Identifying deer antler uhrf1 proliferation and s100a10 mineralization genes using comparative RNA-seq

Abstract

BackgroundDeer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration.MethodsTo identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (> 60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies.ResultsCell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6–11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10.ConclusionUsing a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-1027-6) contains supplementary material, which is available to authorized users.

Deciphering the potential pharmaceutical mechanism of Guzhi Zengsheng Zhitongwan on rat bone and kidney based on the “kidney governing bone” theory

“…Nfatc3, a family member of the nuclear factors of activated T cells, plays an important role in regulating bone formation and resorption through the receptor activator of NF-κB ligand (RANKL) [26]. S100a4, S100a6, and S100a10, which belong to the calcium-binding S100 family members, have been shown to regulate bone formation and resorption [27][28][29]. Therefore, these results were consistent with the above findings that the effect of GZZSZTW on inhibiting osteophyte formation during the process of OA might be achieved by enhancing kidney function and then suppressing bone formation and resorption.…”

Tendon‐derived extracellular matrix induces mesenchymal stem cell tenogenesis via an integrin/transforming growth factor‐β crosstalk‐mediated mechanism

“…Sorted hASCs from each donor were routinely validated using colony‐forming unit‐fibroblast (CFU‐F) and tri‐lineage differentiation assay (osteogenesis, adipogenesis, and chondrogenesis) until P8 as previously described 36 . For CFU‐F assay, hASCs were seeded (100 cells/100 mm diameter dish) and cultured in alpha‐Minimum Essential Medium (α‐MEM) for up to 14 days and stained with 0.5% of Crystal Violet solution 36 .…”

“…Sorted hASCs from each donor were routinely validated using colony‐forming unit‐fibroblast (CFU‐F) and tri‐lineage differentiation assay (osteogenesis, adipogenesis, and chondrogenesis) until P8 as previously described 36 . For CFU‐F assay, hASCs were seeded (100 cells/100 mm diameter dish) and cultured in alpha‐Minimum Essential Medium (α‐MEM) for up to 14 days and stained with 0.5% of Crystal Violet solution 36 . For osteogenic differentiation study, hASCs (1.5 × 10 4 cells/cm 2 ) were cultured with osteogenic medium (Dulbecco's Modified Eagle's Medium (DMEM) with 50 µg/mL ascorbic acid (ChemCruz, Dallas, TX, USA), 10 mM of β‐glycerophosphate (Sigma, St. Louis, MO, USA) and 10 nM of dexamethasone (Sigma) or basal control medium and stained for calcium deposits via Alizarin Red S staining (Electron Microscopy Sciences, Hatfield, PA, USA) on day 21 36 .…”

“…For CFU‐F assay, hASCs were seeded (100 cells/100 mm diameter dish) and cultured in alpha‐Minimum Essential Medium (α‐MEM) for up to 14 days and stained with 0.5% of Crystal Violet solution 36 . For osteogenic differentiation study, hASCs (1.5 × 10 4 cells/cm 2 ) were cultured with osteogenic medium (Dulbecco's Modified Eagle's Medium (DMEM) with 50 µg/mL ascorbic acid (ChemCruz, Dallas, TX, USA), 10 mM of β‐glycerophosphate (Sigma, St. Louis, MO, USA) and 10 nM of dexamethasone (Sigma) or basal control medium and stained for calcium deposits via Alizarin Red S staining (Electron Microscopy Sciences, Hatfield, PA, USA) on day 21 36 . For adipogenic differentiation study, hASCs (1.5 × 10 4 cells/cm 2 ) were cultured with adipogenic medium (Adipogenesis Differentiation Kit, Gibco) or basal control medium, and stained for oil deposits using Oil Red O Staining (ChemCruz) at day 14 36 .…”

“…For chondrogenic differentiation study, hASCs (8 × 10 4 cells/5 μL drops) were seeded in 48‐well plate for 2 hours before incubation in chondrogenic medium (Chondrogenesis Differentiation Kit, Gibco) or basal control medium. After 15 days, chondrogenic‐associated ECM (sulfated proteoglycans) was detected using Alcian Blue staining (Electron Microscopy Sciences) 36 . hASCs (P2‐P5) from each donor were used for the following experiments.…”