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doi: 10.1242/10.1242/jcs.00171

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Differentiation plasticity of chondrocytes derived from mouse embryonic stem cells

¹ Department of Medical Molecular Biology, Medical University of Lübeck, D-23538 Lübeck, Germany
² In Vitro Differentiation Group, IPK Gatersleben, D-06466 Gatersleben, Germany

View larger version (44K): [in a new window]   Fig. 1. Alcian-blue-stained areas loose their stainability during differentiation of ES cells in vitro. Alcian blue staining demonstrating differentiation of chondrocyte nodules in ES-cell-derived EBs at 5+21 d (A), 5+26 d (B) and 5+31 d (C). The size of the stained areas was measured during EB cultivation from 5+7 d up to 5+31 d (D). Eleven days after plating (5+11 d) the first stained areas were detected, their number and size increased during cultivation up to 5+18 d and decreased during later stages. Mean values±s.e.m. are shown (n=10). The decrease in the size of Alcian-blue-stained areas was caused by a loss of stainability as demonstrated for nodules from 5+21 d, 5+26 d and 5+31 d (A-C) EBs. Representative areas of EB outgrowths are shown. Bar, 100 µm.

View larger version (29K): [in a new window]   Fig. 2. Genes characteristically expressed during chondrocyte hypertrophy and calcification were found to be differentially expressed during ES cell differentiation in vitro. Genes encoding extracellular matrix proteins of cartilage tissue such as collagen II (A), collagen X (B), osteocalcin (C) and the osteoblast-specific transcription factor Cbfa-1 (D) were expressed in EBs from different cultivation stages from 2 (5+2 d) up to 30 days (5+30 d) after plating as demonstrated by RT-PCR. The primers used to amplify collagen II transcripts detected two splice variants (A), the juvenile variant A and the adult variant B. The transcriptional levels relative to those of the house-keeping gene HPRT are presented. Mean values±s.e.m. derived from independent experiments (n=4) are shown.

View larger version (105K): [in a new window]   Fig. 3. Cells that express markers characteristic for hypertrophic and/or calcifying chondrocytes differentiated in EB outgrowths. Collagen X mRNA (A,B), osteopontin protein (C,D) and bone sialoprotein I + II (E,F) were expressed in cells organized in nodules (A,C,E) or as single cell clusters (B,D,F) in EB outgrowths. Representative areas of EB outgrowths at 5+23 d (A), 5+16 d (B) and 5+30 d (C-F) are shown. Bar, 100 µm.

View larger version (82K): [in a new window]   Fig. 4. Chondrogenic nodules isolated from EBs showed expression of marker proteins characteristic for mature and hypertrophic cartilage. Nodules were cut from 5+16 d EBs, and 10 µm cryosections were analyzed by immunostaining for collagen II (A), osteopontin (B), bone sialoprotein I + II (C) and collagen X (D). Interference contrast microscopy pictures (E-H) demonstrate the typical round-shaped morphology of chondrocytes. Bar, 100 µm.

View larger version (86K): [in a new window]   Fig. 5. Expression of marker genes (I) and proteins (II) during cultivation of chondrogenic cells isolated from EBs, indicating initial dedifferentiation followed by redifferentiation after prolonged cultivation. In chondrocyte cultures obtained from 5+16 d EB outgrowths mRNA levels of chondrocyte marker genes such as the adult splice variant B of collagen II (IA) and collagen X (IB) declined 4 days after isolation and were upregulated again up to 14 days after isolation. Coating of tissue culture plastic either with collagen II or gelatin did not cause significant differences. The level of transcription in relation to HPRT is shown. Significant differences (*P0.05; **P0.01; ***P0.001) in the mRNA levels for collagen IIB: 1-3, 2-4=***; 3-7=**; 4-8 and 3-5=* and for collagen X: 1-3, 2-4, 5-7=**. Mean values±s.e.m. from independent experiments (n=8) are shown. The isolated chondrogenic cells initially showed a fibroblastic morphology after one day in culture (IIA), started to form condensations after 4 days (IID) and eventually showed a round-shaped morphology after 8 days (IIG). These morphological alterations were accompanied by increasing expression of collagen II (IIB,E,H) and loss of collagen I expression (IIC,F,I) after one (IIB,C), four (IIE,F) and eight (IIH,I) days. Representative areas are shown. Bar, 100 µm.

View larger version (35K): [in a new window]   Fig. 6. Additional mesenchymal cell types developed in cultures of chondrogenic cells isolated from EBs. Adipocytes (A), skeletal muscle cells (B) and epithelial cells (C) were detected in cultures of isolated chondrocytes after 12-14 days as demonstrated by histochemical staining with Sudan III (A) and immunostaining for sarcomeric actinin (B) or a mixture of cytokeratins (C), respectively. Bar, 100 µm.

View larger version (32K): [in a new window]   Fig. 7. Application of TGF-ß3 resulted in downregulation of chondrogenic marker genes in cultures of chondrogenic cells isolated from EBs. Semi-quantitative RT-PCR demonstrated that the transcription levels of the juvenile splice variant A (A) as well as the adult splice variant B (B) of collagen II, collagen X (C) and osteocalcin (D) significantly decreased after application of TGF-ß3 at two different concentrations of 10 and 50 ng/ml to cultures of chondrogenic cells isolated from EBs. The mRNA levels in relation to those of HPRT are shown. Mean values±s.e.m. derived from independent experiments (n=2) are presented. Significant differences (in comparison to untreated control cells): *P0.05; **P0.01; ***P0.001.

View larger version (83K): [in a new window]   Fig. 8. Chondrogenic cells isolated from EBs were kept in a dedifferentiated state by treatment with TGF-ß3. After 8 days in culture, initially dedifferentiated chondrogenic cells isolated from EBs formed cell condensations (A) and showed expression of collagen II (B) but only trace amounts of collagen I (C) as demonstrated by immunostaining. Treatment with TGF-ß3 with either 10 ng/ml (D-F) or 50 ng/ml (G-I) resulted in inhibition of cell condensations (D,G) and collagen II expression (E,H) but increased expression of collagen I (F,I). Representative areas are shown. Bar, 100 µm.

View larger version (24K): [in a new window]   Fig. 9. ES-cell-derived chondrocytes display a differentiation plasticity. As shown in this paper ES cells differentiate in vitro into chondrocytes as well as osteocytes and into chondrocytes, which transdifferentiate into osteocytic cells. Chondrocytes isolated from EBs after initial dedifferentiation in culture, redifferentiate into chondrocytes and transdifferentiate mainly into adipocytes.

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