Loss of tenomodulin results in reduced self-renewal and augmented senescence of tendon stem/progenitor cells

Abstract

Tenomodulin (Tnmd) is a well-known gene marker for the tendon and ligament lineage, but its exact functions in these tissues still remain elusive. In this study, we investigated Tnmd loss of function in mouse tendon stem/progenitor cells (mTSPC) by implicating a previously established Tnmd knockout (KO) mouse model. mTSPC were isolated from control and Tnmd KO tail tendons and their stemness features, such as gene marker profile, multipotential, and self-renewal, were compared. Immunofluorescence and reverse transcriptase-polymerase chain reaction analyses for stem cell-, tenogenic-, osteogenic-, and chondrogenic-related genes confirmed their stemness and lineage specificity and demonstrated no profound differences between the two genotypes. Multipotential was not significantly affected since both cell types differentiated successfully into adipogenic, osteogenic, and chondrogenic lineages. In contrast, self-renewal assays validated that Tnmd KO TSPC exhibit significantly reduced proliferative potential, which was also reflected in lower Cyclin D1 levels. When analyzing possible cellular mechanisms behind the observed decreased self-renewability of Tnmd KO TSPC, we found that cellular senescence plays a major role, starting earlier and cumulating more in Tnmd KO compared with control TSPC. This was accompanied with augmented expression of the cell cycle inhibitor p53. Finally, the proliferative effect of Tnmd in TSPC was confirmed with transient transfection of Tnmd cDNA into Tnmd KO TSPC, which rescued their proliferative deficit. Taken together, we can report that loss of Tnmd affects significantly the self-renewal and senescence properties, but not the multipotential of TSPC.

FIG. 1.

Expression profile of mTSPC. (A) Immunocytochemical detection of stem cell markers CD146, CD105, CD90.2, CD73, CD44, Nestin, Nanog, and Sca-1 and tenogenic markers Scx and Tnmd. Insets show nuclear DAP1 stainings. (B) Quantitative PCR for Scx. **P<0.005. Semiquantitative PCR for tendon- (C), bone- and cartilage-related genes (D). Staining and PCRs were performed with mTSPC in passage 3, and were repeated minimum two independent times. mTSPC, mouse tendon stem/progenitor cells; PCR, polymerase chain reaction; KO, knockout; Scx, Scleraxis; Tnmd, tenomodulin; HPRT, hypoxanthine-guanine phosphoribosyltransferase; Col, collagen; Fmod, fibromodulin; Osx, osterix; BSP, bone sialoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Color images available online at www.liebertpub.com/scd

FIG. 2.

Three-lineage differentiation of control and Tnmd KO mTSPC. (A) On the left, representative pictures of adipogenic differentiation. Accumulated lipid vacuoles were detected by Oil Red O staining after 21 days stimulation (lipid vacuoles in red). On the right, quantitative analyses of the Oil Red O-positive areas. (B) On the left, representative pictures of osteogenic differentiation. Deposited calcified matrix was visualized by Alizarin Red staining at day 21 (calcified matrix in red). On the right, quantification of the Alizarin Red accumulation. (C) On the left, representative images of chondrogenic differentiation. Cartilage glycosaminoglycans were visualized by Safranin Orange staining at day 28 (glycosaminoglycans in orange, cell nuclei in dark red). On the right, quantification analyses of positively stained areas. In (A–C), unstimulated mTSPC were used as negative controls. Differentiation protocols were carried out in triplicates with cells in passage 1 and were reproduced twice independently. AD, adipogenic; OS, osteogenic; CH, chondrogenic; −, unstimulated; +, stimulated. Color images available online at www.liebertpub.com/scd

FIG. 3.

Investigation of mTPSC self-renewal capacity. (A) Long-term proliferation was assessed by estimating cumulative population doubling for a period of 50 days (corresponding to eight consecutive passages). (B) Calculation of population doubling time of control and Tnmd KO mTSPCs from early passages (p1–3) and middle passages (4–5). (C) Colony-forming unit assay. Formed colonies were visualized with crystal violet staining after 14 days. (D) CFU efficiency was estimated by counting colony numbers. CFU assays were performed in triplicates and reproduced twice independently. (E) The WST-1 proliferation assay (average of three independent experiments). (F) Semiquantitative PCR for Cyclin D1 (left panel) and densitometric quantification of PCR bands (right panel). PCRs were reproduced three times. *P<0.05, **P<0.005. CPD, cumulative population doubling, PDT, population doubling time; p, passage; CFU, colony forming unit.

FIG. 4.

Cellular senescence analyses. (A) Representative images of mTSPC stained for active β-galactosidase (arrows) at three different passages (p3–5). (B) Quantification of senescent cell numbers. For each passage, β-galactosidase staining was repeated two independent times in triplicates. (C) Quantitative PCR for cell cycle inhibitors and senescent markers p16, p21, and p53. PCRs were performed three independent times. (D) Representative images of p16, p21, and p53 immunofluorescent detection in vivo. Stainings were reproduced twice using Achilles tendon sections from 6-month-old mice. *P<0.05, ***P<0.0005. Color images available online at www.liebertpub.com/scd

FIG. 5.

Rescue of Tnmd expression in Tnmd KO mTSPC. (A) Schematic representation of the Tnmd full length and Tnmd C-terminal plasmids used for transfection. Transfections were carried out two independent times. (B) A representative picture of EGFP-transfected mTSPC, which was used to monitor transfection efficiency. (C) Upper panel: PCR analysis for the expression of the transfected Tnmd FL and Tnmd Cterm cDNA performed with primer sets indicated in (A). Lower panel: PCR for the expression of endogenous Tnmd. PCRs were performed twice independently. (D) The WST-1 proliferation assay with control and Tnmd KO mTSPC after transfection. Fold change was calculated to WST-1 value of nontransfected control mTSPC. The WST-1 experiments were reproduced twice in triplicates. (E) Semiquantitative PCR for Cyclin D1 (right panel) and densitometrical quantification of the PCR product (left panel). PCR were repeated twice independently. *P<0.05, **P<0.005, ***P<0.0005. FL, full length; Cterm, C-terminal; ppt, pre-pro-trypsin secretion signal; nt, nontransfected; LPA, Lipofectamine; Ct w/LPA, C-terminal without Lipofectamine; Ct, C-terminal. Color images available online at www.liebertpub.com/scd