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First published online 1 April 2003
doi: 10.1242/jcs.00417

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Macroscopic cartilage formation with embryonic stem-cell-derived mesodermal progenitor cells

Naoki Nakayama1,*, Diane Duryea2, Raffi Manoukian3, Gwyneth Chow2 and Chun-ya E. Han1

1 Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
2 Department of Pathology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
3 Department of Flow Cytometry Laboratory, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA



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  		Fig. 1. Characterization of the flk-1+ and PDGFR{alpha}+ EB cells generated with BMP4 in the serum-free medium. (A) Generation of the flk-1+ and PDGFR{alpha}+ EB cells. The undifferentiated E14 ES cells (a) and the cells from EBs cultured for 4.6 days in the BMP4-containing serum-free medium (b) were stained with Flk-1-PE and PDGFR{alpha}-bio. The latter group was further stained with SA-APC. Single positive cells, such as flk-1–PDGFR{alpha}+ cells (R3) and flk-1+PDGFR{alpha}– cells (R4), as well as DP cells (R5) and DN cells (R6), were then analyzed (a) and sorted (b) with Vantage SE. Viable cell populations were pre-enriched by propidium iodide exclusion (R1) as described previously (Nakayama et al., 2000Go). For sorting, stained EB cells were pre-gated with R2 to further reduce dead cell contamination (b, left). R6 was determined by the corresponding isotype control staining (a, middle; b, middle). Populations of R3 to R5, indicated as % total EB cells, are: (a) R3: 0.7, R4: 0.0, R5: 0.3 (right) and as for the isotype control, R3: 0.7, R4: 1.3, R5: 0.5 (middle); (b) R3: 26.4, R4: 7.7, R5: 9.2 (right) and as for the isotype control, R3: 0.5, R4: 0.0, R5: 0.2 (middle). These results are representative of 17 independent experiments. (B) Erythro-myeloid CFCs in the flk-1+ and PDGFR{alpha}+ EB cell fractions, with or without culturing on the OP9 cells. Individual FACS-purified cell fractions were either directly plated in the serum-free methylcellulose medium (–) or cultured for 3 days on OP9 (+). The latter cells were then plated into the same medium for hematopoietic colony formation. Total CFC numbers were averaged over four to six independent experiments and are shown with the corresponding s.d. (vertical line). R3: flk-1–PDGFR{alpha}+, R4: flk-1+PDGFR{alpha}–, R5: DP, R6: DN. (C) Lymphoid potentials of the flk-1+ and PDGFR{alpha}+ EB cells. Individual FACS-purified cell fractions were cultured for 12 days on OP9 in the presence of IL-2 and IL-7. The entire culture was then harvested, stained with B220-PE and Sca-1-FITC and analyzed by FACScan. Populations of B220+Sca-1–, B220–Sca-1+, and B220+Sca-1+ cells are indicated as % of total cells. The scatter patterns, the gate settings and the isotype control staining patterns (IgG-FITC/IgG-PE) were essentially the same as described previously (Nakayama et al., 1998Go). Note that the OP9 cells were B220–Sca-1–. Cellularity, reflected by the density of the plot, was highest in (a) and (c), owing to the massive accumulation of lymphocytic cells. In contrast, small numbers of lymphocytic cell foci were observed in d, and no such cells were detected in b and e. These results are representative of six independent experiments. (a), flk-1+PDGFR{alpha}– (isotype control staining for c); (b), flk-1–PDGFR{alpha}+; (c), flk-1+PDGFR{alpha}–; (d), DP; (e), DN.

 


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  		Fig. 2. Requirement of TGFß for the in vitro chondrogenesis of the ES-cell-derived mesodermal cells. The sorted flk-1–PDGFR{alpha}+ cells (A,B), flk-1+PDGFR{alpha}– cells (C,D), DP cells (E,F), and DN cells (G) were subjected to the pellet culture with (B,D,F,G) or without (A,C,E) TGFß3. Particles formed in 15 days of culture were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. These results are representative of three independent experiments.

 


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  		Fig. 3. TGFß3-dependent chondrogenesis causes COL2-containing cartilage matrix accumulation and induces cartilage-specific gene expression. (A) Cartilage-like particle formation with single positive EB cells in vitro. The sorted flk-1– PDGFR{alpha}+ cells (a-c) and flk-1+PDGFR{alpha}– cells (d-f) were individually subjected to the pellet culture in the presence of TGFß3. Particles formed in 17 days of culture were fixed with Gendre's fluid, paraffin-embedded, sectioned and stained with Toluidine blue (a,d). Two contiguous sections were also immunostained with AB765P (b,e) and 2B1.5 (c,f). Brown areas indicate the accumulation of COL1 (b,e) and COL2 (c,f). Immunostaining with rabbit IgG and mouse IgG did not yield any signals (data not shown). These results are representative of seven independent experiments. (B) Expression of cartilage-specific mRNAs during the TGFß-dependent generation of cartilage-like particles from single positive EB cells. The sorted flk-1–PDGFR{alpha}+ cells and flk-1+PDGFR{alpha}– cells were individually subjected to the TGFß3-containing pellet culture. On the day indicated, three aggregates were collected and RNAs were isolated for nested RT-PCR. No signals were observed without reverse transcription or when the first 21 cycles were done with a primer set for a different gene, indicating that the positive signals originated from the corresponding transcripts (data not shown). Essentially the same results were obtained from three independent experiments.

 


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  		Fig. 4. TGFß3 enhances the accumulation of sulfated glycosaminoglycans and COL2 during micromass culture. (A) Cartilage nodule formation in the absence (a-c) and presence (d-f) of TGFß3. The sorted flk-1–PDGFR{alpha}+ cells (a,d), flk-1+PDGFR{alpha}– cells (b,e) and DP cells (c,f) were individually spotted in two wells of a 24-well plate at 1.5x105 cells/7.5 µl/spot/well and were subjected to serum-free micromass culture. On day 8, cultures were formalin-fixed and stained with Alcian blue. (B) Higher power view of a-c. Cartilage nodules with a dark blue appearance were noted in the culture of flk-1–PDGFR{alpha}+ cells (a) and DP cells (c). The Alcian-blue-positive nodules were counted, and the numbers were normalized per 2x105 initial seeding cells and are displayed in the right graph. From three independent experiments, average numbers were calculated and are shown with the corresponding s.d. (vertical line). (C) COL2 accumulation during the micromass culture. (a) The sorted flk-1–PDGFR{alpha}+ cells (lanes 2,5), DP cells (lanes 3,6), and flk-1+PDGFR{alpha}– cells (lanes 4,7) were individually spotted in 2 wells of a 24-well plate at 1.5x105 cells/7.5 µl/spot/well, and were subjected to the serum-free micromass culture with 50 ng/ml PDGF-BB (lanes 2-4, PB) or TGFß3 and 50 ng/ml PDGF-BB (lanes 5-7, TPB). (b) The sorted flk-1–PDGFR{alpha}+ cells (lanes 3-5, F–P+), DP cells (lanes 6-8, F+P+), and flk-1+PDGFR{alpha}– cells (lanes 9-11, F+P–) were also cultured in the same way with TGFß3 (lanes 3,6,9), with TGFß3 and 50 ng/ml PDGF-AA (lanes 4,7,10) or with TGFß3 and 50 ng/ml PDGF-BB (lanes 5,8,11). On day 8, the cultures were harvested with 300 µl/well of loading buffer, and 40 µl aliquots were separated by SDS-PAGE, blotted and developed with 6B3. Soluble COL1 (pepsin-treated) was loaded in lane 1 (b), and soluble COL2 (pepsin-treated) was loaded in lane 1 (a) and lane 2 (b).

 


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  		Fig. 5. Effects of PDGF-BB on cartilage particle development. The sorted flk-1–PDGFR+ cells (A) and flk-1+PDGFR– (B) were subjected to the pellet culture in the presence of TGFß3 (a-c,g-i), or TGFß3 and 50 ng/ml PDGF-BB (d-f,j-l). Particles formed after 17 days of culture were fixed with Gendre's fluid, paraffin-embedded, sectioned and stained with Toluidine blue (a,d,g,j). A contiguous section of each particle was also immunostained with AB765P (b,e,h,k) or 2B1.5 (c,f,i,l). Brown areas indicate the accumulation of collagens. The immunostaining with control IgGs showed negative results (data not shown). Note that the addition of PDGF-BB resulted in a large aggregate filled with cartilage matrix and COL2 (d,f,j,l). These results are representative of four independent experiments.

 


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  		Fig. 6. Involvement of a BMP activity during cartilage particle development in the presence of TGFß or TGFß and PDGF-BB. (A) The sorted flk-1–PDGFR{alpha}+ cells were subjected to the pellet culture in the presence of TGFß3+1 µg/ml IgG-Fc (a) and TGFß3+1 µg/ml noggin-Fc (b). The sorted flk-1+PDGFR{alpha}– cells were also subjected to the pellet culture in the presence of TGFß3+1 µg/ml IgG-Fc (c), TGFß3+0.1 µg/ml noggin-Fc (d) and TGFß3+1 µg/ml noggin-Fc (e). Particles formed in 15 day (a,b) or 18 day (c-e) cultures were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. These results are representative of three (c-e) to five (a,b) independent experiments. Note that the addition of 1 µg/ml IgG-Fc did not have any effect on the growth and maturation of the cartilage-containing particle (a,c). The addition of noggin-Fc resulted in a small aggregate and sometimes left part of the initial cell pellet unincorporated into the round cell aggregate, which occasionally attached to the particle throughout the culture (top and bottom cell debris in e). (B) The sorted flk-1–PDGFR{alpha}+ cells were subjected to the pellet culture with TGFß3 and 50 ng/ml PDGF-BB in the presence of 1 µg/ml IgG-Fc (a,c) and 1 µg/ml noggin-Fc (b,d). On day 18, particles were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. A contiguous section of each particle was also immunostained with 2B1.5 (c,d). Brown areas indicate the accumulation of COL2. The immunostaining with mouse IgG showed negative results (data not shown). Note that the addition of 1 µg/ml noggin-Fc resulted in a small aggregate (b,d). These results are representative of two independent experiments.

 


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  		Fig. 7. Effects of BMP4 on cartilage particle development. (A) The sorted flk-1–PDGFR{alpha}+ cells were subjected to the pellet culture in the presence of TGFß3 (a) or 50 ng/ml BMP4 (b). (B) In a separate experiment, the same cells were cultured with TGFß3 (a), TGFß3+5 ng/ml BMP4 (b), TGFß3+20 ng/ml BMP4 (c) or TGFß3+50 ng/ml BMP4 (d). The sorted flk-1+PDGFR{alpha}– cells were also subjected to the pellet culture in the presence of TGFß3 (e) or TGFß3+50 ng/ml BMP4 (f). Particles formed during 16 days (Aa-b, Be-f) or 17 days (Ba-d) of culture were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. These results are representative of three independent experiments. (C) Late removal of TGFß3 and replacement with BMP4 during the in vitro development of cartilage particles. The sorted flk-1–PDGFR{alpha}+ cells were individually subjected to the pellet culture in the presence of TGFß3. On day 10, the TGFß3 was removed (c), or was substituted with 1 µg/ml noggin-Fc (b) or 50 ng/ml BMP4 (d) in some cultures. On day 18, particles were formalin-fixed, paraffin-embedded, sectioned and stained with Toluidine blue. These results are representative of five independent experiments.

 


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  		Fig. 8. Hyaline cartilage formation and mineralization. (A) Late removal of TGFß3 and PDGF-BB, and replacement with BMP4 during the in vitro development of cartilage particles. The sorted flk-1–PDGFR{alpha}+ cells were subjected to the pellet culture in the presence of TGFß3 and 50 ng/ml PDGF-BB. On day 10, the TGFß3and PDGF-BB were removed (d-f) or were substituted with 50 ng/ml BMP4 (g-i) in some cultures. On day 18, particles were fixed with Gendre's fluid, paraffin-embedded, sectioned and stained with Toluidine blue (a,d,g) or immunostained with AB765P (b,e,h) or 2B1.5 (c,f,i). The results are representative of five independent experiments. (B) The sorted flk-1–PDGFR{alpha}+ cells (a-c) and flk-1+PDGFR{alpha}– cells (d-f) were pellet-cultured with TGFß3 and 50 ng/ml PDGF-BB for 10 days, with 50 ng/ml BMP4 for 6 days and then in the hypertrophic differentiation medium for 5 days. Particles were formalin-fixed, paraffin-embedded, sectioned and stained individually with von Kossa (a,d), with X53 (b,e) and with 2B1.5 (c,f). The results are representative of three independent experiments. Black staining indicates mineral deposition (Ba,d), and brown areas indicate the accumulation of collagens. Immunostaining with control IgGs showed negative results (data not shown).

 

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© The Company of Biologists Ltd 2003