Analysis of the effects of five factors relevant to in vitro chondrogenesis of human mesenchymal stem cells using factorial design and high throughput mRNA-profiling

Abstract

The in vitro process of chondrogenic differentiation of mesenchymal stem cells for tissue engineering has been shown to require three-dimensional culture along with the addition of differentiation factors to the culture medium. In general, this leads to a phenotype lacking some of the cardinal features of native articular chondrocytes and their extracellular matrix. The factors used vary, but regularly include members of the transforming growth factor β superfamily and dexamethasone, sometimes in conjunction with fibroblast growth factor 2 and insulin-like growth factor 1, however the use of soluble factors to induce chondrogenesis has largely been studied on a single factor basis. In the present study we combined a factorial quality-by-design experiment with high-throughput mRNA profiling of a customized chondrogenesis related gene set as a tool to study in vitro chondrogenesis of human bone marrow derived mesenchymal stem cells in alginate. 48 different conditions of transforming growth factor β 1, 2 and 3, bone morphogenetic protein 2, 4 and 6, dexamethasone, insulin-like growth factor 1, fibroblast growth factor 2 and cell seeding density were included in the experiment. The analysis revealed that the best of the tested differentiation cocktails included transforming growth factor β 1 and dexamethasone. Dexamethasone acted in synergy with transforming growth factor β 1 by increasing many chondrogenic markers while directly downregulating expression of the pro-osteogenic gene osteocalcin. However, all factors beneficial to the expression of desirable hyaline cartilage markers also induced undesirable molecules, indicating that perfect chondrogenic differentiation is not achievable with the current differentiation protocols.

Figure 1. Experimental setup and principal component analysis (PCA).

A. Experimental setup with numbering of the different conditions. When not stated, the cell density was 10⁷ cells per mL. B. PCA on all conditions labelled by days in culture. C. PCA limited to conditions 1–32 on days 0 and 1. D. PCA limited to conditions 1–32 on days 0 and day 7.

Figure 2. Statistical analysis of main effects and interactions at day 7.

A. Normal plot of the standardized effects with the response set to mean expression of wanted markers. B. Corresponding main effects plot of all factors. C and D. Corresponding plots of significant second order interactions. E. Normal plot of the standardized effects with the response set to mean expression of unwanted markers. F. Corresponding main effects plot of all factors. G and H. Corresponding plots of significant second order interactions.

Figure 3. Analysis of wanted and unwanted gene expression.

A. Gene sets used to compute mean expression of wanted and unwanted markers. B. Scatter-plot of the mean expression (studentized values) of wanted (x-axis) and unwanted (y-axis) markers at day 0, 1 and 7 of all conditions. C. Scatter-plot of the mean expression (studentized values) of wanted and unwanted markers at day 0 and 7 for condition 1–32.

Figure 4. Heatmap of top ranking conditions.

Heatmap of wanted and unwanted genes in all conditions significantly enriched for wanted, but not unwanted markers, color coded by the studentized score.

Figure 5. Genes uniformly affected by single factors across conditions 1–32 at day 7.

A. Experimental setup conditions 1–32. B. Heatmap of genes significantly downregulated in all conditions contaning any one of the factors compared to condition 32. C. Heatmap of genes significantly upregulated in all conditions contaning any one of the factors compared to condition 32. Values are log2-transformed mean expressions (n  =  3).

Figure 6. Genes significantly regulated between key conditions (day 7).

A. Top 20 upregulated and top 20 downregulated genes when adding DEX to TGFB1. B. Top 20 upregulated and top 20 downregulated genes when adding DEX to TGFB1+BMP2. C. All regulated genes when adding BMP2 to TGFB1. D. All regulated genes when adding BMP2 to TGFB1+DEX. Values represent log2 to the fold change between the gene expression in the condition without and the condition with the specified factor added.