EXPERIMENTAL CELL RESEARCH 232, 287 –294 (1997)ARTICLE NO. EX973532

In Vitro Characterization of Chondrogenic Cells Isolated fromChick Embryonic Muscle Using Peanut Agglutinin

Affinity Chromatography

Emanuela Stringa, Jane M. Love, Sarah C. McBride, Eiko Suyama, and Rocky S. Tuan1

Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

nificant cellular subpopulation of various differentiatedSpecific binding to the lectin, peanut agglutinin tissues and the marrow stroma (Friedenstein, 1976;

(PNA), has been reported in embryonic precartilage Hall and Watt, 1989) and are responsible for processestissues, including the condensing limb bud blastema such as wound healing, tissue regeneration, and ectopicand the caudal half of the developing somite. The pres- tissue formation, particularly with respect to bone andent study aimed to test the hypothesis that PNA-bind- cartilage (Caplan, 1991; Owen, 1988). The classic stud-ing may be a surface characteristic of chondroprogeni- ies of Urist (1965) and Reddi and Huggins (1972) weretor cells residing within noncartilage tissues, such as the first to demonstrate the presence of chondroprogen-muscle, which have the potential of being induced to itor cells in muscle which undergo ectopic endochondralform cartilage, e.g., in the presence of bone matrix- ossification upon the implantation of demineralizedderived factors. Day-14 chick embryonic pectoral mus- bone matrix, later shown to contain a number of chon-cle, which contained histochemically detectable PNA- dro- and osteo-active growth factors (Wozney et al.,binding cells, was dissociated into single cells (TM

1990). There is, however, relatively little informationcells) and fractionated by PNA affinity chromatogra-on the properties of these chondroprogenitor cells, e.g.,phy into PNA-binding (PNA/) and nonbinding (PNA0)in comparison to myoprogenitor cells (Antin and Or-cells by PNA-Sepharose 6 MB affinity chromatography.dahl, 1991; Emerson, 1993; Ontell et al., 1995), particu-The differentiation potential of the PNA-affinity frac-larly those which reside in connective tissues.tionated cells in vitro was analyzed as a function of

In the developing limb bud, a widely used experimen-culture plating cell density. Immunohistochemistry ofa number of cell-type-specific differentiation markers, tal system for the study of chondrogenesis, the appear-including sarcomeric actin, collagen type II, and ag- ance of cellular condensation in the central region pre-grecan core protein, demonstrated that PNA/ cells, cedes cartilage differentiation, and is the first morpho-when cultured as a micromass at high density (20 1 logical sign of cellular commitment to chondrogenesis106 cells/ml), exhibited a chondrocyte-like phenotype, (Ede, 1983; Thorogood and Hinchliffe, 1975). The cellswhereas the PNA0 cells remained myogenic; however, contributing to this condensation are derived from theboth PNA/ and PNA0 monolayer cultures (4 1 104

subridge mesoderm of the limb bud, which contains acells/ml) behaved as myoblastic cells. The expression highly enriched population of chondrogenically com-of collagen type II mRNA was also confirmed by cou- mited cells (Kosher et al., 1979; Newman et al., 1981;pled reverse transcription/polymerase chain reaction Summerbell, 1973). Aulthouse and Solursh (1987),analysis. These observations suggest that PNA bind- Zimmermann and Thies (1984), and Milaire (1991)ing, i.e., the presence of specific galactose-containing

have shown that the lectin, peanut agglutinin (PNA),cell surface moieties, is likely to be one of the charac-which recognizes the disaccharide Gal(b1,3)GalNAc,teristics of chondrogenic cells residing in mesenchy-binds specifically to the precartilage blastema of themally derived embryonic tissues. q 1997 Academic Pressdeveloping limb bud. Binding appears to be extracellu-lar and is preferential for cellular aggregates comparedto loosely arranged mesenchymal cells. In addition, inINTRODUCTIONthe developing somite of the chick and human embryos,PNA has also been shown to bind only to the caudalIt has been proposed that mesenchymal multipotentsclerotomal region, which contains the cell populationor pluripotent stem cells constitute a small, but sig-that give rise to vertebral cartilage (Bagnall and Sand-ers, 1989; Gotz et al., 1991; Stern et al., 1986).

1 To whom correspondence and reprint requests should be ad- When limb mesenchymal cells are placed in high-dressed at Department of Orthopaedic Surgery, Thomas Jeffersondensity micromass cultures, a commonly used in vitroUniversity, 501 Curtis Building, 1015 Walnut Street, Philadelphia,

PA 19107. Fax: (215) 955-9159. E-mail: Tuan1@jeflin.tju.edu. system to study chondrogenesis (Ahrens et al., 1977;

287 0014-4827/97 $25.00Copyright q 1997 by Academic Press

All rights of reproduction in any form reserved.

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288 STRINGA ET AL.

were seeded directly at 4 1 104 cells/ml and maintained in the sameSan Antonio and Tuan, 1986), the cells first undergomedium.distinct aggregation, and later the aggregates become

cartilage nodules (Solursh et al., 1978). Interestingly,Cell Fractionation by PNA Affinity Chromatography

the pattern of PNA binding in high-density micromassAntibodies against peanut agglutinin (PNA) (Arachis hypogaea lec-culture of limb mesenchymal cells was similar to that

tin; Sigma) were covalently linked to CNBr activated Sepharose 6seen in the limb bud, i.e., associated with condensingMB macrobeads (Pharmacia, Piscataway, NJ) according to the proto-regions (Aulthouse and Solursh, 1987). PNA binding col of the manufacturer, and the conjugated resin was packed into a

coincided with aggregation of cells into condensations, column constructed from a 10cc syringe. Isolated embryonic chickenbreast muscle cells (TM) were incubated at 377C with PNA (200 mgand preceded deposition of cartilaginous extracellularPNA/50 1 106 cells/ml) for 20 min, washed with CMFSG, pelleted tomatrix. In our recent study, we have successfully ap-remove the excess PNA, resuspended in 2 ml of CMFSG, and addedplied cell sorting using the PNA-binding property toto the Sepharose column to allow immunoadsorption to occur for 30isolate a chondrogenic cell subpopulation from the min. The column was then washed once with CMFSG to remove cells

chick embryonic calvarium, an intramembranous bone which did not bind PNA (PNA0) during the first incubation. Theresin was then eluted with 0.1 M galactose to compete for the sugar(Stringa and Tuan, 1996). These findings raise the pos-binding site of the lectin to isolate the PNA-binding (PNA/) cells.sibility that PNA binding, i.e., the presence of specificFinally, the column was regenerated by washing with changes ofgalactose-containing cell surface glycoproteins, is a mo- high pH (10) and low pH (4) buffers and stored at 47C in 0.5 M NaCl.

lecular marker for chondroprogenitor cells of mesen-chymal origins, which are capable of undergoing aggre- Histochemistry and Immunohistochemistrygation and chondrogenesis, if and when provided with

Histochemistry. Some cultures were stained with hematoxylin/the appropriate stimuli.eosin as described in Kiernan (1981).In this study, we aimed to test the hypothesis that

PNA binding. Specimens for PNA binding histochemistry in-PNA-binding is a characteristic of such cells, specifi- cluded paraffin sections of whole chick embryonic muscle (Day 14)cally those residing in nonskeletal and noncartilage tis- and cultured cells. Muscle specimens were fixed in Carnoy’s solu-

tion, embedded in Paraplast, transversely sectioned at 10 mm thick-sues. We have chosen to examine PNA-binding cells inness, deparaffinized, hydrated, and incubated with FITC-conju-muscle, in view of its demonstrated ability to be in-gated PNA (100 mg/ml; Sigma) for 30 min, rinsed with PBS, andduced to undergo endochondral ossification (Reddi and mounted with glycerol/PBS (1:1). For cultured cells, horseradish

Huggins, 1972; Urist, 1965). PNA-binding cells were peroxidase-conjugated PNA (5 mg/ml; Sigma) was also used andselected from Day 14 chick embryonic pectoral muscle histochemistry was done using the Zymed Histostain-SP kit (Zymed

Inc., San Francisco, CA).by means of PNA-affinity chromatography, and theirImmunohistochemistry. The following antibodies (and dilutions/chondrogenic differentiation potential analyzed in vitro

concentration and source) were used to assess cellular differentia-on the basis of morphology and the expression of ation: chick collagen type II (1:50; 12 mg/ml), II-II6B3, mouse mono-

number of cell-type-specific differentiation markers. clonal (Linsenmayer and Hendrix, 1980; Developmental Studies Hy-We report here that the PNA-binding cells derived from bridoma Bank, University of Iowa, Iowa City, IA); chick aggrecan

core protein (1:125), rabbit polyclonal (Pacifici et al., 1986; kind giftpectoral muscle display chondrogenic potential in vitro.of Dr. Pacifici, University of Pennsylvania); muscle sarcomeric a-actin (1:50; 2 mg/ml), rabbit polyclonal (Sigma); and desmin (1:20; 50MATERIALS AND METHODSmg/ml), rabbit polyclonal (Sigma). Cultures fixed in 4% paraformalde-hyde were incubated with the respective antibodies. Detection of a-

Embryos actin and collagen type II was done using biotinylated secondaryantibodies and peroxidase-conjugated streptavidin of the Zymed SPFertilized chicken eggs were obtained from Truslow FarmsReagent kit, or using an FITC-conjugated goat anti-mouse IgG(Chestertown, MD) and incubated at 37.57C in a humidified,(Sigma). Desmin and aggrecan were immunolocalized on muscle sec-forced air, commercial egg incubator for the appropriate period oftions (see above) using TRITC-conjugated goat anti-rabbit IgG anti-time.bodies (Sigma).

Cell Isolation and CultureDetection of Collagen Type II mRNA

Breast muscle (pectoralis) was dissected from Day 14 chicken(Hamburger and Hamilton stage 39), rinsed in Ca-Mg-free saline Total RNA was isolated from micromass and monolayer cultures

of total and fractionated cells, using the single step guanidiniumG (CMFSG), and finely minced. After enzymatic dissociation (0.1%trypsin and 0.1% collagenase, Sigma, St. Louis, MO, with 10% thiocyanate method (Chomczynski and Sacchi, 1987; Total RNA Iso-

lation System, Promega kit), and were treated with RNase-freechicken serum, GIBCO BRL, Gaithersburg, MD, 377C), filtrationthrough Nitex filter, and centrifugation to remove blood cells (mostly DNase (1 U/sample; Promega) for 15 min at 377C. For high sensitivity

detection of collagen type II mRNA, total RNA was subjected to re-erythrocytes), the total muscle cells (TM) were pelleted and checkedfor viability by trypan blue exclusion (ú95%). Cell densities were verse transcription (Gibco BRL kit) and the subsequent cDNAs were

amplified by polymerase chain reaction (Perkin–Elmer kit), as de-then adjusted to the desired values.For micromass cultures, the isolated cells were adjusted to a high scribed recently by Stringa and Tuan (1996). Oligodeoxyribonucleo-

tide primers were designed based on the reported chicken collagendensity (20 1 106 cells/ml) and plated as a 10-ml drop on a plasticculture dish (24-well, Corning, Corning, NY). After attachment for type II cDNA sequence (Nah and Upholt, 1991): the forward primer

located between exons 3 and 4 (from 388 to 411 bp, 5*-TTAAAGATG-1–2 hr, the cultures were flooded with 2 ml of culture medium (Ham’sF-12 containing 10% fetal calf serum, 0.2% chick embryo extract, TTGTAGGACCCCGAG-3*); and the backward primer between exons

5 and 6 (from 593 to 573 bp, 5*-CGCAAAGTTTCCACCAAGTCC-3*).and penicillin/streptomycin) which was changed every 24 h (San An-tonio and Tuan, 1986; San Antonio et al., 1987). Monolayer cultures The PCR conditions included a denaturation step at 947C for 1 min,

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289PNA SELECTION OF CHONDROGENIC CELLS

an annealing step at 557C for 1 min, and an extension step at 727C monolayer cultures of either PNA/ or PNA0 cells. Infor 2 min. The samples underwent 35 cycles after a precycle denatur- both PNA/ and PNA0, sarcomeric a-actin expressionation step at 947C for 5 min. The expected size of the collagen type was seen in micromass cultures of both PNA/ andII RT-PCR product was 167 bp. PCR products were analyzed by

PNA0 populations, although in the former, areas ofagarose gel electrophoresis, ethidium bromide staining, and subse-quent Southern blot and hybridization using collagen type II cDNA ‘‘negative staining’’ were often seen in the midst ofas probe. The integrity and loading of the RNA was examined on the dense staining, perhaps corresponding to the cellularbasis of parallel b-actin RT-PCR (Clontech Inc.; expected size, 293 aggregates described above. In both PNA/ and PNA0bp). Chick embryonic sternal total RNA was used as a positive control

micromass cultures, sarcomeric a-actin expression wasfor collagen type II mRNA.found exclusively associated with multinucleated myo-tubes, similar to those seen in the monolayer cultures.MicroscopyInterestingly, collagen type II staining, which appearedSpecimens were observed with epifluorescence optics (FITC andto correspond to the cellular aggregates (Fig. 2B, micro-TRITC), Nomarski differential interference optics, or bright field op-mass), was seen in the PNA/ micromass cultures; suchtics using an Olympus BH-2 microscope, and photographed either

on Agfa color film, or Kodak T-Max or Ektachrome film. staining was totally absent in the PNA0 cultures (bothmicromass and monolayer).

Figure 3A shows that PNA/ muscle-derived cellsRESULTScultured as micromass expressed collagen type II. The

Presence of PNA-Binding Cells in Embryonic Muscle chondrocytic nature of these cells was further illus-trated by their positive staining for aggrecan core pro-As shown by fluorescence PNA histochemistry (Fig. tein (Fig. 3B), an extracellular matrix molecule charac-1A, arrows), PNA-binding (PNA/) cells were detect- teristic of cartilage. On the other hand, these aggre-able in chick pectoral muscle sections, although they gates were negative for the expression of the musclewere few in number (Ç1%). PNA binding cells did not marker a-actin (Fig. 3C). Figure 3D shows the chondro-readily appear to be associated with any particular his- cyte-like morphology of the cells in the aggregates. Fur-tologically distinct structure. Parallel immunofluores- ther evidence indicating the cartilaginous identity ofcence histochemistry revealed that while the PNA-posi- PNA/ muscle-derived cells cultured as micromass istive cells were also desmin-positive (Fig. 1B, arrows), shown in Fig. 4, where RT-PCR detected collagen typethe majority of the desmin expressing cells (Fig. 1B, II mRNA only in PNA/ micromass cultures, but notarrowheads) did not stain with PNA. As a positive con- in total muscle cells, in PNA0 cells, nor PNA/ selectedtrol, an almost universal staining pattern was seen for cells cultured as a monolayer.muscle sarcomeric a-actin (not shown). Taken together, these observations indicate thatboth the PNA/ and PNA0 populations contained cellsPNA Affinity Fractionation of Muscle Cells and Theirwith genuine myoblastic potential, which was ex-Characteristics in Vitropressed prominently when they were plated as stan-dard monolayer cultures; however, the PNA/ cell pop-Day 14 chick embryonic muscle cells were fraction-

ated into PNA-binding (PNA/) and nonbinding ulation, when cultured at high-density as a micromass,were capable of exhibiting a different phenotype, i.e.,(PNA0) populations by means of PNA affinity chroma-

tography. The PNA/ population, enriched for PNA0 the formation of cellular aggregates which failed to ex-press muscle markers, but instead expressed cartilage-binding cells (Ç50%), represented a minor subpopula-

tion (£5%) of the isolated total muscle (TM) cells. The associated matrix molecules.differentiation potential of these two cell populationswas evaluated in vitro as a function of initial plating DISCUSSIONdensity. When plated as low-density monolayer cul-tures (4 1 104 cells/ml), the myoblastic characteristics In this report, we have demonstrated that a subpopu-

lation of muscle-derived cells, which bind the lectinof both cell types were readily apparent. As shown inFigs. 2A and 2B (monolayer), after 4 days in culture PNA, exhibit chondrogenic potential in vitro, when

maintained as high density micromass cultures, a con-both PNA/ and PNA0 monolayer cultures showedmultinucleated actin-positive cells, but stained nega- dition which favors chondrogenic differentiation of

mesenchymal cells (San Antonio and Tuan, 1986; So-tively for collagen type II.On the other hand, upon high-density micromass cul- lursh et al., 1978). The PNA/ subpopulation, which

is enriched for PNA-binding cells, represents a smallturing, major differences were observed between thePNA/ and PNA0 cell populations. After 4 days in cul- fraction of cells making up the total muscle tissue

(õ5%), consistent with the observation that unfraction-ture, micromass cultures of the PNA/ populationreadily formed histologically distinct aggregates de- ated muscle cells do not display any significant chon-

drogenic potential in vitro.tectable by hematoxylin-eosin staining, whereas thePNA0 cell population did not (compare Figs. 2A and The Arachis hypogea lectin, PNA, has previously

been shown to bind specifically to precartilage blas-2B, micromass). Such aggregates were not seen in

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290 STRINGA ET AL.

FIG. 1. Immunofluorescent detection of PNA/ cells in Day 14 chick embryonic muscle tissue sections. (A) Staining with FITC-labeledPNA. (B) Immunostaining of desmin using TRITC-labeled secondary antibodies. (C) Morphology of muscle section viewed with Nomarskiinterference optics. PNA staining cells (A, arrows) appeared to also express desmin (B, arrows), whereas the majority of desmin-expressingcells (B, arrowheads) did not bind PNA. Bar, 50 mm.

FIG. 2. Effect of micromass culture conditions on the differentiation of (A) PNA/ and (B) PNA0 cells. Immunohistochemistry usingantibodies to sarcomeric a-actin and collagen type II, as well as histological staining using hematoxylin-eosin (H & E), were performed onhigh-density micromass cultures (left column) and low-density monolayer cultures (right) on Culture Day 4. Areas of H & E staining,indicating areas of high cell density, were prominent only in PNA/ micromass culture (A). Actin staining was present throughout bothPNA/ and PNA0 cultures, but was conspicuously absent inside the cell aggregates of PNA/ micromass cultures (A). Collagen type IIstaining was visible only in the PNA/ cell cultures and particularly in areas of cell aggregation (A, micromass). Bar, 50 mm.

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291PNA SELECTION OF CHONDROGENIC CELLS

FIG. 4. RT-PCR detection of collagen type II mRNA expression incultures of PNA-fractionated chick embryonic muscle cells. Cultureswere guanidine-extracted and RNA was processed for collagen typeII RT-PCR and analyzed by agarose gel electrophoresis and Southernblot hybridization as described under Materials and Methods. Lanes:1, Total muscle micromass; 2, PNA0 micromass; 3, PNA/ micro-mass; 4, total muscle monolayer; 5, PNA0 monolayer; 6, PNA/monolayer; 7, Negative control (sternal cartilage minus reverse tran-scription); 8, positive control (sternal cartilage); 10, pGEM molecularsize marker: a, 2.6 kb; b, 1.6 kb; c, 1.2 kb; d, 0.67 kb; e, 0.51 kb; f,0.46 kb; g, 0.39 kb; h, 0.35 kb; i, 0.22 kb; j, 0.17 kb; k, 0.12 kb.Only PNA/ muscle-derived cells cultured as high density micromassexpressed collagen type II mRNA (lane 3, ethidium bromide stainingand Southern blot analysis), giving a 167-bp RT-PCR product. b-actin RT-PCR (product size, 293 bp) was performed as internal con-trol (ethidium bromide staining).

tema in the limb bud (Aulthouse and Solursh, 1987;Zalik et al., 1987; Zimmermann and Thies, 1984), andthe caudal, chondrogenic region of somites (Bagnalland Sanders, 1985; Gotz et al., 1991; Stern et al., 1986).Our finding that PNA-binding cells isolated from a dif-ferentiated, nonskeletal, and noncartilage tissue pos-sess chondrogenic potential strongly suggests thatPNA-binding may be a surface marker of chondrogeniccells from tissues of mesodermal origin. (Note: SincePNA staining is found in only a small number of thecells which are desmin positive, it is unlikely that des-min expression, generally considered an early markerof myogenesis (Babai et al., 1990), is a required markerof chondrogenic differentiation potential in these cells.)It is thus tempting to speculate that the PNA-binding

FIG. 3. Immunofluorescent characterization of PNA/ cells in mi-cromass culture. Cultures were examined after 4 days in culture. (A)Collagen type II; (B, D) Aggrecan; (C, D) Sarcomeric a-actin. Cellsfound in aggregates exhibited positive staining for collagen type II(A) and aggrecan (B), but were negative for a-actin (C). Arrows definethe cellular aggregates. (A, B, C) epifluorescence optics; (D) Nomarskioptics. Bar, 50 mm.

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292 STRINGA ET AL.

cells may correspond to pluripotent mesenchymal stem lated by exogenously introduced chondro-active factors,e.g., bone morphogenetic proteins (BMPs), members ofcells residing within a differentiated tissue and are ca-

pable of undergoing specific differentiation pathways the TGF-b superfamily (Wozney et al., 1990). For exam-ple, Denker et al. (1995) observed the formation of carti-in response to external stimuli (Caplan, 1991; Frie-

denstein, 1976; Hall and Watt, 1989; Nathanson and lage-like spheroids in cultures of the pluripotent mes-enchymal mouse cell line, C3H10T1/2, treated withHay, 1980; Owen, 1988). Interestingly, we have re-

cently found that chick embryonic muscle explants TGF-b1. Katagiri et al. (1994) have also reported thedifferentiation of the myoblast cell line C2C12 into thegrafted onto chick chorioallantoic membrane were

stimulated by poly-L-lysine, an agent previously shown osteoblast lineage after treatment with BMP-2. BMPshave been implicated in heterotopic ossification, suchto enhance chondrogenesis in limb mesenchymal cells

(San Antonio and Tuan, 1986; San Antonio et al., 1992), as that seen in fibrodysplasia ossificans progressiva,where endochondral ossification centers, which in-to express cartilage-like phenotype (Tuan et al., 1991).

This finding again supports the presence of precarti- volves chondrogenesis, proliferate nondiscriminatelywithin skeletal muscles (Kaplan et al., 1994). It is thuslage cells in embryonic muscle. Such chondrogenically

competent, pluripotent cells are also present in adult likely that factors such as BMPs and TGF-bs serve asanother control of the differentiation fate of the PNA-muscle, since intramuscular implantation of deminer-

alized bone matrix produces ectopic cartilage and bone binding cells residing in nonskeletal tissues.What is the functional difference between the PNA-(Reddi and Huggins, 1972; Urist, 1965).

An important morphogenetic event preceding and ac- binding and nonbinding cells? Conceivably, PNA-bind-ing cells are more adhesive cells, i.e., more capable ofcompanying chondrogenesis is cellular condensation

(Ede, 1983; Thorogood and Hinchliffe, 1975), a process condensation. Some examples which illustrate PNA-binding cells of mesodermal origin being coincidentresulting most likely from specific cell– cell and cell–

matrix interactions, involving adhesion molecules (e.g., with differential cell adhesion in early development in-clude the following: (1) PNA has been shown to be aN-CAM, Widelitz et al., 1993; N-cadherin, Oberlender

and Tuan, 1994a and b) and membrane/matrix compo- molecular marker for tissues that act as barriers toaxon and neural crest advance in the avian embryo.nents (e.g., fibronectin, Newman et al., 1987). Thus,

cartilage formation in the micromass cultures in vitro Axon outgrowth is perturbed by certain PNA-bindingtissues and cells which are less tolerant of the growthmay be considered to proceed as a loose collection of

cells that gradually condense to form an aggregate, cone movement and elongation (Asamoto et al., 1990;Oakley and Tosney, 1991; Oakley et al., 1994; Tosney,functionally analogous to a blastema in the developing

limb bud. For cellular condensation to initiate and pro- 1991; Tosney and Oakley, 1990), and the segmentedpattern of neural crest cell migration is crucially depen-ceed, specific subpopulations of cells are likely to ex-

hibit changes in their surface components, particularly dent on functional PNA-binding molecules localized tothe caudal sclerotome (Krull et al., 1995). (2) In kera-those which interact with extracellular matrix ligands

and/or homotypic/heterotypic ligands on other cells; nu- tinocyte cultures undergoing stratification, PNA-bind-ing glycoproteins have been localized to cell surfacecleation of the condensation then results from such

changes in these interactions (Newman et al., 1987; microvilli and are especially concentrated at the bound-aries between cells (Watt et al., 1989). (3) In the devel-Steinberg and Takeichi, 1994). In micromass culture,

first cellular aggregation and condensation occur. Once oping somite, PNA binding is localized to the caudalregion, which undergoes overt condensation and givesthe initial aggregate is established, a recruitment

phase begins in which nearby cells join the aggregate. rise to the vertebral cartilage (Bagnall and Sanders,1989; Gotz et al., 1991; Stern et al., 1986). (4) Expres-Interactions among cells of the aggregate are presumed

stronger than those with the matrix or cells outside the sion of the cell adhesion molecules, N-CAM (Widelitzet al., 1993), and N-cadherin (Oberlender and Tuan,aggregate, possibly due to differential changes in the

cellular adhesion occurring between cells in the aggre- 1994a and b) in the limb bud has been shown to imme-diately precede the expression of PNA-binding activity.gate and those surrounding the aggregate (Foty et al.,

1996; Steinberg and Takeichi, 1994). Such cellular ag- Our observations are thus consistent with these re-ports, in that the PNA-binding cells of the embryonicgregates are seen in high density micromass cultures

of muscle-derived PNA/ cells and express a matrix muscle are likely to behave in a more cell–cell adhesivemanner during the early stages of chondrogenesis, i.e.,containing collagen type II and aggrecan, characteristic

of cartilaginous extracellular matrix. rather than being restricted to binding to the extracel-lular matrix, they may contribute to the aggregatingIt is noteworthy that isolated total muscle cells,

which contain the PNA-binding cells, do not undergo centers which eventually form cartilage nodules. It isnoteworthy that mild guanidine treatment extracts thespontaneous chondrogenesis under normal culture con-

ditions. In vivo, noncartilage connective tissues, such PNA-binding moiety from cultured mesenchymal cells(Aulthouse and Solursh, 1987), suggesting that theas muscle, containing the chondrogenically competent

PNA-binding cells, also do not chondrify unless stimu- PNA-binding protein is a cell surface component. Inter-

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293PNA SELECTION OF CHONDROGENIC CELLS

receptors on somites control the behavior of neural cells? Dev.estingly, it was recently reported that the principalGrowth Differ. 32, 91–96.PNA-binding component on the surface of keratino-

Aulthouse, A. L., and Solursh, M. (1987) The detection of a precarti-cytes is the hyaluronan receptor, CD44 (Hudson et al.,lage, blastema-specific marker. Dev. Biol. 120, 377–384.1995), and CD44 has indeed been shown to be involved

Babai, F., Musevi-Aghdam, J., Schurch, W., Royal, A., and Gabbiani,in chondrogenesis (Knudson et al., 1993) and cartilage G. (1990) Coexpression of a-sarcomeric actin, a-smooth musclematuration (Pavasant et al., 1994). In addition, Davies actin and desmin during myogenesis in rat and mouse embryos.

I. Skeletal muscle. Differentiation 44, 132–142.et al. (1990) have demonstrated that PNA binds twoglycoproteins within the caudal sclerotomes; whether Bagnall, K. M., and Sanders, E. J. (1989) The binding pattern of

peanut lectin associated with sclerotome migration and the for-similar cell surface proteins are involved in defining themation of the vertebral axis in the chick embryo. Anat. Embryol.chondroprogenitor nature of the muscle-derived PNA-180, 504–513.binding cells remains to be determined. Furthermore,

Caplan, A. I. (1991) Mesenchymal stem cells. J. Orthop. Res. 9, 641–it would be most interesting to investigate whether the 650.function of such proteins are modulated by endogenous Chomczynsky, P., and Sacchi, N. (1987) Single step method of RNAlectins (Zalik, 1991). isolation by acid guanidium thiocyanate-phenol-chlorophorm

extraction. Anal. Biochem. 162, 156–159.In conclusion, there exist a small but significant pop-ulation of cells in the chick embryonic pectoral muscle Davies, R. J., Cook, G. M. W., Stern, C. D., and Keynes, R. J. (1990)

Isolation from chick somites of a glycoprotein fraction thatwhich bind PNA with high affinity. These PNA-bindingcauses collapse of dorsal root ganglion growth cones. Neuron 4,cells possess chondrogenic potential and possibly repre-11–20.sent a more pluripotent cell population within the mus-

Denker, A. E., Nicoll, S. B., and Tuan, R. S. (1995) Formation of carti-cle capable of undergoing altered differentiation path- lage-like spheroids by micromass cultures of murine C3H10T1/ways under the appropriate conditions. However, be- 2 cells upon treatment with transforming growth factor-b1. Dif-

ferentiation 59, 25–34.cause PNA affinity chromatography is capable of onlythe ‘‘enrichment,’’ not the ‘‘purification,’’ of PNA/ cells, Ede, D. A. (1983) in Cartilage: Development, Differentiation, and

Growth (Hall, B. K., Ed.), Vol. 2, pp. 143–185. Academic Press,it remains possible that their culture condition-depen-Orlando, FL.dent, chondrogenic versus myogenic abilities, as ob-

Emerson, C. P. (1993) Embryonic signals for skeletal myogenesis:served here, are attributable to a different subpopula-Arriving at the beginning. Curr. Opin. Cell. Biol. 5, 1057–1064.tion of cells in the heterogeneous PNA/ fraction. Thus,

Foty, R. A., Pfleger, C. M., Forgacs, G., and Steinberg, M. S. (1996)high and low density platings may have selected for Surface tensions of embryonic tissues predict their mutual en-the proliferation or expansion of different specific cell velopment behavior. Development 122, 1611–1620.subpopulations to give rise to the phenotypes observed Friedenstein, A. J. (1976) Precursor cells of mechanocytes. Int. Rev.here. Ultimately, clonal analysis is required for un- Cytol. 47, 327–355.equivocal lineage information on a given cell. Finally, Gotz, W., Fischer, G., and Herken, R. (1991) Lectin binding pattern

in the embryonal and early fetal human vertebral column. Anat.it is of interest to note that we have recently isolated,Embryol. 184, 345–353.by means of Percoll gradient centrifugation, a subpopu-

Hall, P. A., and Watt, F. M. (1989) Stem cells: The generation andlation of cells from the chick embryonic calvarium, amaintenance of cellular diversity. Development 106, 619–633.normally totally bone tissue, which are capable of un-

Hamburger, V., and Hamilton, H. L. (1951) A series of normal stagesdergoing chondrogenesis in vitro (Wong and Tuan,in development of the chick embryo. J. Morphol. 88, 49 –92.

1995). Our most recent study has revealed that theseHudson, D. L., Sleeman, J., and Watt, F. M. (1995) CD44 is the major

cells also possess PNA-binding activity (Stringa and peanut lectin-binding glycoprotein of human epidermal kera-Tuan, 1996). It remains to be established what specific tinocytes and plays a role in intercellular adhesion. J. Cell. Sci.

108, 1959–1970.characteristics of such cells from nonskeletal, noncarti-Kaplan, F. S., Hahn, G. V., and Zasloff, M. A. (1994) Heterotopic ossi-lage tissues are responsible for their expanded differen-

focation: Two rare forms and what they can teach us. J. Am.tiation potential.Acad. Orthop. Surg. 2, 288–296.

Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N.,We are grateful to Gretchen Meller for assistance with some of the Ikeda, T., Wozney, J. M., Fujisawa-Sehara, A., and Suda, A.

intitial micromass culture. This work was supported in part by (1994) Bone morphogenetic protein-2 converts the differentia-grants from the Orthopaedic Research and Education Foundation, tion pathway of C2C12 myoblasts into osteoblast lineage. J.the Arcadia Foundation, and the NIH (HD 15822, HD 29937, ES Cell Biol. 127, 1755–1765.07005, and DE11327).

Kiernan, J. (1981) in Histological and Histochemical Methods: The-ory and Practice, p. 74 and pp. 150 –151. Pergamon Press, Ox-

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Received January 22, 1997Revised version received February 10, 1997

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