A population of stem cells with strong regenerative potential discovered in deer antlers.

The annual regrowth of deer antlers provides a valuable model for studying organ regeneration in mammals. We describe a single-cell atlas of antler regrowth. The earliest-stage antler initiators were mesenchymal cells that express the paired related homeobox 1 gene (PRRX1+ mesenchymal cells). We also identified a population of "antler blastema progenitor cells" (ABPCs) that developed from the PRRX1+ mesenchymal cells and directed the antler regeneration process. Cross-species comparisons identified ABPCs in several mammalian blastema. In vivo and in vitro ABPCs displayed strong self-renewal ability and could generate osteochondral lineage cells. Last, we observed a spatially well-structured pattern of cellular and gene expression in antler growth center during the peak growth stage, revealing the cellular mechanisms involved in rapid antler elongation.

Integrated analysis of miRNA and mRNA transcriptomic reveals antler growth regulatory network.

The growth of antler is driven by endochondral ossification in the growth center of the apical region. Antler grows faster than cancer tissues, but it can be stably regulated and regenerated periodically. To elucidate the molecular mechanisms of how antler grows rapidly without carcinogenesis, in this study, we used RNA-seq technology to evaluate the changes of miRNA and mRNA profiles in antler at four different developmental stages, including 15, 60, 90, and 110 days. We identified a total of 55004 unigenes and 246 miRNAs of which, 10182, 13258, 10740 differentially expressed (DE) unigenes and 35, 53, 27 DE miRNAs were identified in 60-day vs. 15-day, 90-day vs. 60-day, and 110-day vs. 90-day. GO and KEGG pathway analysis indicated that DE unigenes and DE miRNA were mainly associated with chondrogenesis, osteogenesis and inhibition of oncogenesis, that were closely related to antler growth. The interaction networks of mRNA-mRNA and miRNA-mRNA related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler were constructed. The results indicated that mRNAs (COL2A1, SOX9, WWP2, FGFR1, SPARC, LOX, etc.) and miRNAs (miR-145, miR-199a-3p, miR-140, miR-199a-5p, etc.) might have key roles in chondrogenesis and osteogenesis of antler. As well as mRNA (TP53, Tpm3 and ATP1A1, etc.) and miRNA (miR-106a, miR-145, miR-1260b and miR-2898, etc.) might play important roles in inhibiting the carcinogenesis of antler. In summary, we constructed the mRNA-mRNA and miRNA-mRNA regulatory networks related to chondrogenesis, osteogenesis and inhibition of oncogenesis of antler, and identified key candidate mRNAs and miRNAs among them. Further developments and validations may provide a reference for in-depth analysis of the molecular mechanism of antler growth without carcinogenesis.

S100A4: a novel partner for heat shock protein 47 in antler stem cells and insight into the calcium ion-induced conformational changes.

S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma.

Identification of proteins that mediate the role of androgens in antler regeneration using label free proteomics in sika deer (Cervus nippon).

Deer antlers offer a unique model to study organ regeneration in mammals. Antler regeneration relies on the pedicle periosteum (PP) cells and is triggered by a decrease in circulating testosterone (T). The molecular mechanism for antler regeneration is however, unclear. Label-free liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify differentially-expressed proteins (DEPs) in the regeneration-potentiated PP (under low T environment) over the non-regeneration-potentiated PP (under high T environment). Out of total 273 DEPs, 189 were significantly up-regulated and 84 were down-regulated from these comparisons: after castration vs before castration, natural T vs before castration, and exogenous T vs before castration. We focused on the analysis only of those DEPs that were present in fully permissive environment to antler regeneration (low T). Nine transduction pathways were identified through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, including the estrogen signaling pathway. A total of 639 gene ontology terms were found to be significantly enriched in regeneration-potentiated PP (low T) from the DEPs. Reliability of the label free LC-MS/MS was determined by qRT-PCR to estimate the expression level of selected genes. The results suggest that up-regulated heat shock proteins (HSP90AB1, HSP90B1), peptidyl-prolyl cis-trans isomerase 4 (FKBP4), mitogen-activated protein kinase 3 (MAPK3) and calreticulin (CALR) and down-regulated SHC-transforming protein 1 (SHC1), heat shock protein family A member 1A (HSPA1A) and proto-oncogene tyrosine-protein kinase (SRC) may be associated directly or indirectly with antler regeneration. Further studies are required to investigate the roles of these proteins in regeneration using appropriate in vivo models.

Deer antler stem cells are a novel type of cells that sustain full regeneration of a mammalian organ-deer antler.

Deer antlers are extraordinary mammalian organs that can fully regenerate annually. Antler renewal is a stem cell-based epimorphic process and antler stem (AS) cells can initiate de novo generation of antlers in postnatal mammals. However, although being called stem cells, the AS cells have not been characterized at molecular level based on the stem cell criteria. Comprehensive characterization of the AS cells would undoubtedly help to decipher the mechanism underlying the full regeneration of deer antlers, the only case of stem cell-based epimorphic regeneration in mammals. In the present study, three types of AS cells (antlerogenic periosteal cells APCs, for initial pedicle and first antler formation; pedicle periosteal cells PPC, for annual antler regeneration; and reserve mesenchyme cells RMCs, for rapid antler growth), were isolated for comprehensive molecular characterization. A horn-growth-related gene, RXFP2, was found to be expressed only in AS cells lineages but not in the facial periosteal cells (FPCs, locates geographically in the vicinity of the APCs or PPCs), suggesting the RXFP2 might be a specific marker for the AS cell lineage in deer. Our results demonstrated that AS cells expressed classic MSC markers including surface markers CD73, CD90, CD105 and Stro-1. They also expressed some of the markers including Tert, Nestin, S100A4, nucleostemin and C-Myc, suggesting that they have some attributes of the ESCs. Microinjection of male APC into deer blastocysts resulted in one female foetus (110 days gestation) recovered with obvious pedicle primordia with both male and female genotype detected in the ovary. In conclusion, the AS cells should be defined as MSCs but with partial attributes of ESCs.

Single-cell transcriptome provides novel insights into antler stem cells, a cell type capable of mammalian organ regeneration.

Antler regeneration, a stem cell-based epimorphic process, has a potential as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler development is located in the antlerogenic periosteum (AP). However, whether this ASC pool is homogenous or heterogeneous has not been fully evaluated. In this study, we produced a comprehensive transcriptome dataset at the single-cell level for the ASCs based on the 10× Genomics platform (scRNA-seq). A total of 4565 ASCs were sequenced and classified into a large cell cluster, indicating that the ASC resident in the AP are likely to be a homogeneous population. The scRNA-seq data revealed that tumor-related genes were highly expressed in these homogeneous ASCs, i.e., TIMP1, TMSB10, LGALS1, FTH1, VIM, LOC110126017, and S100A4. Results of screening for stem cell markers suggest that the ASCs may be considered as a special type of stem cell between embryonic (CD9) and adult (CD29, CD90, NPM1, and VIM) stem cells. Our results provide the first comprehensive transcriptome analysis at the single-cell level for the ASCs and identified only one major cell type resident in the AP and some key stem cell genes, which may hold the key to why antlers, the unique mammalian organ, can fully regenerate once lost.

Expression and Functional Analysis of Tumor-Related Factor S100A4 in Antler Stem Cells.

Annual antler renewal is a stem cell-based epimorphic process driven by antler stem cells (ASCs) resident in antlerogenic periosteum (AP). Antlerogenic periosteal cells express a high level of S100A4, a metastasis-associated protein, which intrigued us to explore what role S100A4 could play in antler regeneration. The present study set out to investigate expression and effects of S100A4 in the ASCs and their progeny. The results showed that not only did cells from the AP express a high level of S100A4, but also the pedicle periosteum and the antler growth center. In the antler growth center, we found S100A4-positive cells were specifically located in blood vessel walls and in vascularized areas. In vitro, recombinant deer S100A4 protein stimulated the proliferation of the AP cells, promoted proliferation, migration and tube formation of human vascular endothelial cells, and enhanced migration of Hela cells, but not AP cells. These findings demonstrated that S100A4 in the ASCs may play a significant role in stimulating angiogenesis, proliferation, but not motility, of ASCs. Deer antlers offer a unique model to explore how rapid cell proliferation with a high level of S100A4 expression is elegantly regulated without becoming cancerous.

Characterization of a virulent dog-originated rabies virus affecting more than twenty fallow deer (Dama dama) in Inner Mongolia, China.

Rabies has emerged as a serious problem in the most recent years in northern China. A rabies virus (RABV) isolate, IMDRV-13, was recovered from brain samples of dog-bitten rabid fallow deer (Dama dama) in a farm in Hohhot, Inner Mongolia. We tested the susceptibility of mouse neuroblastoma (MNA) cells and BSR cells as well as that of adult mice to IMDRV-13. The isolate was found to be a virulent isolate with an equivalent pathogenicity index (0.12) and a slight lower neurotropism index (1.07) compared with those of challenge virus standard, CVS-24, which was 0.13 and 1.23, respectively. The complete genome of IMDRV-13 was determined subsequently and found to be 11,924 nucleotides (nt) in length with the same genomic organization as other RABVs. Phylogenetic tree based on complete genome sequences of 43 RABV isolates and strains indicated that IMDRV-13, along with other two isolates in Inner Mongolia, CNM1101C and CNM1104D, clustered within the dog-associated China I clade, which is also the dominant lineage in the current rabies epidemic in China. In addition, sequence analysis of the glycoprotein G identified an amino acid substitution (I338→T338) unique to the IMDRV-13 within antigenic sites III (330-338), this mutation also leads to an additional potential N-glycosylation site (N336), which may represent a useful model to study relationship of N-glycosylation in G protein and specific properties such as pathogenicity or host adaption of RABV.