Schematic summary of the stages of chondrogenesis of ESCs.

<p>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.</p

Condensation of differentiated ESCs.

<p>(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 <i>t</i>-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 <i>t</i>-test.</p

<i>In vitro</i> hypertrophy and degradation of cartilage, and bone replacement.

<p>(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.</p

Adipogenesis during ESC osteoblast differentiation in static culture.

<p>(A) Using Oil Red O staining, lipids with dark red appearance were observed in re-attached cells on day 10 of differentiation in static: KSR-basic (a), KSR-Dex (b), KSR-VD3 (c). Oil Red O co-staining with Alizarin Red S shows both lipid and calcification within re-attached cells in KSR-Dex (d) and KSR-VD3 (e); scale bars represent 20 µm. (B) Lipid accumulation per micro-mass spot was determined on day 10 of differentiation (a). Lipid accumulation was also normalized against DNA content to determine lipid synthesis per cell (b); data is expressed as mean ± SD (n = 3) per well. * represents a significant difference between undifferentiated and differentiated ESCs; P<0.05 with Student's <i>t-</i>test. (C) The expression of adipocyte-related mRNAs (PPARγ, aP2) was analyzed using real-time RT-PCR at 0 (ESCs), 7 and 14 days; data is expressed as means ± SD (n = 3) per point.</p

<i>In vitro</i> cartilage formation, and <i>In vivo</i> cartilage and bone formation.

<p>(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 <i>in vitro</i> 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.</p

Micro-mass directed ESC differentiation towards the osteoblast lineage.

<p>(A) Morphological changes on day 4 of differentiation between the six media conditions tested: FBS-basic (a), FBS-Dex (b), FBS-VD3 (c), KSR-basic (d), KSR-Dex (e), KSR-VD3 (f). Scale bars represent 100 µm. (B) Apoptotic cells were observed using TUNEL on day 4 of differentiation in KSR-Dex (b) and KSR-VD3 (d). The corresponding brightfield channels demonstrates cell position (a and c respectively); scale bars represent 100 µm. (C) Re-attached aggregates on day 7 show significant differences at day 7 between the six media types: FBS-basic (a), FBS-Dex (b), FBS-VD3 (c), KSR-basic (d), KSR-Dex (e), KSR-VD3 (f). (D) After trypsinization on day 7 of differentiation, aggregates also show differences between the six media types: FBS-basic (a), FBS-Dex (b), FBS-VD3 (c), KSR-basic (d), KSR-Dex (e), KSR-VD3 (f); scale bars represent 100 µm. (E) Expression of osteoblast-related proteins, COL 1 and osteocalcin using immunofluorescence on day 7 of differentiation: FBS-Dex (a, m), FBS-VD3 (d, p), KSR-Dex (g, s) and KSR-VD3 (j, v). Brightfield (b, e, h, k, n, g, t, w) and DAPI (c, f, i, l, o, r, u, x) are also shown; scale bars represent 50 µm. (F) Using Alizarin Red S staining, calcification with red appearance was observed in culture dish on day 7 and 14 of differentiation: FBS-basic (a, d), FBS-Dex (b, e), FBS-VD3 (c, f), KSR-basic (g, j), KSR-Dex (h, k), KSR-VD3 (i, l); scale bars represent 50 µm. (G) Calcium content per micro-mass spot (a) and normalized against DNA (b) was determined; data is expressed as mean ± SD (n = 3) per well. ^# represents a significant difference between FBS-Dex and VD3 or KSR-Dex and VD3; P<0.05 with Student's <i>t-</i>test. * represents a significant difference between FBS and KSR, P<0.05 with Student's <i>t-</i>test. (H) Expression of osteoblast-related mRNAs (Cbfa1, osteocalcin) was analyzed using real-time PCR at 0 (ESCs), 7 and 14 days. Data is expressed as means ± SD (n = 3) per lane.</p

Primer sequences.

<p>Primer sequences.</p

Bone-like tissue formation <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The ultra-structure of aggregates was analyzed by SEM on day 15 of stirred suspension culture: KSR-basic (a, d), KSR-Dex (b, e), KSR-VD3 (c, f). Low magnification (a, b, c), scale bars represent 50 µm; high magnification (d, e, f), scale bars represent 10 µm. (B) Histological sections of aggregates were analyzed by hematoxylin and eosin (H&E) staining after 15 days of differentiation: KSR-basic (a), KSR-Dex (b), KSR-VD3 (c); scale bars represent 20 µm. (C) Expression of osteoblast-related mRNAs (Cbfa1, osteocalcin) was analyzed using real-time PCR during ESC differentiation in KSR-VD3 static suspension culture. (D) Calcium content was analyzed during ESC differentiation in KSR-VD3 static suspension culture and normalized against DNA content. (E) Expression of adipocyte-related mRNAs (PPARγ, aP2) was analyzed using real-time RT-PCR during ESC differentiation in KSR-VD3 static suspension culture; data is expressed as means ± SD (n = 3) per lane. * represents a significant difference between two conditions tested; P<0.05 with Student's <i>t-</i>test. (F) Following 15 days of differentiation in static suspension culture, bone (a, b, e, f) and epithelium (c, d, g, h)-like tissues were observed <i>in vivo</i> upon transplantation into SCID mice: KSR-Dex (a, b, c, d) and KSR-VD3 (e, f, g, h); H&E (a, c, e, g) and Methylene Blue (b, d, f, h) staining, scale bars represent 20 µm.</p

Gene expression during chondrogenesis in suspension culture.

<p>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 <i>t</i>-test, * represents a significant difference between two.</p

Chondrocyte differentiation.

<p>(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.</p