Results: 5

1.
Figure 1

Figure 1. Schematic diagram of a double objective FSC-DE.. From: Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

(a) Feedback system control (FSC) applied for the identification of combinatory multiple extrinsic factors to determine the differentiation fate of MSC. (b) Differential evolution (DE) used as the search algorithm in this project. Each color represents the concentration of each of the seven extrinsic factors, selected from a scale ranging from 1 to 10 or 0 to 12. The combination of these factors resulted in 107 (10 million) or 137 (62.7 million) theoretical combinations in the present study.

Yoshitomo Honda, et al. Sci Rep. 2013;3:3420.
2.
Figure 5

Figure 5. Osteogenic effect of m4T5 and m8T11 for primary human MSC.. From: Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

(a) Alizarin red-stained human MSC cultures treated with TB (containing 100 ng/mL BMP-2), FSC cocktails m4T5 or m8T11, or conventional osteogenic media (Conv-OM: containing 10 nM or 100 nM Dex). On culture day 15, the m8T11 cocktail induced alizarin red-positive mineralized nodules, while m4T5 did not. (b) The alizarin red-positive area measurement (excluding the peripheral ring) suggested that the effect of m8T11 on the osteogenic differentiation of human MSC was approximately 50% of that with TB and greater than 300% of that with Conv-OM.

Yoshitomo Honda, et al. Sci Rep. 2013;3:3420.
3.
Figure 2

Figure 2. FSC-DE with ALP assay.. From: Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

(a) Effect of prospective drug cocktails identified with FSC-DE with ALP assay from 10 million potential candidates for the osteoblastogenesis of D1 cells. The prospective cocktails did not induce in vitro mineralization, despite high ALP activity. (b) Correlation between doses of extrinsic factors in each drug cocktail elicited and used in FSC-DE with ALP assay against ALP expression in D1 cells. Dots: each drug cocktail. All data represent the actual measurement values in the FSC-DE process. The highest concentration codes 9 and 10 of RA were involved in 70% of all FSC-generated cocktails. D1 cells were seeded at 3125/cm2, and the ALP activity was measured on day 3. Note that the prospective drug cocktails in Figure 2a were narrowed using setting gates with the following criteria: BMP-2 code less than 9 and ALP activity above 1.5. (a, b) Bar graphs and dots show the mean with/without s.d. of three independent determinations. Each concentration of extrinsic factors represents the one in drug cocktails and does not include the one in FBS.

Yoshitomo Honda, et al. Sci Rep. 2013;3:3420.
4.
Figure 4

Figure 4. Osteoblastogenesis induced by m4T5 and m8T11 cocktail and BMP-SMAD signaling pathway.. From: Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

(a) Taqman-based real-time reverse transcription polymerase chain reaction for Runx2, Osx and Ocn. **: p < 0.01 compared with the control (ANOVA with a Dunnett's test). (b) ALP expression in the well measured on day 7. *: p < 0.05 and **: p < 0.01 compared with the control (ANOVA with a Dunnett's test). (c) Alizarin red staining of D1 cells treated with drug cocktails with/without the following inhibitors: 500 ng/ml of Noggin and 5 μM Dorsomorphin (Dorso). The mineralized matrix was stained with Alizarin red S on Day 7. (d) Quantitative mineralization assay with BMP-SMAD pathway inhibitors. **: p < 0.01 compared with each inhibitor (−) (ANOVA with a Dunnett's test). (e) Effect of drug cocktails on mRNA expression of Bmp2, Bmpr2 and Bmpr1A. *: p < 0.05 and **: p < 0.01 compared with the control (ANOVA with a Dunnett's test). In all experiments, D1 cells were seeded at 45,000/cm2; once the cells became confluent, the media were changed to basal media and subsequently changed to media containing each drug cocktail. The bar graphs show the mean with s.d. of three independent determinations.

Yoshitomo Honda, et al. Sci Rep. 2013;3:3420.
5.
Figure 3

Figure 3. Identification of prospective drug cocktails using FSC-DE with mineralization assay.. From: Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

(a) Arrangement of the drug cocktails in the order of each mineralization index. The break line represents the threshold mineralization index of 0.5. D1 cells were seeded at 45,000/cm2; once the cells were confluent, the media were changed to basal media and subsequently changed to media containing each drug cocktail. The Ca2+ content was measured on day 7. (b) Mineralization assay using the prospective cocktails within the following gate and the diluted cell-seeding condition. D1 cells were seeded at 3125/cm2; the media were changed to media containing each drug cocktail on day 1, and the Ca2+ content in the well was measured on day 15. Gate: BMP-2 code less than 8 and mineralization index above 0.5. (c) Correlation between the doses of the extrinsic factors in each drug cocktail against the mineralization index. Dots: each drug cocktail elicited and used in FSC-DE with mineralization assay. All data represent actual measurement values in the FSC-DE process. The dots show the mean of three independent determinations. R: Correlation coefficient. Line: Linear correlation. (d) The effect of VD3 modifications in m8T11. Bar graph: quantitative mineralization assay with original m8T11 (VD3 code 2) and modified m8T11 (VD3 code 5 and 7). D1 cells were seeded at 45,000/cm2; once the cells were confluent, the media were changed to basal media and subsequently changed to media containing each drug cocktail. The mineralized area was measured on day 7. **: P < 0.01 (ANOVA with a Dunnett's test). Representative Alizarin red-stained images. (a, b, d) The bar graphs shows mean with/without s.d. of three independent determinations. Each concentration of extrinsic factors represents the one in drug cocktails and does not include the one in FBS.

Yoshitomo Honda, et al. Sci Rep. 2013;3:3420.

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