Abstract
Tendon cells (TCs) are important for homeostatic maintenance in the healthy tendon and to promote tissue healing after injury. Further, resident and rare populations of tendon stem/progenitor cells, located at various sites within the tendon, contribute to tendon recovery by differentiating into repairing TCs. Gene expression analysis, through quantitative reverse-transcription polymerase chain reaction (qRT-PCR), constitutes a useful tool to study cellular responses, including the transition from initial inflammation to healing processes. A critical step required for data normalization is the choice of reliable reference genes (RGs), a process highly underestimated in tendon biology. In this study, the suitability of five commonly used RGs (ACTB, B2M, GAPDH, HPRT1, and RPLP0) was evaluated using TCs samples cultured in both standard and progenitor-enriching conditions, as well as under either inflammatory (IFNγ + TNFα) or pro-fibrotic/healing (CTGF) stimulation. The stability of the candidate RGs was computationally determined using NormFinder, geNorm, BestKeeper, and DeltaCt applets. Overall, ACTB resulted as the most stable RG on the basis of the integration of each gene weight, whereas B2M and RPLP0 performed poorly. To further validate ACTB’s optimal performance, we evaluated the expression of ICAM1, coding for an immune-related cell surface glycoprotein, and COL1A1, encoding collagen type I that is the main component of the tendon extracellular matrix (ECM), both known to be modulated by inflammation. The expression of both genes was heavily affected by the RGs used. Consequently, when analyzing gene expression in tendon-derived cells subjected to various stimulatory protocols, the use of a suitable RG should be considered carefully. On the basis of our results, ACTB can be reliably used when analyzing different TC types exposed to pathological conditions.
Highlights
Tendinopathies can be described as a “continuum” of disturbances historically associated with progressive “wear and tear” of the tendon resulting from aging, overuse, and other causes [1]
A rare (
Human tendon cells were isolated from discarded fragments of the semitendinosus and gracilis tendons harvested from three de-identified patients (n = 3, males, 33 ± 9 years old) who underwent elective anterior cruciate ligament (ACL) reconstruction using hamstring tendons and provided their written informed consent (M-SPER-015- Ver. 2 - 04.11.2016 for the use of surgical waste material)
Summary
Tendinopathies can be described as a “continuum” of disturbances historically associated with progressive “wear and tear” of the tendon resulting from aging, overuse, and other causes [1]. Cells 2019, 8, 1188 loading can lead to tendon injury, through either an acute trauma or a degenerative process resulting from an accumulation of micro-damage and an altered cell/matrix balance [2]. In this frame, pioneering works completed a roadmap of cellular and molecular cascades involved in tendon mechanobiology for both healthy and injured tissues [2]. Tendon injuries, along with mechanical stress, disturb the homeostatic balance between specialized tendon cells (TCs), named tenocytes, and immune cell compartments within the tissue [3]. Various strategies have helped elucidate their identification, including cell surface marker selection (e.g., CD146 positivity) [5]
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