Protein discovery platform using deer antler as a model of mammalian regeneration

Abstract

The ability to activate and regulate stem cells during wound healing and tissue regeneration is a promising field which could bring innovative approaches into regenerative medicine. The regenerative capacity of invertebrates has been well documented, however in mammals, stem cells that drive organ regeneration are rare. Deer antler is the only known mammalian structure that can annually regenerate to produce a complex tissue. The neural crest derived stem cells that drive this process result in antler growing at up to 2 cm/day. Deer antler appears to outweigh lower-order animal models when investigating the regulation of stem cell-based regeneration. Pleiotrophin (PTN) is a multifunctional heparin-binding growth factor; the PTN gene is found highly expressed within the active antler stem cell tissues. The studies presented in this thesis aimed to examine the location of stem cells during antler growth, the proteomic profiles of different pools of stem cells involved in antler generation and regeneration, and the role of the PTN growth factor family in stem cell regulation.

The differentially expressed proteins between cells derived from stem cell niches involved in antlerogenesis (antlerogenic periosteum) and regeneration (potentiated and dormant pedicle periosteum), and cells derived from deer facial periosteum as a control (n = 3) were identified using 2D-DIGE-based quantitative proteomics. Label-free mass spectrometry was further used to detect the protein expression profiles of antler stem cell tissues under different stages of activation and included: dormant pedicle periosteum, growth centre, post-active stem cells from mid-beam periosteum, and control facial periosteum (n = 3). Mesenchymal stem cell markers CD73, CD90 and CD105, along with PTN/midkine (MDK) growth factors and their receptors (PTPRZ, ALK, NOTCH2, ITGAV and ITGB3), were examined in antler tissues using immunohistochemistry. In vitro effects of PTN on proliferation and osteogenic differentiation of antler stem cells were also investigated.

Ninety-two differentially expressed proteins were identified by 2D-DIGE. Bioinformatic analysis indicated the epithelial-mesenchymal transition process may participate in the initiation of wound healing and subsequent antler regeneration; cell mobility was highly involved during antler regeneration; energy and nucleotide metabolism may however be less active in antler regeneration as compared to that in antler generation phase. Immunohistochemistry confirmed the central role of stem cells in the development of this mammalian structure by localising the mesenchymal stem cell markers within the antler growth centre. Label-free quantification distinguished unique markers of dormant (6), active (87) and post-active (3) antler stem cells showing that the greatest number of proteins was exclusively found in the active stem cell tissue. There were only 12 proteins detected with expression levels that significantly differed between tissue with dormant stem cells and the control tissues. Protein profiles of these two groups showed that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. One hundred fifty-three significantly regulated proteins were found between antler stem cell tissues under different activation stages; activation of antler stem cells was associated with up-regulation of a number of canonical pathways and molecular/cellular functions such as Hippo and canonical Wnt signalling. PTN was identified as the dominant growth factor in the PTN/MDK family with higher expression levels in the antler growth centre. High expression of PTPRZ and ALK co-localised with PTN suggested their potential interactions. The high levels of PTN and PTPRZ also reflected the antler stem cell activation status during the regenerative process. When antler stem cells were cultured in vitro under the normoxic condition, no PTN was expressed and exogenous PTN did not induce differentiation or proliferation but rather stem cell maintenance.

In summary, this research project explores potential biomarkers for mammalian stem cells, as well as the key proteins, biological processes and pathways involved in stem cell maintenance, development and activation during antler generation and regeneration.