Phenol Red Inhibits Chondrogenic Differentiation and Affects Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro

Abstract

The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P < 0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P < 0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P < 0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P < 0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P < 0.05). The deposition of calcium was decreased on day 21 (P < 0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.

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Acknowledgements

This project was granted from the Velux Foundation and the A.P. Møller Foundation for the Advancement of Medical Science.

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The authors declare no potential conflicts of interest.

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Correspondence to Helle Lysdahl.

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Supplementary Fig. 1
figure10

DNA content after 2, 7, 14, and 21 days of osteogenic differentiation of hMSCs. Vertical axis represents the DNA content expressed by 700 nm channel intensity. Horizontal axis represents the medium type and time points. Data are expressed as mean ± SD (n = 12). (JPEG 19 kb)

Supplementary Fig. 2
figure11

DNA content and alkaline phosphatase activity after 2, 7, 14, and 21 days culture of hMSCs. Vertical axes represent DNA content expressed by 700 nm channel intensity and alkaline phosphatase activity expressed by nmol nitrophenol x ml−1 x min−1, respectively. Horizontal axes represent medium type and time points. Data are expressed as mean ± SD (n = 12). * Significant difference between DMEM wPR and DMEM woPR within the given time point, P < 0.05. (JPEG 40 kb)

Supplementary Fig. 3
figure12

Relative gene expression levels of runx2, alkaline phosphatase, collagen type 1, and osteocalcin after 2, 7, 14, and 21 days of osteogenic differentiation of hMSCs. Vertical axes represent the relative gene expression level and horizontal axes represent the medium type and time points. Data are expressed as mean ± SD (n = 6). * Significant difference between DMEM woPR + and DMEM woPR + D within the given time point, P < 0.05. Vitamin D significantly increased the gene expression of runx2 at all time points (P = 0.0000), alkaline phosphatase on day 2 and 7 (P = 0.0000), collagen type 1 at all time points (P = 0.000), and osteocalcin at all time points (P = 0.000) (Supplementary figure 3). On day 14, vitamin D significantly decreased the gene expression of alkaline phosphatase (P = 0.0019). (JPEG 82 kb)

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Lysdahl, H., Baatrup, A., Nielsen, A.B. et al. Phenol Red Inhibits Chondrogenic Differentiation and Affects Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro. Stem Cell Rev and Rep 9, 132–139 (2013). https://doi.org/10.1007/s12015-012-9417-0

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Keywords

  • Human mesenchymal stem cells
  • Culture media
  • Phenol red
  • Chondrogenic differentiation
  • Osteogenic differentiation