Figure 1.
Condensation of differentiated ESCs.
(A) The morphology of ESC chondrogenic aggregates cultured at day 5 (a) and 15 (b), Alcian Blue at day 15 (c). Scale bars represent 100 µm. (B) The ultra-structure of static suspension aggregates was analyzed by SEM on day 15 (a, b, c). (C) The H&E histological section of chondrogenic aggregate cultured at day 15 (a), Higher magnification (b), Alcian Blue (c). Scale bars represent 20 µm. (D) Expression of Sox9, Aggrecan, COL 2 and COL 10 mRNAs were analyzed by semi-quantitative RT-PCR at 0 (ESCs), 15 and 30 days of differentiation. β-actin was used as an internal control. (E) GAG content was determined. Data is expressed as mean ± SD (n = 4) per well. * Significant difference from day 0 (ESCs). P<0.05 with Student's t-test. (F) Calcium accumulation during differentiation was determined. Data is expressed as mean ± SD (n = 4) per well. * Significant difference from day 0 (ESCs). # Significant difference from day 15 P<0.05 with Student's t-test.
Table 1.
Primer Sequences.
Figure 2.
(A) The ultra-structure of static suspension aggregate was analyzed by SEM at day 15. (B) The cells shed from aggregates displayed two types, round-shaped with fibrils (a) and flat fibroblastic cells (b). Scale bars represent 20 µm. (C) The round-shaped cells formed network spontaneously. Scale bars represent 50 µm. (D) The expression of chondrocyte-related proteins (Aggrecan, COL 2, COL 10) was analyzed at day 20 of differentiation by immunofluorescence (a, d, g), DAPI (b, e, h), Bright field (c, f, i). Scale bars represent 50 µm. (E) Alcian Blue stained chondrogenic cells at day 20 of differentiation (a, b). These cells were induced to form the aggregates spontaneously (c, d). Scale bars represent 50 µm. (F) The expression of chondrocyte-related mRNAs (Sox9, Aggrecan, COL 2, COL 10) and fibroblast-related mRNA (COL 1) were analyzed at day 20 by semi-quantitative RT-PCR. β-actin was used as a loading control.
Figure 3.
In vitro cartilage formation, and In vivo cartilage and bone formation.
(A) The morphology and ultra-structure of aggregate cultured at day 40. Morphology (a), Scale bars represent 100 µm. SEM (b, c) (B) The histological section of ESC chondrogenic aggregate cultured at day 30 and 40, H&E staining (a, d). Higher magnification (b, e). Alcian Blue (c, f). COL 2 (g, h). Scale bars represent 20 µm. (C) Transplantation of aggregates. Cells were cultured in vitro for 30 days under the chondrogenic differentiation protocol. The aggregates were then transplanted into SCID mice. After removal of transplanted tissue, both cartilage and bone tissues were observed. Cartilage was indicated by Toluidine Blue (b, e). Bone was indicated by Methylene Blue (f, i). Scale bars represent 50 µm.
Figure 4.
In vitro hypertrophy and degradation of cartilage, and bone replacement.
(A) The morphology of aggregate cultured at day 50. Scale bars represent 100 µm. (B) The histological section of aggregate cultured at day 50 or 60 was stained by H&E. Hypertrophic cartilage (a), Degraded cartilage (b), Degraded cartilage and bone replacement (c) and bone replacement (d). Scale bars represent 20 µm. (C) Apoptotic cells were indicated by TUNEL staining. TUNEL (a), Bright field (b). Scale bars represent 20 µm. (D)The section of bone replacement cultured at day 60. H&E (a), Alcian Blue (b), Alizarin Red S (c), Alcian Blue & Alizarin Red S (d), Methylene Blue (e). Scale bars represent 20 µm. (E) The section of bone cultured at day 100. H&E (a), Alizarin Red S (b), COL 1 (c). Scale bars represent 20 µm.
Figure 5.
Gene expression during chondrogenesis in suspension culture.
The expression of chondrocyte and osteoblast-related genes was analyzed from 0 to 60 days of differentiation by real-time RT-PCR. Sox9 (a), Aggrecan (b), COL 2 (c), COL 10 (d), MMP13 (e), Cbfa1 (f), Osteocalcin (g). Data is expressed as means ± SD (n = 3) per lane. With a P<0.05 using the Student's t-test, * represents a significant difference between two.
Figure 6.
Schematic summary of the stages of chondrogenesis of ESCs.
The relative temporal aspects of five stages of chondrogenesis are denoted by cellular, extracellular and molecular events: (1) Condensation of differentiated ESCs, (2) Differentiation into chondrocytes and fibril scaffold formation, (3) ECM deposition and cartilage formation, (4) Hypertrophy and degradation of cartilage, and (5) Bone replacement. Scale bars represent 20 µm.