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Tendon proper- and peritenon-derived progenitor cells have unique tenogenic properties.

ABSTRACT: Multipotent progenitor populations exist within the tendon proper and peritenon of the Achilles tendon. Progenitor populations derived from the tendon proper and peritenon are enriched with distinct cell types that are distinguished by expression of markers of tendon and vascular or pericyte origins, respectively. The objective of this study was to discern the unique tenogenic properties of tendon proper- and peritenon-derived progenitors within an in vitro model. We hypothesized that progenitors from each region contribute differently to tendon formation; thus, when incorporated into a regenerative model, progenitors from each region will respond uniquely. Moreover, we hypothesized that cell populations like progenitors were capable of stimulating tenogenic differentiation, so we generated conditioned media from these cell types to analyze their stimulatory potentials.Isolated progenitors were seeded within fibrinogen/thrombin gel-based constructs with or without supplementation with recombinant growth/differentiation factor-5 (GDF5). Early and late in culture, gene expression of differentiation markers and matrix assembly genes was analyzed. Tendon construct ultrastructure was also compared after 45 days. Moreover, conditioned media from tendon proper-derived progenitors, peritenon-derived progenitors, or tenocytes was applied to each of the three cell types to determine paracrine stimulatory effects of the factors secreted from each of the respective cell types.The cell orientation, extracellular domain and fibril organization of constructs were comparable to embryonic tendon. The tendon proper-derived progenitors produced a more tendon-like construct than the peritenon-derived progenitors. Seeded tendon proper-derived progenitors expressed greater levels of tenogenic markers and matrix assembly genes, relative to peritenon-derived progenitors. However, GDF5 supplementation improved expression of matrix assembly genes in peritenon progenitors and structurally led to increased mean fibril diameters. It also was found that peritenon-derived progenitors secrete factor(s) stimulatory to tenocytes and tendon proper progenitors.Data demonstrate that, relative to peritenon-derived progenitors, tendon proper progenitors have greater potential for forming functional tendon-like tissue. Furthermore, factors secreted by peritenon-derived progenitors suggest a trophic role for this cell type as well. Thus, these findings highlight the synergistic potential of including these progenitor populations in restorative tendon engineering strategies.

SUBMITTER: Mienaltowski MJ

PROVIDER: S-EPMC4230637 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Tendon proper- and peritenon-derived progenitor cells have unique tenogenic properties.

Mienaltowski Michael J MJ Adams Sheila M SM Birk David E DE

Stem cell research & therapy 20140708 4

<h4>Introduction</h4>Multipotent progenitor populations exist within the tendon proper and peritenon of the Achilles tendon. Progenitor populations derived from the tendon proper and peritenon are enriched with distinct cell types that are distinguished by expression of markers of tendon and vascular or pericyte origins, respectively. The objective of this study was to discern the unique tenogenic properties of tendon proper- and peritenon-derived progenitors within an in vitro model. We hypothe ...[more]

PMID: 25005797

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Project description:BackgroundTendon healing is clinically challenging largely due to its inferior regenerative capacity. We have previously prepared a soluble, DNA-free, urea-extracted bovine tendon-derived extracellular matrix (tECM) that exhibits strong pro-tenogenic bioactivity on human adipose-derived stem cells (hASCs). In this study, we aimed to elucidate the mechanism of tECM bioactivity via characterization of tECM protein composition and comparison of transcriptomic profiles of hASC cultures treated with tECM versus collagen type I (Col1) as a control ECM component.MethodsThe protein composition of tECM was characterized by SDS-PAGE, hydroxyproline assay, and proteomics analysis. To investigate tECM pro-tenogenic bioactivity and mechanism of action, differentiation of tECM-treated hASC cultures was compared to serum control medium or Col1-treated groups, as assessed via immunofluorescence for tenogenic markers and RNA Sequencing (RNA-Seq).ResultsUrea-extracted tECM yielded consistent protein composition, including collagens (20% w/w) and at least 17 non-collagenous proteins (< 100 kDa) based on MS analysis. Compared to current literature, tECM included key tendon ECM components that are functionally involved in tendon regeneration, as well as those that are involved in similar principal Gene Ontology (GO) functions (ECM-receptor interaction and collagen formation) and signaling pathways (ECM-receptor interaction and focal adhesion). When used as a cell culture supplement, tECM enhanced hASC proliferation and tenogenic differentiation compared to the Col1 and FBS treatment groups based on immunostaining of tenogenesis-associated markers. Furthermore, RNA-Seq analysis revealed a total of 584 genes differentially expressed among the three culture groups. Specifically, Col1-treated hASCs predominantly exhibited expression of genes and pathways related to ECM-associated processes, while tECM-treated hASCs expressed a mixture of ECM- and cell activity-associated processes, which may explain in part the enhanced proliferation and tenogenic differentiation of tECM-treated hASCs.ConclusionsOur findings showed that urea-extracted tECM contained 20% w/w collagens and is significantly enriched with other non-collagenous tendon ECM components. Compared to Col1 treatment, tECM supplementation enhanced hASC proliferation and tenogenic differentiation as well as induced distinct gene expression profiles. These findings provide insights into the potential mechanism of the pro-tenogenic bioactivity of tECM and support the development of future tECM-based approaches for tendon repair.

Project description:BACKGROUND:Tendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Exogenous growth factors that activate TSPC proliferation have emerged as a promising approach for treatment. Here, we evaluated the pigment epithelial-derived factor (PEDF)-derived short peptide (PSP; 29-mer) for treating acute tendon injury and to determine the timing and anatomical features of CD146- and necleostemin-positive TSPC in the tendon healing process. METHODS:Tendon cells were isolated from rabbit Achilles tendons, stimulated by the 29-mer and analyzed for colony-forming capacity. The expression of the TSPC markers CD146, Oct4, and nestin, induced by the 29-mer, was examined by immunostaining and western blotting. Tendo-Achilles injury was induced in rats by full-thickness insertion of an 18-G needle and immediately treated topically with an alginate gel, loaded with 29-mer. The distribution of TSPC in the injured tendon and their proliferation were monitored using immunohistochemistry with antibodies to CD146 and nucleostemin and by BrdU labeling. RESULTS:TSPC markers were enriched among the primary tendon cells when stimulated by the 29-mer. The 29-mer also induced the clonogenicity of CD146+ TSPC, implying TSPC stemness was retained during TSPC expansion in culture. Correspondingly, the expanded TSPC differentiated readily into tenocyte-like cells after removal of the 29-mer from culture. 29-mer/alginate gel treatment caused extensive expansion of CD146+ TSPC in their niche on postoperative day 2, followed by infiltration of CD146+/BrdU- TSPC into the injured tendon on day 7. The nucleostemin+ TSPC were located predominantly in the healing region of the injured tendon in the later phase (day 7) and exhibited proliferative capacity. By 3?weeks, 29-mer-treated tendons showed more organized collagen fiber regeneration and higher tensile strength than control tendons. In culture, the mitogenic effect of the 29-mer was found to be mediated by the phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC. CONCLUSIONS:The anatomical analysis of TSPC populations in the wound healing process supports the hypothesis that substantial expansion of resident TSPC by exogenous growth factor is beneficial for tendon healing. The study suggests that synthetic 29-mer peptide may be an innovative therapy for acute tendon rupture.

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Tendon proper- and peritenon-derived progenitor cells have unique tenogenic properties.

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Tendon proper- and peritenon-derived progenitor cells have unique tenogenic properties.

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