EXPERIMENTAL CELLRESEARCH 200,
26-33 (1992)
Cell Condensation in Chondrogenic Differentiation C. TACCHETTI,I S. TAVELLA,B. DOZIN, R. QUARTO, G. ROBINO, ANDR. CANCEDDA Laboratorio Differenziarnento Cellulare, Istituto Nazionale per la Ricerca sul Cancro, Centro Interuniversitario per la Ricerca sul Cancro, Istituto di Oncologia Clinica e Sperimentale, Universitd di Genova, Genova, Italy
onstrated whether this event, known as precartilage condensation, represents a necessary step in cartilage differentiation and whether it is required for the position-dependent morphogenesis of skeletal structures [3]. Previous reports either have suggested an induction of differentiation in prechondrogenic cells independent of cell condensation [9-14] or have provided evidence for a correlation between the ability of the cells to aggregate in vitro and the formation of cartilage nodules [5, 7, 15-171. T h e role of cell shape for the induction of differentiated function expression in prechondrogenic mesenchymal cells has been investigated. Isolated prechondrogenic mesenchymal cells differentiate when cultured in or upon collagen gels [9], or when exposed to actindisrupting drugs such as cytochalasin D [10]. Similarly, dedifferentiated cells are able to differentiate in agarose cultures [11]. Furthermore, when allowed to spread on a fibronectin-coated substratum, differentiated chondrocytes lose their differentiated phenotype [12]. Spreading inhibition, or induction of chondrocyte rounding, increases sulfate incorporation into proteoglycans [13, 14]. A correlation between the ability of chick limb bud mesenchymal cells to aggregate in vitro and the formation of cartilage nodules has been reported [5]. Furthermore, dibutyryl cAMP or theophylline t r e a t m e n t of cultured chick stage 19 limb bud mesenchymal cells, which are able to aggregate in vitro but unable to differentiate, induces chondrogenesis only if these cells are previously allowed to aggregate [5, 15, 16]. A possible role for cell to cell interactions in the induction of chondrogenesis is INTRODUCTION also suggested by experiments in which prechondrogenic cells have been mixed at several ratios with nonReduction of intercellular space and the consequent chondrogenic cells; the rate of chondrogenic loci formaincrease in cell condensation and cell to cell contacts in tion and their size were directly proportional to the numthe areas of prospective cartilage and bone formation ber of chondrogenic cells [17-18]. are the earliest morphological events associated with T h e molecular mechanisms involved in cell condenlimb skeleton differentiation [1, 2]. It remains to be dem- sation have been suggested to be mediated either directly by cell-cell adhesion molecules or by extracellular matrix proteins, or both. Recently, the cell adhesion 1To whom reprint requests shouldbe addressed at present address: Istituto di Istologia ed Embriologia Generale, Facolth di Medicina e molecule N-CAM has been proposed as a putative effecChirurgia, Universit& di Genova, Via Marsano, 10, 16132 Genova, tor for chondrocyte aggregation [19]. Frenz et al. have suggested t h a t local accumulation of fibronectin could Italy.
R e d u c t i o n o f i n t e r c e l l u l a r s p a c e s in t h e a r e a s o f p r o spective cartilage and bone formation (precartilage condensation) precedes chondrogenesis and may repres e n t an i m p o r t a n t s t e p in t h e p r o c e s s o f c a r t i l a g e differe n t i a t i o n d u r i n g l i m b s k e l e t o g e n e s i s . W e h a v e att e m p t e d to c l a r i f y t h e r o l e o f t h e m i c r o e n v i r o n m e n t e s tablished during cell condensation, taking advantage of a tissue culture model system that allows condensation (i.e., i n c r e a s e d c e l l d e n s i t y d u e to c e l l a g g r e g a t i o n ) a n d c h o n d r o g e n i c d i f f e r e n t i a t i o n (i.e., s y n t h e s i s o f c a r t i l a g e - s p e c i f i c e x t r a c e l l u l a r m a t r i x p r o t e i n s , s u c h as t y p e II c o l l a g e n a n d a c q u i s i t i o n o f a c h o n d r o c y t e m o r phology) of chick embryo cartilage-derived undifferentiated cells. To prevent condensation cells were grown in c a r b o x y m e t h y l c e l l u l o s e a n d c h a n g e s in t h e d i f f e r e n tiation pathway were evaluated. In another series of exp e r i m e n t s , w e h a v e s e p a r a t e d s i n g l e c e l l s f r o m t h e aggregated cells and analyzed their differentiation properties. Morphological analyses and the evaluation of type II c o l l a g e n e x p r e s s i o n , at b o t h t h e p r o t e i n a n d t h e mRNA level, show that a reduced rate of cell clustering a n d c e l l to c e l l c o n t a c t p a r a l l e l s a r e d u c t i o n o f c e l l rec r u i t m e n t i n t o t h e d i f f e r e n t i a t i o n p r o g r a m . On t h e basis of our results, we suggest that the following cascade of events regulates the early stages of chondrocyte d i f f e r e n t i a t i o n : (a) t h e a c q u i s i t i o n o f t h e a b i l i t y to e s t a b l i s h c e l l to c e l l c o n t a c t s , (b) t h e f o r m a t i o n o f a p e r missive environment capable of activating the different i a t i o n p r o g r a m , a n d (c) t h e e x p r e s s i o n o f d i f f e r e n t i a t i o n m a r k e r s . 9 1 9 9 2 Academic Press, Inc.
0014-4827/92$3.00 Copyright9 1992by AcademicPress, Inc. All rights of reproductionin any formreserved.
26
CELL CONDENSATION IN CHONDROGENIC DIFFERENTIATION draw cells in the c o n d e n s a t i o n a r e a by m e a n s of adhesive forces b e t w e e n the a m i n o - t e r m i n a l d o m a i n of fibron e c t i n a n d h e p a r i n - l i k e cell s u r f a c e m o l e c u l e s [20-22]. Interference with cell-fibronectin interactions, exerted b y s y n t h e t i c p e p t i d e s o r m o n o c l o n a l a n t i b o d i e s , signific a n t l y reduces cell c o n d e n s a t i o n a n d t h e p r o d u c t i o n of highly sulfated proteoglycans characteristic of differentiating cartilage in m i c r o m a s s culture of limb bud mese n c h y m a l cel l s [23]. We have previously described a tissue culture model s y s t e m t h a t a l l o w s c o n d e n s a t i o n a n d c h o n d r o g e n i c diff e r e n t i a t i o n o f d e d i f f e r e n t i a t e d c e l ls [6]. W h e n c h i c k e m b r y o tibiae are dissociated by e n z y m a t i c t r e a t m e n t , a n d the cells are allowed to a d h e r e to tissue c u l t u r e dishes, they assume a fibroblast-like phenotype. Under these culture conditions, cells proliferate rapidly a n d secrete fibronectin and type I collagen into the culture medium. When these dedifferentiated fibroblast-like ce ll s a r e t r a n s f e r r e d i n t o s u s p e n s i o n c u l t u r e s t h e y r a p i d l y a g g r e g a t e a n d e v e n t u a l l y s e c r e t e t y p e I I c o l l a g e n (a c h o n d r o c y t e - s p e c i f i c m a r k e r ) . U p o n t i m e i n c u l t u r e , cell aggregates loosen a n d release single h y p e r t r o p h i c chond r o c y t e s in t h e c u l t u r e m e d i u m . C e l l s a t t h i s s t a g e a l s o secrete type X collagen, a specific m a r k e r of h y p e r t r o p h i c c h o n d r o c y t e s [6, 7, 23]. I n t h e p r e s e n t s t u d y we s h o w t h a t c e l l c o n d e n s a t i o n , in t h i s m o d e l s y s t e m , is r e l e v a n t for the e x p r e s s i o n of d i f f e r e n t i a t e d f u n c t i o n s in c h o n d r o g e n i c cells, a n d w e s u g g e s t t h a t a c a s c a d e o f events occurs during the early stages of chondrocyte differentiation.
MATERIALS AND METHODS Cell culture. Primary cultures and suspension cultures of dedifferentiated cells were performed as described previously [6], from stage H.H. 28-30 [24] chick embryo tibiae. In brief, tibiae were isolated from embryos and subjected to enzymatic treatment with trypsin collagenase in order to remove perichondral tissues. Rudiments were then transferred to a fresh solution of collagenase mixture in order to recover chondrocytes in a single cell suspension. Culture medium was Coon's modified Ham F-12 [25] supplemented with 10% fetal calf serum (FCS) (Flow Laboratories, Irvine, Scotland), 50 IU/ml penicillin, and 50 #g/ml streptomycin (GIBCO, Paisley, Scotland). Particular care was taken to use populations of dedifferentiated cells containing less than 6% differentiated chondrocytes for these studies. Suspension cultures were established by plating cells (2.5 • 105/ml) on agarose-coated dishes. When indicated, cells were cultured in complete medium containing 1.3% final concentration of carboxymethylcellulose (4000 cP, Sigma Chemical Co., St. Louis, MO) prepared as previously described [26]. Recovery of cells from carboxymethylcellulose containing medium was obtained by dilution with fresh carboxymethylcellulose-free culture medium, followed by 20 min centrifugation at 2500 rpm; this procedure was repeated three to four times. In order to separate aggregated cells from nonaggregated cells, cultures were established at a concentration of 4-4.5 • 105 cells/ml. At different time intervals, cells were harvested and filtered through a 15-#m mesh nylon membrane mounted on a funnel with a stopcock. Aggregated cells were collected by stopping the flow through the funnel and by rinsing the top surface of the membrane. Nonaggregated cells were
27
recovered from the medium filtered through the funnel. Homogeneity of both cell populations was checked by phase contrast microscopy. Cell viability of the nonaggregated cells was assessed by trypan blue dye exclusion and was always greater than 95%. Nonaggregated cells were counted and this number was subtracted from the total number of plated cells to estimate the percentages of aggregated cells in each experiment. Light and electron microscopy. Samples were processed as previously described [7]. Briefly, samples were fixed for 20 min with 2.5% glutaraldehyde (Polysciences, Inc., Warrington, PA) in 0.1 Mcacodylate buffer, pH 7.3, postfixed for 20 min in 1% osmium tetroxide (Polysciences) in 0.1 M cacodylate buffer, pH 7.3, en bloc stained with uranyl acetate, and embedded in Poly-bed 812 (Polysciences). One-micrometer-thick sections were stained with toluidine blue and observed with a Zeiss Axiophot photomicroscope. Silver-gray sections were stained with uranyl acetate and lead citrate and observed with a Philips 400T electron microscope. Immunofluorescence. Cells maintained for various times in suspension culture of dedifferentiated cells grown adherent to dishes were collected and washed in PBS. Cell aggregates were dissociated by incubation with ECMF (0.8% NaC1, 0.02% KCI, 0.019% Na2HPO4, 0.1% NaHCO3, 0.1% glucose, 5 mM EDTA, 0.005% phenol red) or by digestion with a solution of 0.25% trypsin, 2 mg/ml of collagenase I in PBS for 10 min at 37~ followed by gentle pipetting. Cells (2 x l0 s) in 30 #l of PBS were plated on polylysine-coated wells of multitest slides (Flow Laboratories). Slides were incubated for 45 min at 4~ washed in PBS and fixed for 5 min in 3.7% formaldehyde with PBS, washed, and permeabilized with 0.1% Triton X-100 in PBS. Samples were then incubated with rabbit antiserum to chick type II collagen (diluted 1:100) [7] for 1 h at room temperature and washed in PBS, followed by incubation with rhodamine (TRITC)-labeled goat antirabbit IgG (H + L) (Jackson Immunochemical Research Laboratories, Inc., Avondale, PA) for 30 min at room temperature. All of the antibody solutions contained 4 mg/ml goat 3~-globulin. Slides were mounted with 80% glycerol in PBS and analyzed with a Zeiss Axiophot photomicroscope equipped with epifluorescence illumination. The total number of cells was determined by interference contrast illumination, and the number of positive cells in the same microscopic field was assessed using epifluorescence illumination. At least 200 positive cells were scored in each sample. R N A dot blot. Total RNA was extracted from cells using the guanidinium isothiocyanate method [27]. RNA aliquots (10 ttg) were denatured in 7.4% formaldehyde [28] and blotted on Hybond N (Amersham) membranes with a 96-well manifold (Schleicher and Schuell, Inc., Keene, NH). Recombinant DNA for chicken al(II) collagen (clone pCS1) [29] was labeled with (a-32P)dCTP using the randomprimed labeling system (Amersham), with final specific activities ranging from 0.5 to 2.0 X 109 cpm/~g DNA. Only the DNA insert was used for random labeling after digestion with the appropriate restriction enzyme and purification on agarose gel/NA45 membrane (Schleicher and Schuell). Filters were prehybridized and hybridized at 54~ for 16 h, according to Amersham protocols for Hybond N membranes. However, in order to decrease cross-hybridization between collagen sequences, the concentration of formamide in the hybridization mixture was raised to 60%. After hybridization, filters were washed under stringent conditions, the last wash being in 0.1% SSC (1 • SSC = 0.15 M NaCl and 0.015 M Na-citrate) and 0.1% SDS at 54~ for 30 min. Membranes were autoradiographed between intensifying screens at -80~ Half of duplicate or quadruplicate samples were counted to determine the amount of radioactivity retained on individual dots. The remaining dots were treated to remove the collagen probe and rehybridized with the Xenopus laevis rRNA genomic DNA probe pXCr7 (kindly provided by Professor F. Amaldi, Universith Tor Vergata, Rome, Italy). The whole plasmid containing the probe was labeled by nick translation (BRL, Cambridge, UK). Prehybridization and hybridization were performed at 42~ for 16 h accord-
28
TACCHETTI ET AL. ing to Amersham protocols for Hybond N membranes. The radioactivity retained on each dot was measured by ~-scintillation counting. RESULTS
Carboxymethylcellulose Reduces the Rate of Cell Clustering Cells c u l t u r e d in the absence of c a r b o x y m e t h y l c e l l u lose aggregate within 1-2 h. However, w h e n the single dedifferentiated cells were t r a n s f e r r e d into suspension culture in a carboxymethylcellulose c o n t a i n i n g culture medium, t h e y began to f o r m cell aggregates only after 48-72 h in culture (Fig. 1). Sections of cell pellets from c a r b o x y m e t h y l c e l l u l o s e cultures show isolated cells in the virtual absence of cell to cell interactions. T h e s e cells are r o u n d in shape a n d do not exhibit any evidence of damage (Fig. 2D). T h e s e findings have been c o n f i r m e d by electron microscopy (not shown). Sections of control cell pellets d e m o n strate tight cell-cell i n t e r a c t i o n s which occur within a few h o u r s of suspension culture (Fig. 2A). T h i s is also shown by electron microscopy (Fig. 3), which, f u r t h e r more, d e m o n s t r a t e s the p r e s e n c e of a d h e r e n c e junctions. As previously shown [7], a p e r i p h e r a l layer o f elongated cells appears b e t w e e n 24 a n d 72 h of suspension culture, whereas internally r o u n d e d cells b e c a m e s u r r o u n d e d and s e p a r a t e d by extracellular m a t r i x (Figs. 2B and 2C). Cell to cell i n t e r a c t i o n s are m a i n t a i n e d in the outer layer of elongated cells until the whole aggregate releases individual h y p e r t r o p h i c cells [8].
The Decreased Rate of Cell Clustering Is Associated with a Reduced Recruitment of Cells into the Differentiation Program
FIG. 1. Dedifferentiated ceils transferred to suspension culture aggregate within a few hours. Carboxymethylcellulose slows down the process of aggregation. (A, B, C, D) Phase contrast microscopy of dedifferentiated cells plated in suspension culture. (A', B', C', D') Phase contrast microscopy of dedifferentiated cells plated in suspension culture in the presence of carboxymethylcellulose. (A, A') 1 h
E x p r e s s i o n of type II collagen, a characteristic chondrocyte differentiation m a r k e r , has been analyzed b o t h u n d e r conditions where cell clustering occurs freely a n d u n d e r conditions where it is impaired. Cells were stained by indirect i m m u n o f l u o r e s c e n c e with an antibody to type II collagen at different times in culture (Fig. 4), a n d p e r c e n t a g e s of positive cells were determ i n e d (Table 1). An initial rapid increase in the n u m b e r of positive cells was observed in b o t h c a r b o x y m e t h y l c e l lulose a n d control cultures. In control cultures the n u m ber of positive cells increased steadily over a longer culture period up to 70-85% after I week. In contrast, the rate of increase of positive cells, over a longer culture period, was lower in carboxymethylcellulose, the percentage of positive cells reaching only 30 to 50% a f t e r 1 week. T h e s e findings suggest t h a t in the initial population, cells with at least two different levels of sensitivity
after plating; (B, B') 24 h after plating; (C, C') 48 h after plating; (D, D') 1 week after plating. Bar: 200 #m.
CELL CONDENSATION IN CHONDROGENIC DIFFERENTIATION
29
to stimuli which switch on the differentiation program may be present, i.e., cells capable of initiating the differentiation program regardless of cell aggregation, and cells which require aggregation in order to express differentiation markers.
Early Differentiating Cells Possess Faster Kinetics of Aggregation Transferred cells were allowed to aggregate in suspension cultures for 3, 6, and 9 h. At each time point, both the single cell population and the cell aggregates were recovered separately by filtration and analyzed for type II collagen mRNA levels. The percentage of cell aggregation was also determined (Table 2). Type II collagen mRNA levels in single nonaggregated cells were always comparable to that of starting cells. Type II collagen mRNA levels in the cell aggregates were at least fourfold higher in the initial aggregates, but with time in culture they diminished proportionally to the increase in the percentage of cells recovered in the aggregates (i.e., a 1.3-fold increase in the percentage of cell aggregation, between 3 and 6 h of suspension culture, corresponds to a 1.4-fold decrease in the amount of mRNA in the aggregated cell population; a 1.1-fold increase in the percentage of cell aggregation, between 6 and 9 h of suspension culture, corresponds to a 1.2-fold decrease in the amount of mRNA in the aggregated cell population). Previous work has shown, analyzing the whole cell population, that the type II collagen expression progressively increases after 24 h of suspension culture [6, 23]. Furthermore the number of type II collagen-positive cells progressively increases with time in suspension culture (Table 1). These data suggest t h a t the decrease in the amount of type II collagen mRNA is not due to a transient activation of its expression but to the recruitment of nonexpressing cells into the aggregates. This observation indicates t h a t (a) cells expressing type II collagen within the first hour of suspension culture, with or without carboxymethylcellulose (11-14% of the total population), were all in the aggregated cell population after 3 h of culture; (b) nonaggregated cells were not expressing type II collagen mRNA; and (c) the population of single cells eventually aggregates, with a slower kinetics, as shown by the rate of increase in the percentage of cells recovered in the aggregates (40% of cells aggregated within the first 3 h of suspension culture and an increase of only 17 and 5%, namely, after 6 and 9 h). These single cells have not yet begun type II collagen synthesis, as shown by the corresponding decrease in
FIG. 2. Histologicalsections through pellets of chondrogenic cells fixed and embedded at differenttimes after plating in suspension culturein the absence (A, B, C) or in the presence (D) of carboxymethylcellulose.(A, D) 4~2h after plating; (B) 24 h after plating; (C) 72 h after plating. Bar: 95 #m.
30
TACCHETTI E T AL.
FIG. 3. Electron micrograph of a section through a cell aggregate fixed and embedded 412 h after plating in suspension culture in the absence of carboxymethylcellulose. Note the tight interactions between the membranes of adjacent cells. The boxed area is enlarged in the inset to show two junctional complexes of adherens junctions (arrowheads). Stars indicate the outer limits of the cell aggregate. Bar: 2.3 #m; inset: 0.65 t~m.
CELL CONDENSATION IN CHONDROGENIC D I F F E R E N T I A T I O N
31
the levels of type II collagen mRNA in the aggregated cell population. Because the number of type II collagenpositive cells reaches 70-85% after 1 week in control cultures, we conclude that late differentiating cells need to aggregate before expressing differentiated functions. Furthermore, as the percentage of type II collagen-positive cells is always lower in the carboxymethylcellulose cultures than in control cultures, we argue that interaction with other cells or with the microenvironment created by other cells appears to be necessary for the activation of the differentiation program. DISCUSSION
F I G . 4. Indirect immunofluorescence detection of type II collagen. Micrographs show the type II collagen producing chondrogenic cells at different times after the beginning of suspension culture, both in the presence and in the absence of carboxymethylcellulose. (A, B, C, D, E) Interference contrast microscopy of single cells on polylysine-coated slides (see Materials and Methods) and (A', B', C', D', E') corresponding field in epifluorescence microscopy. (A, A') Cells at the time of the transfer into suspension culture; (B, B') cells after 1 h of suspension culture in the absence of carboxymethylcellulose; (C, C') cells after 1 h of suspension culture in the presence of carboxymethylcellulose; (D, D') cells after 36 h of suspension culture in the absence of carboxymethylcellulose; (E, E') cells after 36 h of suspension culture in the presence of carboxymethylcellulose. Note t h a t the increase in the n u m b e r of positive cells is paralleled by an increase in the intensity of fluorescence.
Prechondrogenic cell condensation is the earliest morphological event associated with the differentiation of limb skeleton [1, 2] and it is necessary for the positiondependent morphogenesis of skeletal structures [3]. The process of cell condensation takes place at stage H.H. 23 in the leg bud (12 h later in the wing bud, stage H.H. 25) in the proximal portion of the limb and proceeds distally [24]. The first areas of chondrogenesis are detected in the central portion of the blastema and extend peripherally; cells acquire a rounded shape in the area of overt chondrogenesis whereas in the periphery of the condensed area, they retain a more elongated fibroblast-like phenotype [2]. We have previously described a tissue culture model system that allows condensation and chondrogenic differentiation of dedifferentiated cells. Here we report experiments aimed at understanding whether cell condensation plays a role in the activation and/or stabilization of the expression of the chondrocyte phenotype. Taking advantage of the increased viscosity produced in the culture medium by carboxymethylcellulose, the spontaneous process of cell aggregation, which occurs when dedifferentiated cells are cultured in suspension, has been slowed down. Although our results indicate a role of cell aggregation for the activation of the differentiation program in chondrogenic cells, a note of caution should be made in transferring these results to the in vivo situation, because it remains to be established whether in vitro dedifferentiated cells and prechondrogenic mesenchymal cells in vivo are functionally equivalent. Experiments similar to those described in this report, using cells dissociated from limb buds at the precondensation stage, are currently in progress in our laboratory to address this point. Analyses at the ultrastructural level suggest that a first step in the process of the in vitro condensation of dedifferentiated cells is the adhesion of cells to each other by means of membrane to membrane contacts. This probably involves cell adhesion molecules [18], as suggested by the presence of adherens junctions, and a second step involves matrix secretion which is primarily responsible for holding cells together.
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TACCHETTI ET AL.
Analysis of the number of cells expressing the chondrocyte marker type II collagen suggests that in the original population cells may be present with two different levels of sensitivity to anchorage-independent culture c o n d i t i o n s (i.e., t o s t i m u l i w h i c h i n i t i a t e t h e d i f f e r e n t i a t i o n p r o g r a m ) [6, 10, 11]. T h e s e i n c l u d e c e l l s c a p a b l e o f entering the differentiation program regardless of the cell aggregation and cells which need to aggregate in order to express type II collagen. The latter cells may require some signal (of unknown nature) derived from the microenvironment existing in the condensed area, whereas the former cells may have already received such information. The kinetics of aggregation and type II collagen m R N A e x p r e s s i o n in s o r t e d s i n g l e a n d a g g r e g a t e d cell populations indicates that late differentiating cells have slower aggregation kinetics compared to that of control cells. A g g r e g a t i o n a p p e a r s t o b e n e c e s s a r y f o r t h e e x p r e s s i o n o f d i f f e r e n t i a t e d m a r k e r s in t h e s e l a t e d i f f e r e n t i a t i n g cells, a s t y p e I I c o l l a g e n s y n t h e s i s is r e d u c e d i n carboxymethylcellulose cultures. This suggests that a cascade of events and the acquisition of a step by step competence to differentiate must occur. These events m a y i n c l u d e : (a) a c q u i s i t i o n o f t h e a b i l i t y t o e s t a b l i s h cell t o cell c o n t a c t s , (b) f o r m a t i o n o f a p e r m i s s i v e e n v i ronment capable of activating the differentiation prog r a m , a n d (c) e x p r e s s i o n o f d i f f e r e n t i a t i o n m a r k e r s . I t is o f n o t e t h a t cell d i f f e r e n t i a t i o n i n v i v o b e g i n s a t t h e center of the chick embryo limb chondrogenic blastema a n d p r o c e e d s t o w a r d t h e p e r i p h e r y [2], s u g g e s t i n g a wave of differention events. As shown by filtration exp e r i m e n t s , t h e r a t e o f cell a g g r e g a t i o n c o n s t a n t l y inc r e a s e s ; it is t h e r e f o r e p o s s i b l e t h a t a c o o p e r a t i v e e f f e c t s u c h a s t h a t d e s c r i b e d b y G u r d o n [31] ( c o m m u n i t y effect) m a y o c c u r . W e h a v e n o i n d i c a t i o n a s t o t h e m e c h a -
TABLE 1 Percentage of T y p e II Collagen-Positive Cells D e t e r m i n e d by Indirect Immunofluorescence a Experiment A
Experiment B
% Positive cells Time (hours)
Control
Methocel
0 1 12 36
5.1 11.0 20.6 33.3
5.1 11.6 18.4 22.3
168
71.2
30.4
% Positive ceils Time (hours)
Control
Methocel
0 1 6 20 48 120 168
5.6 12.8 24.5 30.2 44.2 69.8 84.2
5.6 14.0 16.1 21.6 21.5 34.7 48.1
Because of the experimental variability in the timing of the onset of cell aggregation in carboxymethylcellulosecultures, statistical analyses of the results would be misleading. Consequently the results of two representative experiments are shown.
TABLE 2 Correlation between the Ability of Dedifferentaited Cells to Aggregate a n d the Levels of T y p e II Collagen mRNA Expression a Time (hours)
Culture conditions
Cell type
Cell (%)
mRNA (cpm)
0 3
Adherent Suspension
6
Suspension
9
Suspension
Dediff. Single Aggreg. Single Aggreg. Single Aggreg.
-60 40 43 57 38 62
529 570 2446 578 1846 637 1474
a All values are means of two separate determinations. The values in cpm for type II collagen mRNA are expressed as amount of cpm per cell, since they have been normalized for the amount of rRNA in the same slots.
n i s m s i n v o l v e d i n t h i s p r o c e s s . I t is p o s s i b l e t h a t w i t h i n t h e m i c r o e n v i r o n m e n t o f t h e cell a g g r e g a t e s , c e l l t o c e l l a d h e s i o n , cell t o e x t r a c e l l u l a r m a t r i x i n t e r a c t i o n s [18, 26], a n d a l o c a l l y p r o d u c e d g r o w t h f a c t o r [30] m a y p l a y a role. We thank Drs. C. E. Grossi and D. Noonan for helpful discussions and critical revision of the manuscript and Ms. D. Giacoppo for its editing. This work was supported by funds from CNR Progetto Finalizzato "Invecchiamento" (911025) and "Ingegneria Genetica" and from the Associazione Italiana Ricerca sul Cancro (AIRC). REFERENCES 1. Ede, D. A. (1983) in Cartilage (Hall, B. K., Ed.) pp. 143-185. Academic Press, New York. 2. Thorogood, P. V., and Hinchliffe, J. R. (1975) J. Embryol. Exp. Morphol. 33,581-606. 3. Newman, S. A. (1988) Trends Genetics 4, 329-332. 4. Ahrens, P. B., Solursh, M., and Reiter, R. S. (1977) Dev. Biol. 60, 69-82. 5. vonder Mark, K., and vonder Mark, H. (1977) J. Cell Biol. 73, 736-747. 6. Castagnola, P., Moro, G., Descalzi-Cancedda, F., and Cancedda, R. (1986) J. Cell Biol. 102, 2310-2317. 7. Tacchetti, C., Quarto, R., Nitsch, L., Hartmann, D. J., and Cancedda, R. (1987) J. Cell Biol. 105, 999-1006. 8. Solursh, M., Linsenmayer, T. F., and Jensen, K. L. (1982) Dev. Biol. 94, 259-264. 9. Zanetti, N. C., and Solursh, M. (1984) J. Cell Biol. 99, 115-123. 10. Benya, P. D., and Shaffer, J. D. (1982) Cell 30, 215-224. 11. West, C. M., Lanza, R., Rosenbloom, J., Lowe, M., and Holtzer, H. (1979) Cell 17, 491-501. 12. Newman, S. A., and Watt, F. M. (1988) Exp. Cell Res. 178, 199210. 13. Glowacki, J., Trepman, E., and Folkman, J. (1983) Proc. Soc. Exp. Biol. Med. 172, 93-98. 14. Hunter, J. S., and Caplan, A. I. (1983) in Cartilage. (Hall, B. K., Ed.), pp. 87-119. Academic Press, New York.
CELL CONDENSATION IN CHONDROGENIC DIFFERENTIATION 15. Solursh, M. {1983) Cartilage (Hall, B. K., Ed.), pp. 121-141. Academic Press, New York. 16. Moscona, A. (1956) Proc. Soc. Exp. Biol. Med. 92,410-416. 17. Solursh, M., and Reiter, R. S. (1980) Dev. Biol. 78, 141-150. 18. Chuong, C. M., Jiang, T. X., and Chen, H. M. (1990) J. Cell. Biol. 111(2), 270a. 19. Frenz, D. A., Akiyama, S. K., Paulsen, D. F., and Newman, S. A. (1989) Dev. Biol. 136, 87-96. 20. Dessau, W., vonder Mark, H., vonder Mark, K., and Fischer, S. (1980) J. Embryol. Exp. Morphol. 57, 51-60. 21. Tomasek, J. J., Mazurkiewicz, J. E., and Newman, S. A. (1982) Dev. Biol. 90, 118-126. 22. Frenz, D. A., Jaikaria, N. S., and Newman, S. A. (1989) Dev. Biol. 136, 97-103. Received January 7, 1991 Revised version received October 30, 1991
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23. Castagnola, P., Dozin, B., Moro, G., and Cancedda, R. {1988) J. Cell Biol. 106, 461-467. 24. Hamburger, V., and Hamilton, H. L. (1951) J. Morphol. 88, 4992. 25. Ambesi-Impiombato, F. S., Parks, L. A., and Coon, H. G. {1980) Proc. Natl. Acad. Sci. USA 77, 3455-3459. 26. Rheinwald, J., and Green, H. (1974) Cell 2, 287-293. 27. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 28. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205. 29. Young, M. F., Vogeli, G., Nunez, A. M., Fernandez, M. P., Sullivan, M., and Sobel, M. (1984) Nucl. Acids Res. 12, 4207-4228. 30. Solursh, M. (1989) Curr. Opinion Cell Biol. 1,989-994. 31. Gurdon, J. B. (1988) Nature 336, 772-774.
26-33 (1992)
Cell Condensation in Chondrogenic Differentiation C. TACCHETTI,I S. TAVELLA,B. DOZIN, R. QUARTO, G. ROBINO, ANDR. CANCEDDA Laboratorio Differenziarnento Cellulare, Istituto Nazionale per la Ricerca sul Cancro, Centro Interuniversitario per la Ricerca sul Cancro, Istituto di Oncologia Clinica e Sperimentale, Universitd di Genova, Genova, Italy
onstrated whether this event, known as precartilage condensation, represents a necessary step in cartilage differentiation and whether it is required for the position-dependent morphogenesis of skeletal structures [3]. Previous reports either have suggested an induction of differentiation in prechondrogenic cells independent of cell condensation [9-14] or have provided evidence for a correlation between the ability of the cells to aggregate in vitro and the formation of cartilage nodules [5, 7, 15-171. T h e role of cell shape for the induction of differentiated function expression in prechondrogenic mesenchymal cells has been investigated. Isolated prechondrogenic mesenchymal cells differentiate when cultured in or upon collagen gels [9], or when exposed to actindisrupting drugs such as cytochalasin D [10]. Similarly, dedifferentiated cells are able to differentiate in agarose cultures [11]. Furthermore, when allowed to spread on a fibronectin-coated substratum, differentiated chondrocytes lose their differentiated phenotype [12]. Spreading inhibition, or induction of chondrocyte rounding, increases sulfate incorporation into proteoglycans [13, 14]. A correlation between the ability of chick limb bud mesenchymal cells to aggregate in vitro and the formation of cartilage nodules has been reported [5]. Furthermore, dibutyryl cAMP or theophylline t r e a t m e n t of cultured chick stage 19 limb bud mesenchymal cells, which are able to aggregate in vitro but unable to differentiate, induces chondrogenesis only if these cells are previously allowed to aggregate [5, 15, 16]. A possible role for cell to cell interactions in the induction of chondrogenesis is INTRODUCTION also suggested by experiments in which prechondrogenic cells have been mixed at several ratios with nonReduction of intercellular space and the consequent chondrogenic cells; the rate of chondrogenic loci formaincrease in cell condensation and cell to cell contacts in tion and their size were directly proportional to the numthe areas of prospective cartilage and bone formation ber of chondrogenic cells [17-18]. are the earliest morphological events associated with T h e molecular mechanisms involved in cell condenlimb skeleton differentiation [1, 2]. It remains to be dem- sation have been suggested to be mediated either directly by cell-cell adhesion molecules or by extracellular matrix proteins, or both. Recently, the cell adhesion 1To whom reprint requests shouldbe addressed at present address: Istituto di Istologia ed Embriologia Generale, Facolth di Medicina e molecule N-CAM has been proposed as a putative effecChirurgia, Universit& di Genova, Via Marsano, 10, 16132 Genova, tor for chondrocyte aggregation [19]. Frenz et al. have suggested t h a t local accumulation of fibronectin could Italy.
R e d u c t i o n o f i n t e r c e l l u l a r s p a c e s in t h e a r e a s o f p r o spective cartilage and bone formation (precartilage condensation) precedes chondrogenesis and may repres e n t an i m p o r t a n t s t e p in t h e p r o c e s s o f c a r t i l a g e differe n t i a t i o n d u r i n g l i m b s k e l e t o g e n e s i s . W e h a v e att e m p t e d to c l a r i f y t h e r o l e o f t h e m i c r o e n v i r o n m e n t e s tablished during cell condensation, taking advantage of a tissue culture model system that allows condensation (i.e., i n c r e a s e d c e l l d e n s i t y d u e to c e l l a g g r e g a t i o n ) a n d c h o n d r o g e n i c d i f f e r e n t i a t i o n (i.e., s y n t h e s i s o f c a r t i l a g e - s p e c i f i c e x t r a c e l l u l a r m a t r i x p r o t e i n s , s u c h as t y p e II c o l l a g e n a n d a c q u i s i t i o n o f a c h o n d r o c y t e m o r phology) of chick embryo cartilage-derived undifferentiated cells. To prevent condensation cells were grown in c a r b o x y m e t h y l c e l l u l o s e a n d c h a n g e s in t h e d i f f e r e n tiation pathway were evaluated. In another series of exp e r i m e n t s , w e h a v e s e p a r a t e d s i n g l e c e l l s f r o m t h e aggregated cells and analyzed their differentiation properties. Morphological analyses and the evaluation of type II c o l l a g e n e x p r e s s i o n , at b o t h t h e p r o t e i n a n d t h e mRNA level, show that a reduced rate of cell clustering a n d c e l l to c e l l c o n t a c t p a r a l l e l s a r e d u c t i o n o f c e l l rec r u i t m e n t i n t o t h e d i f f e r e n t i a t i o n p r o g r a m . On t h e basis of our results, we suggest that the following cascade of events regulates the early stages of chondrocyte d i f f e r e n t i a t i o n : (a) t h e a c q u i s i t i o n o f t h e a b i l i t y to e s t a b l i s h c e l l to c e l l c o n t a c t s , (b) t h e f o r m a t i o n o f a p e r missive environment capable of activating the different i a t i o n p r o g r a m , a n d (c) t h e e x p r e s s i o n o f d i f f e r e n t i a t i o n m a r k e r s . 9 1 9 9 2 Academic Press, Inc.
0014-4827/92$3.00 Copyright9 1992by AcademicPress, Inc. All rights of reproductionin any formreserved.
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CELL CONDENSATION IN CHONDROGENIC DIFFERENTIATION draw cells in the c o n d e n s a t i o n a r e a by m e a n s of adhesive forces b e t w e e n the a m i n o - t e r m i n a l d o m a i n of fibron e c t i n a n d h e p a r i n - l i k e cell s u r f a c e m o l e c u l e s [20-22]. Interference with cell-fibronectin interactions, exerted b y s y n t h e t i c p e p t i d e s o r m o n o c l o n a l a n t i b o d i e s , signific a n t l y reduces cell c o n d e n s a t i o n a n d t h e p r o d u c t i o n of highly sulfated proteoglycans characteristic of differentiating cartilage in m i c r o m a s s culture of limb bud mese n c h y m a l cel l s [23]. We have previously described a tissue culture model s y s t e m t h a t a l l o w s c o n d e n s a t i o n a n d c h o n d r o g e n i c diff e r e n t i a t i o n o f d e d i f f e r e n t i a t e d c e l ls [6]. W h e n c h i c k e m b r y o tibiae are dissociated by e n z y m a t i c t r e a t m e n t , a n d the cells are allowed to a d h e r e to tissue c u l t u r e dishes, they assume a fibroblast-like phenotype. Under these culture conditions, cells proliferate rapidly a n d secrete fibronectin and type I collagen into the culture medium. When these dedifferentiated fibroblast-like ce ll s a r e t r a n s f e r r e d i n t o s u s p e n s i o n c u l t u r e s t h e y r a p i d l y a g g r e g a t e a n d e v e n t u a l l y s e c r e t e t y p e I I c o l l a g e n (a c h o n d r o c y t e - s p e c i f i c m a r k e r ) . U p o n t i m e i n c u l t u r e , cell aggregates loosen a n d release single h y p e r t r o p h i c chond r o c y t e s in t h e c u l t u r e m e d i u m . C e l l s a t t h i s s t a g e a l s o secrete type X collagen, a specific m a r k e r of h y p e r t r o p h i c c h o n d r o c y t e s [6, 7, 23]. I n t h e p r e s e n t s t u d y we s h o w t h a t c e l l c o n d e n s a t i o n , in t h i s m o d e l s y s t e m , is r e l e v a n t for the e x p r e s s i o n of d i f f e r e n t i a t e d f u n c t i o n s in c h o n d r o g e n i c cells, a n d w e s u g g e s t t h a t a c a s c a d e o f events occurs during the early stages of chondrocyte differentiation.
MATERIALS AND METHODS Cell culture. Primary cultures and suspension cultures of dedifferentiated cells were performed as described previously [6], from stage H.H. 28-30 [24] chick embryo tibiae. In brief, tibiae were isolated from embryos and subjected to enzymatic treatment with trypsin collagenase in order to remove perichondral tissues. Rudiments were then transferred to a fresh solution of collagenase mixture in order to recover chondrocytes in a single cell suspension. Culture medium was Coon's modified Ham F-12 [25] supplemented with 10% fetal calf serum (FCS) (Flow Laboratories, Irvine, Scotland), 50 IU/ml penicillin, and 50 #g/ml streptomycin (GIBCO, Paisley, Scotland). Particular care was taken to use populations of dedifferentiated cells containing less than 6% differentiated chondrocytes for these studies. Suspension cultures were established by plating cells (2.5 • 105/ml) on agarose-coated dishes. When indicated, cells were cultured in complete medium containing 1.3% final concentration of carboxymethylcellulose (4000 cP, Sigma Chemical Co., St. Louis, MO) prepared as previously described [26]. Recovery of cells from carboxymethylcellulose containing medium was obtained by dilution with fresh carboxymethylcellulose-free culture medium, followed by 20 min centrifugation at 2500 rpm; this procedure was repeated three to four times. In order to separate aggregated cells from nonaggregated cells, cultures were established at a concentration of 4-4.5 • 105 cells/ml. At different time intervals, cells were harvested and filtered through a 15-#m mesh nylon membrane mounted on a funnel with a stopcock. Aggregated cells were collected by stopping the flow through the funnel and by rinsing the top surface of the membrane. Nonaggregated cells were
27
recovered from the medium filtered through the funnel. Homogeneity of both cell populations was checked by phase contrast microscopy. Cell viability of the nonaggregated cells was assessed by trypan blue dye exclusion and was always greater than 95%. Nonaggregated cells were counted and this number was subtracted from the total number of plated cells to estimate the percentages of aggregated cells in each experiment. Light and electron microscopy. Samples were processed as previously described [7]. Briefly, samples were fixed for 20 min with 2.5% glutaraldehyde (Polysciences, Inc., Warrington, PA) in 0.1 Mcacodylate buffer, pH 7.3, postfixed for 20 min in 1% osmium tetroxide (Polysciences) in 0.1 M cacodylate buffer, pH 7.3, en bloc stained with uranyl acetate, and embedded in Poly-bed 812 (Polysciences). One-micrometer-thick sections were stained with toluidine blue and observed with a Zeiss Axiophot photomicroscope. Silver-gray sections were stained with uranyl acetate and lead citrate and observed with a Philips 400T electron microscope. Immunofluorescence. Cells maintained for various times in suspension culture of dedifferentiated cells grown adherent to dishes were collected and washed in PBS. Cell aggregates were dissociated by incubation with ECMF (0.8% NaC1, 0.02% KCI, 0.019% Na2HPO4, 0.1% NaHCO3, 0.1% glucose, 5 mM EDTA, 0.005% phenol red) or by digestion with a solution of 0.25% trypsin, 2 mg/ml of collagenase I in PBS for 10 min at 37~ followed by gentle pipetting. Cells (2 x l0 s) in 30 #l of PBS were plated on polylysine-coated wells of multitest slides (Flow Laboratories). Slides were incubated for 45 min at 4~ washed in PBS and fixed for 5 min in 3.7% formaldehyde with PBS, washed, and permeabilized with 0.1% Triton X-100 in PBS. Samples were then incubated with rabbit antiserum to chick type II collagen (diluted 1:100) [7] for 1 h at room temperature and washed in PBS, followed by incubation with rhodamine (TRITC)-labeled goat antirabbit IgG (H + L) (Jackson Immunochemical Research Laboratories, Inc., Avondale, PA) for 30 min at room temperature. All of the antibody solutions contained 4 mg/ml goat 3~-globulin. Slides were mounted with 80% glycerol in PBS and analyzed with a Zeiss Axiophot photomicroscope equipped with epifluorescence illumination. The total number of cells was determined by interference contrast illumination, and the number of positive cells in the same microscopic field was assessed using epifluorescence illumination. At least 200 positive cells were scored in each sample. R N A dot blot. Total RNA was extracted from cells using the guanidinium isothiocyanate method [27]. RNA aliquots (10 ttg) were denatured in 7.4% formaldehyde [28] and blotted on Hybond N (Amersham) membranes with a 96-well manifold (Schleicher and Schuell, Inc., Keene, NH). Recombinant DNA for chicken al(II) collagen (clone pCS1) [29] was labeled with (a-32P)dCTP using the randomprimed labeling system (Amersham), with final specific activities ranging from 0.5 to 2.0 X 109 cpm/~g DNA. Only the DNA insert was used for random labeling after digestion with the appropriate restriction enzyme and purification on agarose gel/NA45 membrane (Schleicher and Schuell). Filters were prehybridized and hybridized at 54~ for 16 h, according to Amersham protocols for Hybond N membranes. However, in order to decrease cross-hybridization between collagen sequences, the concentration of formamide in the hybridization mixture was raised to 60%. After hybridization, filters were washed under stringent conditions, the last wash being in 0.1% SSC (1 • SSC = 0.15 M NaCl and 0.015 M Na-citrate) and 0.1% SDS at 54~ for 30 min. Membranes were autoradiographed between intensifying screens at -80~ Half of duplicate or quadruplicate samples were counted to determine the amount of radioactivity retained on individual dots. The remaining dots were treated to remove the collagen probe and rehybridized with the Xenopus laevis rRNA genomic DNA probe pXCr7 (kindly provided by Professor F. Amaldi, Universith Tor Vergata, Rome, Italy). The whole plasmid containing the probe was labeled by nick translation (BRL, Cambridge, UK). Prehybridization and hybridization were performed at 42~ for 16 h accord-
28
TACCHETTI ET AL. ing to Amersham protocols for Hybond N membranes. The radioactivity retained on each dot was measured by ~-scintillation counting. RESULTS
Carboxymethylcellulose Reduces the Rate of Cell Clustering Cells c u l t u r e d in the absence of c a r b o x y m e t h y l c e l l u lose aggregate within 1-2 h. However, w h e n the single dedifferentiated cells were t r a n s f e r r e d into suspension culture in a carboxymethylcellulose c o n t a i n i n g culture medium, t h e y began to f o r m cell aggregates only after 48-72 h in culture (Fig. 1). Sections of cell pellets from c a r b o x y m e t h y l c e l l u l o s e cultures show isolated cells in the virtual absence of cell to cell interactions. T h e s e cells are r o u n d in shape a n d do not exhibit any evidence of damage (Fig. 2D). T h e s e findings have been c o n f i r m e d by electron microscopy (not shown). Sections of control cell pellets d e m o n strate tight cell-cell i n t e r a c t i o n s which occur within a few h o u r s of suspension culture (Fig. 2A). T h i s is also shown by electron microscopy (Fig. 3), which, f u r t h e r more, d e m o n s t r a t e s the p r e s e n c e of a d h e r e n c e junctions. As previously shown [7], a p e r i p h e r a l layer o f elongated cells appears b e t w e e n 24 a n d 72 h of suspension culture, whereas internally r o u n d e d cells b e c a m e s u r r o u n d e d and s e p a r a t e d by extracellular m a t r i x (Figs. 2B and 2C). Cell to cell i n t e r a c t i o n s are m a i n t a i n e d in the outer layer of elongated cells until the whole aggregate releases individual h y p e r t r o p h i c cells [8].
The Decreased Rate of Cell Clustering Is Associated with a Reduced Recruitment of Cells into the Differentiation Program
FIG. 1. Dedifferentiated ceils transferred to suspension culture aggregate within a few hours. Carboxymethylcellulose slows down the process of aggregation. (A, B, C, D) Phase contrast microscopy of dedifferentiated cells plated in suspension culture. (A', B', C', D') Phase contrast microscopy of dedifferentiated cells plated in suspension culture in the presence of carboxymethylcellulose. (A, A') 1 h
E x p r e s s i o n of type II collagen, a characteristic chondrocyte differentiation m a r k e r , has been analyzed b o t h u n d e r conditions where cell clustering occurs freely a n d u n d e r conditions where it is impaired. Cells were stained by indirect i m m u n o f l u o r e s c e n c e with an antibody to type II collagen at different times in culture (Fig. 4), a n d p e r c e n t a g e s of positive cells were determ i n e d (Table 1). An initial rapid increase in the n u m b e r of positive cells was observed in b o t h c a r b o x y m e t h y l c e l lulose a n d control cultures. In control cultures the n u m ber of positive cells increased steadily over a longer culture period up to 70-85% after I week. In contrast, the rate of increase of positive cells, over a longer culture period, was lower in carboxymethylcellulose, the percentage of positive cells reaching only 30 to 50% a f t e r 1 week. T h e s e findings suggest t h a t in the initial population, cells with at least two different levels of sensitivity
after plating; (B, B') 24 h after plating; (C, C') 48 h after plating; (D, D') 1 week after plating. Bar: 200 #m.
CELL CONDENSATION IN CHONDROGENIC DIFFERENTIATION
29
to stimuli which switch on the differentiation program may be present, i.e., cells capable of initiating the differentiation program regardless of cell aggregation, and cells which require aggregation in order to express differentiation markers.
Early Differentiating Cells Possess Faster Kinetics of Aggregation Transferred cells were allowed to aggregate in suspension cultures for 3, 6, and 9 h. At each time point, both the single cell population and the cell aggregates were recovered separately by filtration and analyzed for type II collagen mRNA levels. The percentage of cell aggregation was also determined (Table 2). Type II collagen mRNA levels in single nonaggregated cells were always comparable to that of starting cells. Type II collagen mRNA levels in the cell aggregates were at least fourfold higher in the initial aggregates, but with time in culture they diminished proportionally to the increase in the percentage of cells recovered in the aggregates (i.e., a 1.3-fold increase in the percentage of cell aggregation, between 3 and 6 h of suspension culture, corresponds to a 1.4-fold decrease in the amount of mRNA in the aggregated cell population; a 1.1-fold increase in the percentage of cell aggregation, between 6 and 9 h of suspension culture, corresponds to a 1.2-fold decrease in the amount of mRNA in the aggregated cell population). Previous work has shown, analyzing the whole cell population, that the type II collagen expression progressively increases after 24 h of suspension culture [6, 23]. Furthermore the number of type II collagen-positive cells progressively increases with time in suspension culture (Table 1). These data suggest t h a t the decrease in the amount of type II collagen mRNA is not due to a transient activation of its expression but to the recruitment of nonexpressing cells into the aggregates. This observation indicates t h a t (a) cells expressing type II collagen within the first hour of suspension culture, with or without carboxymethylcellulose (11-14% of the total population), were all in the aggregated cell population after 3 h of culture; (b) nonaggregated cells were not expressing type II collagen mRNA; and (c) the population of single cells eventually aggregates, with a slower kinetics, as shown by the rate of increase in the percentage of cells recovered in the aggregates (40% of cells aggregated within the first 3 h of suspension culture and an increase of only 17 and 5%, namely, after 6 and 9 h). These single cells have not yet begun type II collagen synthesis, as shown by the corresponding decrease in
FIG. 2. Histologicalsections through pellets of chondrogenic cells fixed and embedded at differenttimes after plating in suspension culturein the absence (A, B, C) or in the presence (D) of carboxymethylcellulose.(A, D) 4~2h after plating; (B) 24 h after plating; (C) 72 h after plating. Bar: 95 #m.
30
TACCHETTI E T AL.
FIG. 3. Electron micrograph of a section through a cell aggregate fixed and embedded 412 h after plating in suspension culture in the absence of carboxymethylcellulose. Note the tight interactions between the membranes of adjacent cells. The boxed area is enlarged in the inset to show two junctional complexes of adherens junctions (arrowheads). Stars indicate the outer limits of the cell aggregate. Bar: 2.3 #m; inset: 0.65 t~m.
CELL CONDENSATION IN CHONDROGENIC D I F F E R E N T I A T I O N
31
the levels of type II collagen mRNA in the aggregated cell population. Because the number of type II collagenpositive cells reaches 70-85% after 1 week in control cultures, we conclude that late differentiating cells need to aggregate before expressing differentiated functions. Furthermore, as the percentage of type II collagen-positive cells is always lower in the carboxymethylcellulose cultures than in control cultures, we argue that interaction with other cells or with the microenvironment created by other cells appears to be necessary for the activation of the differentiation program. DISCUSSION
F I G . 4. Indirect immunofluorescence detection of type II collagen. Micrographs show the type II collagen producing chondrogenic cells at different times after the beginning of suspension culture, both in the presence and in the absence of carboxymethylcellulose. (A, B, C, D, E) Interference contrast microscopy of single cells on polylysine-coated slides (see Materials and Methods) and (A', B', C', D', E') corresponding field in epifluorescence microscopy. (A, A') Cells at the time of the transfer into suspension culture; (B, B') cells after 1 h of suspension culture in the absence of carboxymethylcellulose; (C, C') cells after 1 h of suspension culture in the presence of carboxymethylcellulose; (D, D') cells after 36 h of suspension culture in the absence of carboxymethylcellulose; (E, E') cells after 36 h of suspension culture in the presence of carboxymethylcellulose. Note t h a t the increase in the n u m b e r of positive cells is paralleled by an increase in the intensity of fluorescence.
Prechondrogenic cell condensation is the earliest morphological event associated with the differentiation of limb skeleton [1, 2] and it is necessary for the positiondependent morphogenesis of skeletal structures [3]. The process of cell condensation takes place at stage H.H. 23 in the leg bud (12 h later in the wing bud, stage H.H. 25) in the proximal portion of the limb and proceeds distally [24]. The first areas of chondrogenesis are detected in the central portion of the blastema and extend peripherally; cells acquire a rounded shape in the area of overt chondrogenesis whereas in the periphery of the condensed area, they retain a more elongated fibroblast-like phenotype [2]. We have previously described a tissue culture model system that allows condensation and chondrogenic differentiation of dedifferentiated cells. Here we report experiments aimed at understanding whether cell condensation plays a role in the activation and/or stabilization of the expression of the chondrocyte phenotype. Taking advantage of the increased viscosity produced in the culture medium by carboxymethylcellulose, the spontaneous process of cell aggregation, which occurs when dedifferentiated cells are cultured in suspension, has been slowed down. Although our results indicate a role of cell aggregation for the activation of the differentiation program in chondrogenic cells, a note of caution should be made in transferring these results to the in vivo situation, because it remains to be established whether in vitro dedifferentiated cells and prechondrogenic mesenchymal cells in vivo are functionally equivalent. Experiments similar to those described in this report, using cells dissociated from limb buds at the precondensation stage, are currently in progress in our laboratory to address this point. Analyses at the ultrastructural level suggest that a first step in the process of the in vitro condensation of dedifferentiated cells is the adhesion of cells to each other by means of membrane to membrane contacts. This probably involves cell adhesion molecules [18], as suggested by the presence of adherens junctions, and a second step involves matrix secretion which is primarily responsible for holding cells together.
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TACCHETTI ET AL.
Analysis of the number of cells expressing the chondrocyte marker type II collagen suggests that in the original population cells may be present with two different levels of sensitivity to anchorage-independent culture c o n d i t i o n s (i.e., t o s t i m u l i w h i c h i n i t i a t e t h e d i f f e r e n t i a t i o n p r o g r a m ) [6, 10, 11]. T h e s e i n c l u d e c e l l s c a p a b l e o f entering the differentiation program regardless of the cell aggregation and cells which need to aggregate in order to express type II collagen. The latter cells may require some signal (of unknown nature) derived from the microenvironment existing in the condensed area, whereas the former cells may have already received such information. The kinetics of aggregation and type II collagen m R N A e x p r e s s i o n in s o r t e d s i n g l e a n d a g g r e g a t e d cell populations indicates that late differentiating cells have slower aggregation kinetics compared to that of control cells. A g g r e g a t i o n a p p e a r s t o b e n e c e s s a r y f o r t h e e x p r e s s i o n o f d i f f e r e n t i a t e d m a r k e r s in t h e s e l a t e d i f f e r e n t i a t i n g cells, a s t y p e I I c o l l a g e n s y n t h e s i s is r e d u c e d i n carboxymethylcellulose cultures. This suggests that a cascade of events and the acquisition of a step by step competence to differentiate must occur. These events m a y i n c l u d e : (a) a c q u i s i t i o n o f t h e a b i l i t y t o e s t a b l i s h cell t o cell c o n t a c t s , (b) f o r m a t i o n o f a p e r m i s s i v e e n v i ronment capable of activating the differentiation prog r a m , a n d (c) e x p r e s s i o n o f d i f f e r e n t i a t i o n m a r k e r s . I t is o f n o t e t h a t cell d i f f e r e n t i a t i o n i n v i v o b e g i n s a t t h e center of the chick embryo limb chondrogenic blastema a n d p r o c e e d s t o w a r d t h e p e r i p h e r y [2], s u g g e s t i n g a wave of differention events. As shown by filtration exp e r i m e n t s , t h e r a t e o f cell a g g r e g a t i o n c o n s t a n t l y inc r e a s e s ; it is t h e r e f o r e p o s s i b l e t h a t a c o o p e r a t i v e e f f e c t s u c h a s t h a t d e s c r i b e d b y G u r d o n [31] ( c o m m u n i t y effect) m a y o c c u r . W e h a v e n o i n d i c a t i o n a s t o t h e m e c h a -
TABLE 1 Percentage of T y p e II Collagen-Positive Cells D e t e r m i n e d by Indirect Immunofluorescence a Experiment A
Experiment B
% Positive cells Time (hours)
Control
Methocel
0 1 12 36
5.1 11.0 20.6 33.3
5.1 11.6 18.4 22.3
168
71.2
30.4
% Positive ceils Time (hours)
Control
Methocel
0 1 6 20 48 120 168
5.6 12.8 24.5 30.2 44.2 69.8 84.2
5.6 14.0 16.1 21.6 21.5 34.7 48.1
Because of the experimental variability in the timing of the onset of cell aggregation in carboxymethylcellulosecultures, statistical analyses of the results would be misleading. Consequently the results of two representative experiments are shown.
TABLE 2 Correlation between the Ability of Dedifferentaited Cells to Aggregate a n d the Levels of T y p e II Collagen mRNA Expression a Time (hours)
Culture conditions
Cell type
Cell (%)
mRNA (cpm)
0 3
Adherent Suspension
6
Suspension
9
Suspension
Dediff. Single Aggreg. Single Aggreg. Single Aggreg.
-60 40 43 57 38 62
529 570 2446 578 1846 637 1474
a All values are means of two separate determinations. The values in cpm for type II collagen mRNA are expressed as amount of cpm per cell, since they have been normalized for the amount of rRNA in the same slots.
n i s m s i n v o l v e d i n t h i s p r o c e s s . I t is p o s s i b l e t h a t w i t h i n t h e m i c r o e n v i r o n m e n t o f t h e cell a g g r e g a t e s , c e l l t o c e l l a d h e s i o n , cell t o e x t r a c e l l u l a r m a t r i x i n t e r a c t i o n s [18, 26], a n d a l o c a l l y p r o d u c e d g r o w t h f a c t o r [30] m a y p l a y a role. We thank Drs. C. E. Grossi and D. Noonan for helpful discussions and critical revision of the manuscript and Ms. D. Giacoppo for its editing. This work was supported by funds from CNR Progetto Finalizzato "Invecchiamento" (911025) and "Ingegneria Genetica" and from the Associazione Italiana Ricerca sul Cancro (AIRC). REFERENCES 1. Ede, D. A. (1983) in Cartilage (Hall, B. K., Ed.) pp. 143-185. Academic Press, New York. 2. Thorogood, P. V., and Hinchliffe, J. R. (1975) J. Embryol. Exp. Morphol. 33,581-606. 3. Newman, S. A. (1988) Trends Genetics 4, 329-332. 4. Ahrens, P. B., Solursh, M., and Reiter, R. S. (1977) Dev. Biol. 60, 69-82. 5. vonder Mark, K., and vonder Mark, H. (1977) J. Cell Biol. 73, 736-747. 6. Castagnola, P., Moro, G., Descalzi-Cancedda, F., and Cancedda, R. (1986) J. Cell Biol. 102, 2310-2317. 7. Tacchetti, C., Quarto, R., Nitsch, L., Hartmann, D. J., and Cancedda, R. (1987) J. Cell Biol. 105, 999-1006. 8. Solursh, M., Linsenmayer, T. F., and Jensen, K. L. (1982) Dev. Biol. 94, 259-264. 9. Zanetti, N. C., and Solursh, M. (1984) J. Cell Biol. 99, 115-123. 10. Benya, P. D., and Shaffer, J. D. (1982) Cell 30, 215-224. 11. West, C. M., Lanza, R., Rosenbloom, J., Lowe, M., and Holtzer, H. (1979) Cell 17, 491-501. 12. Newman, S. A., and Watt, F. M. (1988) Exp. Cell Res. 178, 199210. 13. Glowacki, J., Trepman, E., and Folkman, J. (1983) Proc. Soc. Exp. Biol. Med. 172, 93-98. 14. Hunter, J. S., and Caplan, A. I. (1983) in Cartilage. (Hall, B. K., Ed.), pp. 87-119. Academic Press, New York.
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