Anat Embryol (2005) 210: 175–185 DOI 10.1007/s00429-005-0020-x
O R I GI N A L A R T IC L E
Jan Kramer Æ Matthias Klinger Æ Charli Kruse Marius Faza Æ Gunnar Hargus Æ Ju¨rgen Rohwedel
Ultrastructural analysis of mouse embryonic stem cell-derived chondrocytes
Accepted: 20 July 2005 / Published online: 7 October 2005 Springer-Verlag 2005
Abstract Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via J. Kramer (&) Æ M. Faza Æ G. Hargus Æ J. Rohwedel Department of Medical Molecular Biology, University of Lu¨beck, 23538 Lu¨beck, Germany E-mail:
[email protected] Tel.: +49-451-5003404 Fax: +49-451-5006579 J. Kramer Medical Department 1, Division of Nephrology Transplantation unit, University Clinics of Schleswig-Holstein, Campus Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany M. Klinger Institute of Anatomy, University of Lu¨beck, 23538 Lu¨beck, Germany C. Kruse Fraunhofer Institute of Biomedical Engineering, Group of Cell Differentiation and Cell Technology, University of Lu¨beck, 23538 Lu¨beck, Germany Present address: G. Hargus Centre for Molecular Neurobiology, University of Hamburg, 20251 Hamburg, Germany
progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue. Keywords ES cells Æ Chondrocytes Æ Collagen Æ Cartilage Æ Chondrogenic differentiation
Introduction The formation of the skeleton during embryogenesis is a complex multi-step process that is still incompletely understood (for review: Cancedda et al. 2000; Provot and Schipani 2005; Sandell and Adler 1999). During vertebrate skeletogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous template. This process, called endochondral ossification, is characterized by a precise series of events. First, mesenchymal cells aggregate and form mesenchymal condensations at the sites where later on, the skeletal elements are generated. The mesenchymal cells differentiate into chondroblasts producing the extracellular matrix of the cartilage anlagen. Later on, this cartilage template will be replaced by bone through a process called endochondral ossification including proliferation, hypertrophy, and apoptosis of chondrocytes (de Crombrugghe et al. 2001; Karsenty 2003; Karsenty and Wagner 2002; Olsen et al. 2000). However, in a few skeletal elements, the mesenchymal cells will bypass the chondrogenic stage and will directly differentiate into osteoblast cells. This process called intramembranous ossification occurs in the lateral parts of the clavicle and parts of the skull. Furthermore, chondrocytes form the hyaline joint (Mitrovic 1977), the tracheal and nasal cartilage (Pavlov et al. 2003), as well as the elastic cartilage of the external ear (Moskalewski 1976). The chondrogenic subpopulations differ in their gene-expression profiles and their morphology according to their specific function. For example, the main
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subtypes of cartilage tissue—hyaline and elastic—differ in the composition of their ectracellular matrix. Elastic cartilage is characterized by the expression of elastic fibrils, e.g. elastin (Moskalewski 1976), in contrast to hyaline chondrogenic tissue. Different stages of chondrogenic differentiation can be distinguished by their specific patterns of markermolecule expression. Mesenchymal cells express marker molecules such as Sox5 and Sox6 (Lefebvre et al. 1998) after the formation of condensations indicating the initiation of chondrogenesis. These chondrogenic precursors differentiate into chondroblasts expressing the cartilage proteoglycan aggrecan and collagen II. Finally, the chondrocytes become hypertrophic as indicated by the expression of collagen X, undergo apoptosis and are replaced by invading osteoblasts or alternatively, transdifferentiate into osteoblasts. Transdifferentiation of hypertrophic chondrocytes into osteoblasts is also discussed as an in vivo pathway participating in initial bone formation (Bianco et al. 1998; Cancedda et al. 2000). Moreover, transdifferentiation of hypertrophic chondrocytes into osteogenic cells has been demonstrated in vitro (Erenpreisa and Roach 1996). However, osteoblast invasion after vascularization seems to be the general pathway for enchondral ossification (Karsenty 2003). Differentiation of embryonic stem (ES) cells in vitro can serve as a model system to study cartilage differentiation. Permanent lines of ES cells exhibit the capacity to differentiate into cell types of all the three germ layers (for review: Rodda et al. 2002) due to their origin from the inner cell mass of blastocysts (Evans and Kaufman 1981; Martin 1981). Previously, we have demonstrated that pluripotent mouse ES cells cultivated via cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into chondrogenic cell types in vitro (Hegert et al. 2002; Kramer et al. 2000). Using the EScell-model system, chondrogenesis can be recapitulated from the undifferentiated stem cell via mesenchymal and chondrogenic progenitor cells up to mature and hypertrophic chondrocytes. Finally, osteogenic cells can be detected in the nodules. Initially, the mesenchymal cells form condensations in the EBs expressing mesenchymal marker molecules such as scleraxis (Kramer et al. 2000). Later on, these cellular aggregates become more and more compact and collagen II expression indicates cartilage-nodule formation. Besides this process of endochondral ossification, direct differentiation of precursors into osteoblasts bypassing the chondrogenic stage was observed in EB-outgrowths (Hegert et al. 2002). Furthermore, we and others have previously reported that the influence of growth factors and signaling molecules on chondrogenic/osteogenic differentiation can be studied using the ES-cell-model system (Kawaguchi et al. 2005; Kramer et al. 2000; Phillips et al. 2001; zur Nieden et al. 2005). A time-window of ES cell culture (from 2–5 day) was found to be sensitive for induction of cartilage nodule formation in EBs. This early stage of ES cell differentiation is a period of early mesodermal development characterized by the expression of
Brachyury and BMP-4 (Johansson and Wiles 1995; Rohwedel et al. 1998; Yamada et al. 1994). In this paper we ask the questions as to whether EScell-derived pre-cartilaginous condensations and cartilage nodules resemble their embryonic counterparts in particular at the ultrastructural level, and which type of cartilage tissue these nodules may represent.
Materials and methods ES cell cultivation and differentiation of ES cells via EBs ES cells of line BLC6 (Wobus et al. 1988) were cultivated on a feeder layer of primary mouse embryonic fibroblasts in Dulbecco’s modified Eagle’s medium (Invitrogen, Karlsruhe, Germany) supplemented with 15% fetal calf serum (FCS, selected batches, Invitrogen, Karlsruhe, Germany), 2 mM L-glutamine (Invitrogen, Karlsruhe, Germany), 5·10 5 Mb mercaptoethanol (Serva, Heidelberg, Germany), and non-essential amino acids (Invitrogen, Karlsruhe, Germany; stock solution diluted 1 : 100) to keep the cells in the undifferentiated stage as described previously. The embryonic fibroblasts were growth-inactivated by treatment with Mitomycin C (Serva, Heidelberg, Germany). In addition, leukemiainhibitory factor (LIF; 5 ng/ml; Invitrogen, Karlsruhe, Germany) was supplemented to the media for the maintenance of the ES cell pluripotency. For differentiation, aliquots of 20 ll differentiation medium (with 20% FCS instead of 15%) containing 800 cells were cultivated in ‘hanging drops’ for 2 days (0–2 day) and subsequently in suspension on bacteriological petri dishes for additional 3 days (2–5 day) as described (Kramer et al. 2000). The 5-day-old EBs were plated onto (0.1%) gelatin-coated 6-cm tissue culture plates for micro-dissection, and onto (0.1%) gelatin-coated twowell Lab-Tek chamber slides (Nunc, Wiesbaden, Germany) for indirect immunostaining and in situ hybridization. Alcian blue staining was used to quantify cartilage-nodule formation in EB-outgrowths as described previously (Kramer et al. 2003). Fluorescence in situ hybridization For fluorescence in situ hybridization, we performed a modified procedure of Yamada et al. (1994). Ten EBs were plated per chamber slide and analyzed at different developmental stages. The chambers were rinsed twice with phosphate buffer solution (PBS) and cells were fixed with 4% (w/v) paraformaldehyde, 4% (w/v) sucrose in PBS for 20 min at room temperature. Prior to incubation at 70C in 2· SSC for 15 min, the specimens were washed twice with PBS for 5 min. After rinsing once again with PBS for 5 min followed by 2· SSC, the EBs were fixed once again for 5 min and the washing step with PBS and 2· SSC was repeated. The cells were subsequently dehydrated at room temperature for 2 min
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each in 50%, 70%, 95% ethanol and twice in 100% ethanol. Prehybridization was performed in prehybridization buffer containing 5X SSC, 5x Denhardt‘s, 50% formamide, 250 mg/ml yeast t-RNA, 250 mg/ml denatured salmon sperm DNA, and 4 mM EDTA for 3 h in a humidified chamber at 45C. Hybridization with digoxigenin-labeled sense and antisense probes against the below-designated marker molecule (1 ng/ll) was done in prehybridization buffer without salmon sperm DNA at 45C overnight. After hybridization, specimens were washed twice with 2· SSC for 15 min, once with 0.2· SSC for 15 min, and twice with 0.1· SSC for 15 min at 45C, and then rinsed in PBS. The monoclonal antibody II-II6B3 against collagen II (diluted 1 : 20 in PBS) was applied, followed by incubation in a humidified chamber for 1 h at 37C. Specimens were washed three times with PBS at room temperature and FITC-conjugated sheep F(ab) fragments against digoxigenin (Boehringer, Mannheim, Germany) and Cy3-conjugated goat anti-mouse secondary antibodies (Dianova) for detection of collagen II (diluted 1 : 800 in PBS) were added. After incubation for 1 h at 37C, the slides were washed three times in PBS and once in distilled water, embedded in Vectashield mounting medium, and analyzed with the Axioskop Fluorescence microscope (Zeiss, Germany). Control slides that were incubated with the sense probe or only with the FITC-conjugated sheep F(ab) fragments against digoxigenin or the Cy3-conjugated goat anti-mouse secondary antibodies showed no staining demonstrating the specificity of the responses. Hybridization probe The design of the hybridization probes against the marker molecules, scleraxis (Kramer et al. 2000) and collagen X (Hegert et al. 2002) have been previously described. The Sox5 and Sox6 cDNAs were a kind gift of Veronique Lefebvre, Department of Biomedical Engineering (Cleveland, Ohio, USA). Digoxigenin-labeled RNA probes of either sense or antisense orientation of Sox5 or Sox6 (Lefebvre et al. 1998) were synthesized from linearized plasmids of the cloned cDNA fragment by in vitro transcription using the polymerase of T7-RNA (New England Biolabs, Frankfurt, Germany) or T3-RNA (Roche, Mannheim, Germany) following the protocol supplied by the manufacturer. Immunostaining and confocal microscopy EBs were cultivated on chamber slides and rinsed two times with PBS. They were fixed for 5 min with methanol : acetone (7:3) at room temperature, washed three times with PBS again, and incubated at 37C for 30 min with goat serum (1 : 10 dilution). Specimens were then incubated for 1 h with the first antibody in a humidified
chamber at 37C. The monoclonal antibodiy specific for collagen type II (II-II-6B3; (Linsenmayer and Hendrix 1980) was obtained from the Developmental Studies Hybridoma Bank, University of Iowa, USA. PBS was used to dilute the antibodies 1 : 20. After rinsing three times with PBS, slides were incubated for 45 min at 37C with Cy3-conjugated goat anti-mouse secondary antibodies (Dianova, Hamburg, Germany), diluted 1:200 times. Slides were washed three times in PBS and briefly in distilled water. Specimens were embedded in Vectashield mounting medium (Vector, Burlinggame, CA, USA) and analyzed with the laser scanning microscope MRC600 (BIORAD, Munich, Germany). Control specimens that were incubated only with the FITC-labeled anti-mouse IgG antibodies showed no staining indicating the specificity of the response. Isolation of mesenchymal condensations and cartilage nodules from EBs To analyze mesenchymal condensations and cartilage nodules by electron-microscopy, these structures were isolated from EBs. The EBs were cultivated as described above. The mesenchymal condensations were selected 8– 10 days after EB-plating and the cartilage nodules were cut from the EB-outgrowths (Fig. 1) at 17–21 days after EB-plating using a micro-dissector (Eppendorf, Hamburg, Germany). The specimens were collected in 4Ccold fixation solution for electron-microscopy. The criteria used to discriminate between condensations and nodules were (1) their morphology (non-round shaped mesenchymal cells which have close cell contacts versus round-shaped cartilage cells seperated by large amounts of extracellular matrix), (2) the data from the immunostainings (strong expression of Sox5/6 in mesenchymal condensations at 8–10 days after EB plating versus strong expression of collagen type II at 17–21 days after EB plating), and (3) data from Alcian blue staining (mesenchymal condensations show no staining whereas cartilage nodules are intensively stained; see also Fig. 4). Cupromeronic blue staining for transmission electron microscopy Glycosaminoglycans were stained with cupromeronic blue according to Scott’s critical electrolyte concentration technique (Scott 1985), modified by Stoeckelhuber et al. (2002). In this method, the cationic dye cupromeronic blue and Mg2+ ions of MgCl2 compete for the binding to the negatively charged sulfate groups of the glycosaminoglycans. Excised cartilage nodules were fixed by immersion in 3.5% glutaraldehyde in PBS, pH 7.2, for 2 h at 22C. The nodules were washed 3·10 min in 0.2 M acetate buffer, pH 5.6, containing 1% glutaraldehyde and stained overnight in 1% cupromeronic blue in 0.2 M acetate buffer containing 2.5% glutaraldehyde and
178 Fig. 1 Murine ES cells, cultivated via embryoid bodies (EBs) form cartilage nodules in vitro. Mesenchymal cells aggregate and finally form cartilage nodules containing round-shaped chondrocytes (a). These nodules show a distinct structure of roundshaped cells seperated by extracellular matrix surrounded by a circular arranged fibrillar matrix and can easily be detected by light microscopy (b). Isolation of nodules from EBs can be performed without damaging their characteristic cellular structures by microdissection (c). Bar = 100 lm
0.06 M MgCl2. The nodules were then washed in acetate buffer with 1% glutaraldehyde and 0.06 M MgCl2, and were treated with 0.5% NaWO4 in the same solution for 1 h. Afterward, the nodules were treated with 0.5% NaWO4 overnight in 30% ethanol. After dehydration in a graded ethanol series, nodules were incubated in propylene oxide, and embedded in Araldite (Fluka, Buchs, Switzerland). Ultrathin sections were cut on an Ultracut E (Reichert-Jung, Nußloch, Germany) and were stained for 20 min with uranyl acetate and were examined with a Philips EM 400electron microscope. For transmission electron-microscopy without cupromeronic blue staining, the samples were also fixed with 5% glutaraldehyde in 0.1 mol/l cacodylate buffer for 1 h, treated with 1% OsO4 for 2 h, and dehydrated in graded ethanol series. The specimens were embedded and ultrathin sections were cut as described above. Before the analysis with Philips EM 400 was performed, the sections were contrasted with uranyl acetate and lead
citrate (Ultrostainer Carlsberg System, LKB, Bromma, Sweden).
Results Chondrogenic progenitor cells form mesenchymal condensations which further differentiate into cartilage nodules In EB-outgrowths derived from ES cell line BLC6, mesenchymal cells aggregate and form cellular condensations around 10 days after plating of EBs. These mesenchymal condensations were randomly distributed in the EB-outgrowths. Initially, Sox6 and Sox5 were expressed by mesenchymal cells localized in condensations as revealed by in situ hybridization (Fig. 2a, b). These chondrogenic progenitor cells did not express the cartilage-marker molecule collagen II. The cells were tightly packed in the condensations and did not yet show
179 Fig. 2 Chondrogenic differentiation of ES cells in vitro recapitulates cellular events of chondrogenesis from undifferentiated stem cells via progenitor cells up to differentiated, mature and hypertrophic chondrocytes. EScell-derived mesenchymal cells aggregate between 8–10 days after EB plating and express Sox6 (a) and Sox5 (b) as revealed by in situ hybridization. Combination with immunostaining against collagen II showed that this cartilage-marker molecule was not expressed in these mesenchymal aggregates (data not shown). Such cellular condensations consisting of tightly packed cells (c, d) foreshadowed cartilage-nodule development. Collagen II expression in cartilage nodules between 17 and 21 days after EB plating, shown by immunostaining (red, e), indicated further differentiation into the chondrogenic direction. Cells at this stage still expressed the precartilaginous marker molecule scleraxis (green, e) as revealed by in situ hybridization. Finally, in situ hybridization showed the expression of collagen X (green, f) and, in combination with immunostaining, a decreasing expression of collagen II protein (red, f) in hypertrophic chondrocytes within nodules. Bar = 100 lm
the round-shaped phenotype of mature chondrocytes at this stage (Fig. 2c, d). Later, in combination with immunostaining against collagen II, in situ hybridization demonstrated colocalization of scleraxis transcripts and collagen II protein in cartilage nodules (Fig. 2e). Further differentiation resulted in an upregulated expression of collagen II. Cartilage nodules at this stage are highly organized structures representing a small recessus in the EB-outgrowth, which contain typical round-shaped cells separated by extracellular matrix (Fig. 3; see also Fig. 1). During the final stages of cartilage-nodule development the expression of collagen X, a marker molecule for hypertrophic chondrocytes, could be
detected in the cartilage nodules and the amount of collagen II protein decreased (Fig. 2f). The change in the composition of the extracellular matrix was already indicated by a quantitative evaluation of nodule formation using Alcain blue staining (Fig. 4). In EB-outgrowths derived from ES cell line BLC6, chondrogenic cells appeared as intensively Alcian-blue-stained nodules (Fig. 4, inset). The number and size of these nodules initially increased during EB cultivation, but was later accompanied by a reduced intensity of Alcian blue staining. Immunostaining with an antibody against elastin was negative at all the investigated stages, demonstrating
180 Fig. 3 Confocal microscopy of an ES-cell-derived cartilage nodule demonstrating the highly organized structure. Confocal sections of 2.5 lm (a–d) of a nodule in the outgrowth of an EB 17 days after plating, immunostained for type II collagen, demonstrate the threedimensional character. At higher magnification, sections of 1.8 lm (e–h) show that type II collagen is deposited as extracellular matrix between the cells. Bars = 100 lm (a–d) and 25 lm (e–h)
that this component of elastic cartilage tissue was not expressed in the investigated ES-cell-derived mesenchymal condensations and cartilage nodules. Electron microscopic analysis of ES cell-derived chondrogenic progenitors and chondrocytes To study ES-cell-derived chondrogenic cells at an ultrastructural level, mesenchymal condensations and
cartilage nodules were excised from EB-outgrowths and analyzed by electron-microscopy. The cells in the condensations were tightly packed and intercellular spaces were as narrow as in epithelial cell layers. Nuclei were prominent and almost all cells showed a low cytoplasm to nucleus ratio (Fig. 5a) like the proliferating precartilaginous mesenchymal cells in vivo. In contrast, in cartilage nodules, the cells were separated from each other by a loosely woven extracellular matrix (Fig. 5b). Chondrogenic cells were relatively
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(Fig. 6a). Interestingly, some densely packed chondrogenic cells, which were located at the peripheral zone of the cartilage condensations in the EBs, displayed many microvilli (Fig. 6b) resembling an organized cell layer of chondrogenic cells in highly cellular embryonic cartilage. ES cell-derived chondroblasts express large amounts of proteoglycans
small and round, sometimes with irregular contours. Intercellular cartilaginous matrix was composed of small collagen-like fibers (Fig. 5c), relatively shorter structures interwoven to create a three-dimensional network. These collagen-like fibrils were thin and did not show any banding pattern. Some of the chondrogenic cells in the specimens showed a cytoplasm overcrowded with granular endoplasmic reticulum. Its distended cisternae strongly suggest these cells to be actively engaged in synthesis and secretion of extracellular proteins
To test for the presence of proteoglycans in ES-cell-derived nodules at the ultrastructural level, the dye cupromeronic blue was used to stain glycosaminoglycans in electron-microscope (EM) sections. It is known that proteoglycans with their large molecular array generally collapse into small dense structures about 20 nm across as their hydration sheaths are stripped away. Indeed, in our specimens, cupromeronic-blue-positive material was mostly visible as densely staining fibrillar or granular structures presumably representing collapsed assemblies of protein and glycosaminoglycans (Figs. 6a; 7a, b). In the ES-cell-derived cartilage nodules, cells could be detected showing the typical morphology of chondroblasts producing cartilage proteoglycans and collagens. The cells contained large quantities of granular endoplasmatic reticulum with some distended cisternae (Fig. 7a). Their nuclei were rounded, with one or more prominent nucleoli. The chondroblasts in the ES-cell-derived cartilage nodules were separated by their extracellular matrix, but still showed a few intercellular contacts. The matrix around the secreting chondroblasts was filled with proteoglycans predominantly arranged in a crisscross pattern (Fig. 7b). To verify the results of the cupromeronic blue staining, cartilage tissue derived from rat sternum was used as a control. This material contains collagen fibers with
Fig. 5 Mesenchymal aggregates consist of tightly packed cells with narrow intercellular spaces. These cells have prominent nuclei and display a low cytoplasm to nucleus ratio (a) Within cartilage nodules, cells are scattered in a loosely woven extracellular matrix
(b). The matrix is composed of small thin collagen-like fibers without any banding pattern and relatively shorter compact structures that are arranged in a three-dimensional network (c). Magnification: (a) 6000·2.3; (b) 4600·2.3; (c) 46000·2.3
Fig. 4 Fig. 1. The number of Alcian-blue-stained nodules increased during EB cultivation. EBs were stained with Alcian blue at different times from 2 up to 31 days after plating and the number of stained nodules in individual EBs was counted. Fourteen days after plating the first cartilage nodules were detected and their number increased during further cultivation up to 22 days after plating. Later the number of nodules decreased again probably due to a rearrangement in the extracellular matrix. Mean values ± s.d. are shown. The inset shows an Alcian-blue-stained cartilage nodule in a ‘‘5+18 days’’ EB outgrowth (Bar = 100 lm)
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Discussion
Fig. 6 Chondrogenic cells are surrounded by a dense meshwork of cupromeronic-blue-positive material and display a cytoplasm filled with granular endoplasmic reticulum (arrow). Its distended cisternae are indicative for synthesis and secretion of extracellular proteins (a). At the peripheral zone of the cartilage condensations, densely packed chondrogenic cells display many microvilli (arrow) thus resembling an organized cell layer (b). Magnification: 4600·2.3
regularly associated proteoglycan complexes (Fig. 8). These cartilage proteoglycans were arranged either in a parallel pattern or in a non-parallel crisscross arrangement demonstrating the network of cartilaginous extracellular matrix.
ES cells are pluripotent due to their origin from the inner cell mass of early embryonic blastocysts (Evans and Kaufman 1981; Martin 1981). In vitro ES cells differentiate via EBs into various cell types of the three germlayers and it has been shown that ES cell differentiation rather closely recapitulates developmental processes in vivo. Recently, ES-cell-derived chondrogenesis has been described in detail by our group (Hegert et al. 2002; Kramer et al. 2000, 2003, 2005). Here, we confirm at the ultrastructural level that the ES-cell-model system includes all stages of chondrogenesis, starting with pluripotent cells that become limited to the mesenchymal lineage and develop into mature and finally hypertrophic chondrocytes. We provide a detailed ultrastructural analysis by electron-microscopy. Mesenchymal cells in the EB-outgrowths aggregate and in situ hybridization showed the expression of Sox5 and Sox6 as well as scleraxis, marker molecules of pre-cartilaginous cell condensations (Cserjesi et al. 1995; Kramer et al. 2000; Lefebvre et al. 1998). Moreover, we have also shown that Sox9, which is a key regulatory transcription factor for chondrogenic differentiation, was expressed during the whole period of EB cultivation (Kramer et al. 2000). Similar results regarding the expression of Sox9 have been published by other groups (Kawaguchi et al. 2005; Tanaka et al. 2004). The continous expression of Sox9 during EB cultivation may be due to a functional role of Sox9 from mesenchymal up to hypertrophic chondrogenic stages (de Crombrugghe et al. 2001). Electron-microscopic analysis of the mesenchymal aggregates in the EB-outgrowths demonstrated the typical morphological structure of mesenchymal cells initiating chondrogenesis. This stage proceeds with the onset of collagen II expression, characteristic for cartilaginous tissue (Eyre and Muir 1975) as confirmed by immunostaining. Furthermore, we have studied the expression of additional cartilage– and boneassociated molecules including aggrecan, osteocalcin, and cbfa1 and found that they were expressed during EB cultivation (Hegert et al. 2002; Kramer et al. 2000; reviewed by Kramer et al. 2003) . Other groups confirmed our results, and have studied the expression of other molecules such as Col10a1, PTHR1, PTHrP, Link protein, and biglycan (Kawaguchi et al. 2005; Nakayama et al. 2003; Tanaka et al. 2004; zur Nieden et al. 2005). The ES-cell-derived chondrogenic cells form distinct nodules in the EB-outgrowths which can be stained by Alcian blue. Similar structures have been described when limb bud progenitor cells were cultivated as threedimensional aggregates (Sui et al. 2003). Finally, the EScell-derived chondrogenic cells pass over into hypertrophic chondrocytes expressing collagen X, a unique marker molecule for this chondrogenic stage (Sandell and Adler 1999). The change in the extracellular matrix composition of the nodules is accompanied by a loss of Alcian blue staining. These results are in line with our
183 Fig. 7 A cell derived from a cartilage nodule displays the typical morphology of a chondroblast-producing cartilage proteoglycan and collagen. The cell contains granular endoplasmatic reticulum with some distended cisternae (arrow), and the cytoplasm to nucleus ratio is low (a). Chondroblasts from cartilage nodules are surrounded by proteoglycans that are predominantly arranged in a crisscross pattern (b). Magnification: (a) 8000·2.3; (b) 22000·2.3
previous studies, which already demonstrated that the expression pattern of collagen and osteogenic marker genes and proteins during the process of endochondral ossification in vivo is nicely recapitulated during cultivation of EBs (Hegert et al. 2002). Electron-microscopic analysis of ES-cell-derived cartilage nodules showed at least three different types of chondrogenic cells. First, cells located in groups at the peripheral zones of the nodules had many short microvilli reminding of highly cellular embryonic cartilage in vivo. Second, many ES-cell-derived active chondrogenic cells could be described. These chondroblast-like cells showed many characteristics of cells making and secreting proteins. Indeed, large amounts of proteoglycans and collagens filled the intercellular space between
the cells. Third, chondrogenic cells surrounded by this cartilaginous matrix, but without morphological signs of protein synthesis, were detectable. Similar cell types can be found in cartilage tissue. Cells which had many short microvilli reminding of highly cellular embryonic cartilage and chondrogenic cells without morphological signs of protein synthesis were observed very rarely (approximately 10% of the cells) in ES-cell-derived cartilage nodules. Most of the cells were chondrogenic cells which showed characteristics of protein secretion. Immunostaining of cartilage nodules showed collagen II expression, but no expression of elastin, a marker molecule of elastic cartilage tissue. Collagen II is not expressed in fibrocartilage and is the prominent collagen in mature hyaline cartilage tissue (Eyre and Muir 1975).
Fig. 8 Cartilage tissue derived from rat sternum was used to verify the method of cupromeronic blue staining. This material contains single collagen fibers with regularly associated proteoglycan complexes within a meshwork of cupromeronic-blue-positive
material (a), but also both the electron dense meshwork alone (b) or thick bundles of collagen fibers with associated proteoglycans (arrow) arranged in a parallel pattern (c). Magnification: 36000·2.3
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Therefore, we conclude that ES-cell-derived chondrogenic cells belong to the subpopulation of chondrogenic cell types which represent hyaline cartilage in joints and other tissues. Currently, we are generating a reporterand selection-construct with a gene cassette encoding an enhanced green fluorescent protein (EGFP)–puromycin fusion protein under the control of the Col2a1-promoter. ES cell clones stably transfected with such a construct can be used to select pure chondrocyte populations from differentiated EBs. These chondrogenic cells will then be used to test their physiological integration and function in vivo after transplantation into animal models. ES-cell-based regenerative strategies have to be discussed critically due to ethical aspects regarding the origin of the cells. Furthermore, several technical problems have to be resolved such as generation of pure populations of differentiated cell types and generation of autologous cells. However, our results demonstrate that cellular differentiation and specification during chondrogenesis can be studied in vitro using the ES-cellmodel system. This is particularly interesting for in vitro studies using transgenic and gene knock-out ES cell lines as an additional or alternative approach to in vivo studies. Furthermore, the ES cell system may serve as a model to study mechanisms of cell differentiation with important implications for generation and manipulation of somatic stem cells. Acknowledgements The skilful technical assistance of A. Eirich and M. Dose is gratefully acknowledged. The work was supported by the Medical Faculty of the University of Lu¨beck and funded by Intermed Service GmbH&CoKG (Geesthacht, Germany) and Eppendorf AG (Hamburg, Germany).
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