA from adult mouse cardiac and skeletal muscle, respectively.

Cardiac-actin and skeletal-actin are normally coexpressed in fetal heart and skeletal muscle. Both transcripts appeared in differentiating MR16 and MR322 cultures and accumulated to similar levels. The accumulation of the skeletal-actin mRNA appeared to lag slightly behind that of the cardiac-actin mRNA. Transcripts encoding MHC and slow-MLCl also increased during differentiation with kinetics similar to that of skeletal-actin mRNA.

The skeletal-muscle-specific transcripts encoding tro-ponin-T (TNT), MLC2, and MLC1&3 all appeared in differentiating cultures and accumulated with similar kinetics.

MR16 and MR322 cells are bipotential

MR16 and MR322 cells differentiate into skeletal myotubes on reaching confluence, in the same way as myoblast cell lines. However, the proportion of cells that differentiated into myocytes was never more than 20-40% and did not increase in cultures exposed to reduced concentrations of serum or exposed to horse serum, conditions that are normally used to induce extensive differentiation of myoblast cell lines. Nevertheless, it seemed possible that these cells might be myoblasts in which the efficiency of fusion is low. Recently a cDNA, MyoD, has been identified that reacts with a transcript thought to be expressed exclusively in myoblasts and myotubes (Davis et al. 1987). The Northern blot shown in Fig. 7 shows that the exponentially growing MR16 and MR322 cells contain very low levels of MyoD mRNA but that, very early upon reaching confluence, this transcript becomes abundant in these cells. By comparing Figs 6 and 7, it is apparent that the MyoD mRNA increase occurs earlier than any of the transcripts encoding the myofibrillar proteins, an observation consistent with the suspected role of the MyoD product as an intracellular inducer of myogenesis.

Fig. 7.

Differentiating MR16 and MR322 cultures accumulate abundant MyoD and type I collagen mRNAs. RNA was isolated from MR16 cultures at daily intervals (days 0 to 10) following confluence. This RNA was electrophoresed on formaldehyde-agarose gels, blotted to filter paper, and hybridized to probes for (A) MyoD mRNA (Davies et al. 1987), (B) type 1 collagen mRNA (Stacey et al. 1987), and (C) alpha-tubulin mRNA (Lemishka et al. 1981). No signal was obtained when these filters were probed for the chondrocyte-specific type 11 collagen mRNA (Kohno et al. 1984). The postions of the 28S and 18S rRNAs are indicated on the right of each panel.

Fig. 7.

Differentiating MR16 and MR322 cultures accumulate abundant MyoD and type I collagen mRNAs. RNA was isolated from MR16 cultures at daily intervals (days 0 to 10) following confluence. This RNA was electrophoresed on formaldehyde-agarose gels, blotted to filter paper, and hybridized to probes for (A) MyoD mRNA (Davies et al. 1987), (B) type 1 collagen mRNA (Stacey et al. 1987), and (C) alpha-tubulin mRNA (Lemishka et al. 1981). No signal was obtained when these filters were probed for the chondrocyte-specific type 11 collagen mRNA (Kohno et al. 1984). The postions of the 28S and 18S rRNAs are indicated on the right of each panel.

Also shown in Fig. 7 is the presence of type I collagen mRNA in differentiating MR16 and MR322 cultures. Type I collagen is expressed at high levels in cell cultures of prechondrocytes (Castagnola et al. 1988). The blots shown in Fig. 7 were also probed for mRNA encoding type II collagen (Kohno et al. 1984), but no signal was observed. Since type II collagen is chondrocyte-specific, its absence indicated that differentiated chondrocytes are absent or rare in these differentiated MR16 and MR322 cultures.

A large proportion of the nonmuscle cells in differentiated MR16 and MR322 cultures are able to resume proliferation following dispersal of differentiated cultures into single cell suspensions. Clonal lines from such cultures are fibroblast-like in morphology and resemble the MR16 and MR322 cells except that, on reaching confluence, most of these clonal lines do not develop myocytes (data not shown). Since virtually all subclones from exponentially growing cultures of MR16 and MR322 cells are able to develop into both muscle and connective tissue, it appears that, on reaching conflu ence, these MR16 and MR322 cells become irreversibly committed to myogenic or connective tissue lineages and that the loose connective tissue cells are capable of proliferation but not myogenesis.

Studies involving the micromanipulation of cells of the preimplantation mouse embryo have indicated that the first several developmental decisions in these embryos are binary and based on the position of cells within the embryo (Rossant, 1984). These early decisions all involve the formation of extraembryonic tissues. Relatively little is known regarding the formation of the cell types derived from the embryonic ectoderm, the tissue from which the fetus itself is formed, partly because the postimplantation embryo is almost inaccessible to experimental manipulation and partly because of the complexity of the developing fetus. Although there are indirect routes that may be applied to the embryo to trace lineages (Turner and Cepko, 1987; Alpert et al. 1988), a more direct means of learning the relationships between cells of different types is to isolate cells with multiple developmental potentials and compare the cell types that develop from each. For example, the O-A2 cell of the rat optic nerve appears to be a bipotential cell capable of giving rise to both oligodendrocytes and type 2 astrocytes (Raff et al. 1983). The identification of this precursor cell defines the relationship between these two differentiated cell types and allows one to determine the developmental signals involved in the choice between these two possible fates (Noble et al. 1988; Raff et al. 1988).

A number of continuous cell lines have been isolated which, under appropriate conditions, can be induced to differentiate into cells of two differentiated characters (Collins, 1987; Frederiksen and McKay, 1988; Bartlett et al. 1988). The results presented here demonstrate that differentiating cultures of P19 embryonal carcinoma cells can also yield permanent cell lines which are no longer pluripotential but are capable of differentiating into a limited spectrum of cell types.

These P19-derived cell lines, MR16 and MR322, appear capable of differentiating into at least two distinct cell types, skeletal muscle and loose connective tissue. Immunofluorescent staining experiments demonstrated the absence of the cardiac-specific isoforms A-MLC2 and A-MHC within MR16 and MR322 myocytes (Figs 4 and 5). Northern hybridization analysis showed the presence of the skeletal muscle-specific mRNAs, TNT, MLC2 and MLC1&3 (Fig. 6). Thus, the striated muscle cells in these differentiating cultures are skeletal in nature and not cardiac. Since cardiac-actin and skeletal-actin mRNAs are coexpressed, and since the myocytes express the embryonic isoforms, slow-MLC1 and slow type I MHC, the myocytes within MR16 and MR322 resemble embryonic skeletal muscle cells.

P19 cells, when induced to differentiate with DMSO, give rise to a spectrum of cells, many of which are normally derived from the embryonic mesoderm. In these DMSO-treated cultures, cardiac muscle appears relatively synchronously at 5 to 6 days post-treatment while skeletal muscle does not appear until 8 to 9 days (Edwards et al. 1983). This difference in kinetics appears to reflect the rates of maturation of the progenitor cells of these two populations and is similar to the kinetics seen in the embryo. The MR16 and MR322 cell lines were isolated from cultures disaggregated at day 7, so it is not surprising that skeletal but not cardiac muscle cells developed from them.

During embryonic development, segmentation of the lateral mesodermal plate gives rise to the somites from which myoblasts, fibroblasts and chondroblasts develop (Langman, 1981). The stem cell lines we have isolated from differentiating P19 cultures may be analogous to the mesodermal progenitor cells within the embryonic somites since they give rise to skeletal myocytes and loose connective tissue. At the present time, we have no evidence that the loose connective tissue cells are able to further differentiate into chondrocytes or osteocytes, perhaps because the culture conditions we used might not allow for their differentiation into these more mature cell types (Grigoriadis el al. 1988).

Differentiated cultures derived from pluripotent EC cells have been the source of cell lines with a variety of developmental characters (Rheinwald and Green, 1975; Jakob et al. 1978; Nicolas et al. 1980). All of these previously reported cell lines have the ability to develop into only one cell type. The experiments reported here demonstrate that cell lines with limited developmental potential can be isolated from differentiating cultures of pluripotential EC cells. Thus, the normally transient states of reduced developmental potential, anchorageindependence, and immortality may be suspended if differentiating cells are dispersed and allowed to form clonal colonies. The cell lines MR16 and MR322 appear to be analogous to a multipotential mesodermal progenitor cell and thus may prove useful in studies into cell differentiation and gene expression along the mesodermal lineages. By selecting for cells that arise in differentiating cultures of P19 cells earlier during the developmental process we expect to isolate cells with less restricted developmental potential. By determining the spectrum of cell types that arise from cells isolated in this fashion, we may be able to establish the developmental relationships between the various cell types that normally arise in differentiating cultures of EC cells.

We thank Dr Eunice Lee for help with identification of the loose connective tissue, Bethel Borgenson, Laura Hendricks, Karen Jardine, and Jane Craig for technical assistance, and the following colleagues for generously providing materials: E. Lee, J. Aubin, and G. Pringle for antibodies to type II collagen, G. Jackowski for antibodies against V-MHC, A-MLC2 and V-MLC1, and the human V-MLC1 cDNA, S. Schiaffino for antibody against A-MHC, V. Mahdavi for the cDNAs of rat MHCemb, troponin-T, MLC1&3 and MLC2, D. Fischman for MF20, L. Kedes for the human cardiac-actin gene, N. Davidson for the mouse skeletal-actin gene, and S. Tapscott for the MyoD cDNA.

This work was supported by grants from the Muscular Dystrophy Association of Canada and the National Cancer Institute of Canada. M.A.R. was supported by a Steven Fonyo Studentship from the National Cancer Institute of Canada and M.W.M. is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada.

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