Functional & Integrative Genomics

, Volume 19, Issue 4, pp 555–564 | Cite as

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

  • Hengxing Ba
  • Datao Wang
  • Weiyao Wu
  • Hongmei Sun
  • Chunyi LiEmail author
Original Article


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.


Antler Stem cell Antlerogenic periosteum Single cell Transcriptome scRNA-seq 



We wish to thank Drs. Peter Fennessy and Eric Lord for reading through the paper and giving valuable comments.

Author contributions

H.B., D.W., and C.L. conceived the experiment. D.W. collected the samples and performed molecular- and cell-related experiments. H.S. cultured the cell lines. W.W. extracted the RNA samples and prepared them for single-cell 3′ library construction and sequencing. H.B. performed QC and data analysis. H.B., W.W., and C.L. wrote the manuscript. All authors read and approved the final manuscript.

Funding information

This work was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010403), Natural Science Foundation of Jilin Province (No. 20170101003JC) and Central Public-Interest Scientific Institution Basal Research Fund (No. 1610342019026).

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.

Supplementary material

10142_2019_659_MOESM1_ESM.jpg (1.6 mb)
Figure S1. Detection of variable genes across the ASCs. A total of 2415 variable genes were selected by using FindVariableGenes function (x.low.cutoff = 0.01, x.high.cutoff = 4, y.cutoff=0.3) in Seurat R package. The parameters identify ~2943 variable genes. (JPG 1611 kb)
10142_2019_659_MOESM2_ESM.jpg (413 kb)
Figure S2. Standard deviation of principal components. To assess the true dimensionality of our dataset, the first 30 principal components were deemed as a cutoff, as there is a clear elbow in the graph. (JPG 413 kb)
10142_2019_659_MOESM3_ESM.docx (19 kb)
Table S1. List of Antibodies (DOCX 19 kb)
10142_2019_659_MOESM4_ESM.docx (23 kb)
Table S2. Summary of scRNA-seq data quality (DOCX 22 kb)
10142_2019_659_MOESM5_ESM.docx (15 kb)
Table S3. Summary of single cell sequencing data (DOCX 14 kb)
10142_2019_659_MOESM6_ESM.xlsx (13 kb)
Table S4. Summary of top 35 highly expressed genes in the scRNA-seq data. (XLSX 13 kb)


  1. Berg DK, Li C, Asher G, Wells DN, Oback B (2007) Red deer cloned from antler stem cells and their differentiated progeny. Biol Reprod 77:384–394. CrossRefGoogle Scholar
  2. Blondel VD, Guillaume JL, Lambiotte R, Lefebvre E (2008) Fast unfolding of communities in largenetworks. Journal of Statistical Mechanics. 2008:155–168
  3. Bramanti V, Tomassoni D, Avitabile M, Amenta F, Avola R (2010) Biomarkers of glial cell proliferation and differentiation in culture. Front Biosci (Schol Ed) 2:558–570Google Scholar
  4. Butler A, Hoffman P, Smibert P, Papalexi E, Satija R (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36:411–420. CrossRefGoogle Scholar
  5. Chu W, Zhao H, Li J, Li C (2017) Custom-built tools for the study of deer antler biology. Front Biosci (Landmark edition) 22:1622–1633CrossRefGoogle Scholar
  6. Colucci-Guyon E, Portier MM, Dunia I, Paulin D, Pournin S, Babinet C (1994) Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell 79:679–694CrossRefGoogle Scholar
  7. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21. CrossRefGoogle Scholar
  8. Gardiner DM, Endo T, Bryant SV (2002) The molecular basis of amphibian limb regeneration: integrating the old with the new. Semin Cell Dev Biol 13:345–352CrossRefGoogle Scholar
  9. Goldman RD, Khuon S, Chou YH, Opal P, Steinert PM (1996) The function of intermediate filaments in cell shape and cytoskeletal integrity. J Cell Biol 134:971–983CrossRefGoogle Scholar
  10. Goss RJ (1995) Future directions in antler research. Anat Rec 241:291–302. CrossRefGoogle Scholar
  11. Goss RJ, Powel RS (1985) Induction of deer antlers by transplanted periosteum. I Graft size and shape. J Exp Zool 235:359–373. CrossRefGoogle Scholar
  12. Hartwig H, Schrudde J (1974) Experimentelle Untersuchungen zur Bildung der primaren Stirnauswuchse beim Reh (Capreolus capreolus L.). Z Jagdwiss 20:1–13Google Scholar
  13. Houle J, Fedoroff S (1983) Temporal relationship between the appearance of vimentin and neural tube development. Brain Res 285:189–195CrossRefGoogle Scholar
  14. Huang LY, Xu Y, Cai GX, Guan ZQ, Cai SJ (2012) Downregulation of S100A4 expression by RNA interference suppresses cell growth and invasion in human colorectal cancer cells. Oncol Rep 27:917–922. CrossRefGoogle Scholar
  15. Jung S, Yi L, Kim J, Jeong D, Oh T, Kim CH, Kim CJ, Shin J, An S, Lee MS (2011) The role of vimentin as a methylation biomarker for early diagnosis of cervical cancer. Mol Cell 31:405–411. CrossRefGoogle Scholar
  16. Kierdorf U, Kierdorf H, Szuwart T (2007) Deer antler regeneration: cells, concepts, and controversies. J Morphol 268:726–738. CrossRefGoogle Scholar
  17. Kierdorf U, Li CY, Price JS (2009) Improbable appendages: deer antler renewal as a unique case of mammalian regeneration. Semin Cell Dev Biol 20:535–542. CrossRefGoogle Scholar
  18. Kim YS, Kim SH, Kang JG, Ko JH (2012) Expression level and glycan dynamics determine the net effects of TIMP-1 on cancer progression. BMB Rep 45:623–628. CrossRefGoogle Scholar
  19. Kumar P, Tan YQ, Cahan P (2017) Understanding development and stem cells using single cell-based analyses of gene expression. Development 144:17–32. CrossRefGoogle Scholar
  20. Li CY (2012) Deer antler regeneration: a stem cell-based epimorphic Process. Birth Defects Res C 96:51–62. CrossRefGoogle Scholar
  21. Li C, Suttie JM (1994) Light microscopic studies of pedicle and early first antler development in red deer (Cervus elaphus). Anat Rec 239:198–215. CrossRefGoogle Scholar
  22. Li C, Suttie JM (2001) Deer antlerogenic periosteum: a piece of postnatally retained embryonic tissue? Anat Embryol (Berl) 204:375–388CrossRefGoogle Scholar
  23. Li C, Suttie JM (2003) Tissue collection methods for antler research. Eur J Morphol 41:23–30. CrossRefGoogle Scholar
  24. Li C, Littlejohn RP, Suttie JM (1999) Effects of insulin-like growth factor 1 and testosterone on the proliferation of antlerogenic cells in vitro. J Exp Zool 284:82–90CrossRefGoogle Scholar
  25. Li CY, Clark DE, Lord EA, Stanton JAL, Suttie JM (2002) Sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery. Anat Rec 268:125–130. CrossRefGoogle Scholar
  26. Li C, Suttie JM, Clark DE (2005) Histological examination of antler regeneration in red deer (Cervus elaphus). Anat Rec A Discov Mol Cell Evol Biol 282:163–174. CrossRefGoogle Scholar
  27. Li C, Yang F, Li G, Gao X, Xing X, Wei H, Deng X, Clark DE (2007a) Antler regeneration: a dependent process of stem tissue primed via interaction with its enveloping skin. J Exp Zool A Ecol Genet Physiol 307:95–105. CrossRefGoogle Scholar
  28. Li CY, Mackintosh CG, Martin SK, Clark DE (2007b) Identification of key tissue type for antler regeneration through pedicle periosteum deletion. Cell Tissue Res 328:65–75. CrossRefGoogle Scholar
  29. Li C, Yang F, Sheppard A (2009) Adult stem cells and mammalian epimorphic regeneration-insights from studying annual renewal of deer antlers. Curr Stem Cell Res Ther 4:237–251CrossRefGoogle Scholar
  30. Li C, Harper A, Puddick J, Wang W, McMahon C (2012) Proteomes and signalling pathways of antler stem cells. PLoS One 7:e30026. CrossRefGoogle Scholar
  31. Lindstrom MS (2011) NPM1/B23: a multifunctional chaperone in ribosome biogenesis and chromatin remodeling. Biochem Res Int 2011:195–209. CrossRefGoogle Scholar
  32. Liu FT (2005) Regulatory roles of galectins in the immune response. Int Arch Allergy Immunol 136:385–400. CrossRefGoogle Scholar
  33. Liu S, Trapnell C (2016) Single-cell transcriptome sequencing: recent advances and remainingchallenges. F1000research 5
  34. Lord EA, Clark DE, Martin SK, Pedersen GM, Gray JP, Li CY (2004) Profiling genes expressed in the regenerating tip of red deer (cervua elaphus) antler. Paper presented at the In advances in antler science and product technologyGoogle Scholar
  35. Ma X, Yang YX, Wang YF, An GF, Lv G (2010) Small interfering RNA-directed knockdown of S100A4 decreases proliferation and invasiveness of osteosarcoma cells. Cancer Lett 299:171–181. CrossRefGoogle Scholar
  36. Mescher AL (1996) The cellular basis of limb regeneration in urodeles. Int J Dev Biol 40:785–795Google Scholar
  37. Mount JG, Muzylak M, Allen S, Althnaian T, Mcgonnell IM, Price JS (2006) Evidence that thecanonical Wnt signalling pathway regulates deer antler regenerationGoogle Scholar
  38. Park HJ, Lee DH, Park SG, Lee SC, Cho S, Kim HK, Kim JJ, Bae H, Park BC (2004) Proteome analysis of red deer antlers. Proteomics 4:3642–3653. CrossRefGoogle Scholar
  39. Price J, Faucheux C, Allen S (2005a) Deer antlers as a model of mammalian regeneration. Curr Top Dev Biol 67:1–48. CrossRefGoogle Scholar
  40. Price JS, Allen S, Faucheux C, Althnaian T, Mount JG (2005b) Deer antlers: a zoological curiosity or the key to understanding organ regeneration in mammals? J Anat 207:603–618. CrossRefGoogle Scholar
  41. Rabinovich GA, Liu FT, Hirashima M, Anderson A (2007) An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer. Scand J Immunol 66:143–158. CrossRefGoogle Scholar
  42. RJ G (1983) Deer antlers. regeneration, function and evolution. Academic Press, New YorkGoogle Scholar
  43. Rolf HJ, Kierdorf U, Kierdorf H, Schulz J, Seymour N, Schliephake H, Napp J, Niebert S, Wölfel H, Wiese KG (2008) Localization and characterization of STRO-1 cells in the deer pedicle and regenerating antler. PLoS One 3:e2064. CrossRefGoogle Scholar
  44. Santelli G, Califano D, Chiappetta G, Vento MT, Bartoli PC, Zullo F, Trapasso F, Viglietto G, Fusco A (1999) Thymosin beta-10 gene overexpression is a general event in human carcinogenesis. Am J Pathol 155:799–804CrossRefGoogle Scholar
  45. Seo MS, Park SB, Choi SW, Kim JJ, Kim HS, Kang KS (2014) Isolation and characterization of antler-derived multipotent stem cells. Cell Transplant 23:831–843. CrossRefGoogle Scholar
  46. Sherbet GV (2009) Metastasis promoter S100A4 is a potentially valuable molecular target for cancer therapy. Cancer Lett 280:15–30. CrossRefGoogle Scholar
  47. Sribenja S, Li M, Wongkham S, Wongkham C, Yao Q, Chen C (2009) Advances in thymosin beta10 research: differential expression, molecular mechanisms, and clinical implications in cancer and other conditions. Cancer Investig 27:1016–1022. CrossRefGoogle Scholar
  48. Stocum D (2006) Regenerative biology and medicine. Academic Press, New YorkCrossRefGoogle Scholar
  49. Torti SV, Torti FM (2013) Iron and cancer: more ore to be mined. Nat Rev Cancer 13:342–355. CrossRefGoogle Scholar
  50. Wang D, Guo Q, Ba H, Li C (2016) Cloning and characterization of a Nanog pseudogene in sika deer (Cervus nippon). DNA and Cell Biology 35:576–584. CrossRefGoogle Scholar
  51. Wang DT, Chu WH, Sun HM, Ba HX, Li CY (2017) Expression and functional analysis of tumor-related factor S100A4 in antler stem cells. J Histochem Cytochem 65:579–591. CrossRefGoogle Scholar
  52. Yan KS, Gevaert O, Zheng GXY, Anchang B, Probert CS, Larkin KA, Davies PS, Cheng ZF, Kaddis JS, Han A, Roelf K, Calderon RI, Cynn E, Hu X, Mandleywala K, Wilhelmy J, Grimes SM, Corney DC, Boutet SC, Terry JM, Belgrader P, Ziraldo SB, Mikkelsen TS, Wang F, von Furstenberg RJ, Smith NR, Chandrakesan P, May R, Chrissy MAS, Jain R, Cartwright CA, Niland JC, Hong YK, Carrington J, Breault DT, Epstein J, Houchen CW, Lynch JP, Martin MG, Plevritis SK, Curtis C, Ji HP, Li L, Henning SJ, Wong MH, Kuo CJ (2017) Intestinal enteroendocrine lineage cells possess homeostatic and injury-inducible stem cell activity. Cell Stem Cell 21:78–90 e76. CrossRefGoogle Scholar
  53. Yu PJ, Lin W (2016) Single-cell transcriptome study as big data. Genom Proteom Bioinf 14:21–30. CrossRefGoogle Scholar
  54. Zhang X, Ren D, Guo L, Wang L, Wu S, Lin C, Ye L, Zhu J, Li J, Song L, Lin H, He Z (2017) Thymosin beta 10 is a key regulator of tumorigenesis and metastasis and a novel serum marker in breast cancer. Breast Cancer Res 19:15. CrossRefGoogle Scholar
  55. Zhang W, Chu W, Liu Q, Coates D, Shang Y, Li C (2018) Deer thymosin beta 10 functions as a novel factor for angiogenesis and chondrogenesis during antler growth and regeneration. Stem Cell Res Ther 9:166. CrossRefGoogle Scholar
  56. Zheng GX et al (2017) Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Hengxing Ba
    • 1
  • Datao Wang
    • 1
  • Weiyao Wu
    • 2
  • Hongmei Sun
    • 1
  • Chunyi Li
    • 1
    • 3
    Email author
  1. 1.State Key Laboratory for Molecular Biology of Special Wild Economic Animals, Institute of Special Wild Economic Animals and PlantsChinese Academy of Agricultural SciencesChangchunChina
  2. 2.BGI GenomicsBGI-ShenzhenShenzhenChina
  3. 3.Changchun Sci-Tech UniversityChangchunChina

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