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A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes

View Article: PubMed Central - PubMed

ABSTRACT

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

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Box-and-whisker plots showing the distribution of normalised and transformed cycle threshold qPCR values (y-axis, 2^-dCt) for three experimental conditions (x-axis, tendon / monolayer / fibrin cultures) for selected genes (vertical legends).Box and whisker plots show mean, first and third quartiles, and maximum and minimum values. Results for technical triplicates on n = 4 biological replicates. Results of hypothesis testing (p-values) for differences between groups, after post-hoc corrections, are shown where thresholds are met; NSD – no significant difference.
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f3: Box-and-whisker plots showing the distribution of normalised and transformed cycle threshold qPCR values (y-axis, 2^-dCt) for three experimental conditions (x-axis, tendon / monolayer / fibrin cultures) for selected genes (vertical legends).Box and whisker plots show mean, first and third quartiles, and maximum and minimum values. Results for technical triplicates on n = 4 biological replicates. Results of hypothesis testing (p-values) for differences between groups, after post-hoc corrections, are shown where thresholds are met; NSD – no significant difference.

Mentions: Significant reductions in the expression of Col2a1, Acan, and Sox9 for dedifferentiated chondrocytes, Fig. 2, and Tnmd and Serpinf1 for dedifferentiated tenocytes, Fig. 3, were confirmed. The higher expression of Thbs4 in cartilage relative to monolayer chondrocytes was not confirmed by qPCR. Both dedifferentiated chondrocytes and tenocytes were shown to have elevated expression of scleraxis (Scx), prion (Prnp) and Thy1 and a reduction in Sox9 expression. Alginate and fibrin 3D cultures expressed significantly higher levels of Hif1a and Nfe2l2, relative to native tendon or cartilage. Fibrin cultures significantly elevated expression of Atf4; a comparable trend was observed in alginate cultures. Alginate cultures exhibited higher expression of Pitx1 and Sox9, whilst fibrin cultures up-regulated Thbs4, Scx, and Slit3. Expression of tissue-specific differentiation markers (Tnmd, Col2a1) was not restored in three-dimensional cultures.


A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes
Box-and-whisker plots showing the distribution of normalised and transformed cycle threshold qPCR values (y-axis, 2^-dCt) for three experimental conditions (x-axis, tendon / monolayer / fibrin cultures) for selected genes (vertical legends).Box and whisker plots show mean, first and third quartiles, and maximum and minimum values. Results for technical triplicates on n = 4 biological replicates. Results of hypothesis testing (p-values) for differences between groups, after post-hoc corrections, are shown where thresholds are met; NSD – no significant difference.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037390&req=5

f3: Box-and-whisker plots showing the distribution of normalised and transformed cycle threshold qPCR values (y-axis, 2^-dCt) for three experimental conditions (x-axis, tendon / monolayer / fibrin cultures) for selected genes (vertical legends).Box and whisker plots show mean, first and third quartiles, and maximum and minimum values. Results for technical triplicates on n = 4 biological replicates. Results of hypothesis testing (p-values) for differences between groups, after post-hoc corrections, are shown where thresholds are met; NSD – no significant difference.
Mentions: Significant reductions in the expression of Col2a1, Acan, and Sox9 for dedifferentiated chondrocytes, Fig. 2, and Tnmd and Serpinf1 for dedifferentiated tenocytes, Fig. 3, were confirmed. The higher expression of Thbs4 in cartilage relative to monolayer chondrocytes was not confirmed by qPCR. Both dedifferentiated chondrocytes and tenocytes were shown to have elevated expression of scleraxis (Scx), prion (Prnp) and Thy1 and a reduction in Sox9 expression. Alginate and fibrin 3D cultures expressed significantly higher levels of Hif1a and Nfe2l2, relative to native tendon or cartilage. Fibrin cultures significantly elevated expression of Atf4; a comparable trend was observed in alginate cultures. Alginate cultures exhibited higher expression of Pitx1 and Sox9, whilst fibrin cultures up-regulated Thbs4, Scx, and Slit3. Expression of tissue-specific differentiation markers (Tnmd, Col2a1) was not restored in three-dimensional cultures.

View Article: PubMed Central - PubMed

ABSTRACT

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

No MeSH data available.


Related in: MedlinePlus