Retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) restore the germline competence of in vitro cultured chicken blastodermal cells

  • Xiaochuan Tang
  • Jun Shi
  • Xiaolian Qin
  • Ning Xiao
  • Rongyang Li
  • Hao Hu
  • Fengshuo Yang
  • Deshun ShiEmail author
  • Xiaoli WangEmail author


Chicken blastodermal cells (BCs) are pluripotent stem cells derived from early embryos and may be easily obtained and manipulated. However, in vitro cultured BCs have extremely low germline capacity, which may limit their applications. Research on the germ cell differentiation of mammalian pluripotent cells using chemical-inducing agents has gained popularity, and tremendous achievements have been made. Whether chemical-inducing agents allow acquirement of germline competence in BCs is, however, questionable. In this study, retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) promoted the expression of germline-specific genes and restored the germline competence of in vitro cultured BCs. Moreover, BCs induced with RA and BMP4 could efficiently produce gonadal chimeric chick embryos. These results may greatly enhance the potential applications of BCs in biotechnology and basic research.


Chicken blastodermal cells Retinoic acid Bone morphogenetic protein 4 Germline competence Genetically modified chicken 



We thank Zhiqiang Guan for the help in the experiment.

Funding information

This study was supported by Technical Innovation to Guide Specialy of Guangxi Province (2017AD10038) and the National Natural Science Foundation of China (31460644).

Compliance with ethical standards

Ethical compliance

All experiments were in accordance with the guidelines of the regional Animal Ethics Committee, and the Institutional Animal Care and Use Committee of Guangxi University approved all experiments.

Supplementary material

11626_2019_324_MOESM1_ESM.jpg (954 kb)
Supplementary Fig. 1 Comparison of migration efficiency of induced BCs between the left and right sides of the testis and between the sexes. (A) All gonads with obvious visible GFP. (B) Comparison of the number of GFP positive cells between the left and right sides of the testis. (C) Comparison of the number of GFP positive cells between the sexes. GFP positive cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA). *P < 0.05; **P < 0.01. (JPG 954 kb)


  1. Boast S, Stern CD (2013) Simple methods for generating neural, bone and endodermal cell types from chick embryonic stem cells. Stem Cell Res 10(1):20–28Google Scholar
  2. Cao D, Wu H, Li Q, Sun Y, Liu T, Fei J, Zhao Y et al (2015) Expression of recombinant human lysozyme in egg whites of transgenic hens. PLoS One 10(2):e0118626Google Scholar
  3. Carsience RS, Clark ME, Gibbins AMV, Etches RJ (1993) Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 117(2):669–675Google Scholar
  4. Chen GK, Gulbranson DR, Hou ZG, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K et al (2011) Chemically defined conditions for human Ipsc derivation and culture. Nat Methods 8(5):424–U76Google Scholar
  5. Chen W, Jia WW, Wang K, Zhou Q, Leng Y, Duan T, Kang JH (2012) Retinoic acid regulates germ cell differentiation in mouse embryonic stem cells through a Smad-dependent pathway. Biochem Biophys Res Commun 418(3):571–577Google Scholar
  6. Couteaudier M, Trapp-Fragnet L, Auger N, Courvoisier K, Pain B, Denesvre C, Vautherot JF (2015) Derivation of keratinocytes from chicken embryonic stem cells: establishment and characterization of differentiated proliferative cell populations. Stem Cell Res 14(2):224–237Google Scholar
  7. Cunningham TJ, Brade T, Sandell LL, Lewandoski M, Trainor PA, Colas A, Mercola M, Duester G (2015) Retinoic acid activity in undifferentiated neural progenitors is sufficient to fulfill its role in restricting Fgf8 expression for somitogenesis. PLoS One 10(9):e0137894Google Scholar
  8. Cunningham TJ, Duester G (2015) Mechanisms of retinoic acid signalling and its roles in organ and limb development. Nat Rev Mol Cell Biol 16(2):110–123Google Scholar
  9. Eyal-Giladi H, Kochav S (1976) From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick: I. General morphology. Dev Biol 49(2):321–337Google Scholar
  10. Griswold MD, Hogarth CA, Bowles J, Koopman P (2012) Initiating meiosis: the case for retinoic acid. Biol Reprod 86(2):35Google Scholar
  11. Han JY, Lee HC, Park TS (2015) Germline-competent stem cell in avian species and its application. Asian J Androl 17(3):421–426Google Scholar
  12. Imamura M, Hikabe O, Lin ZYC, Okano H (2014) Generation of germ cells in vitro in the era of induced pluripotent stem cells. Mol Reprod Dev 81(1):2–19Google Scholar
  13. Janesick A, Wu SC, Blumberg B (2015) Retinoic acid signaling and neuronal differentiation. Cell Mol Life Sci 72(8):1559–1576Google Scholar
  14. Jean C, Oliveira NMM, Intarapat S, Fuet A, Mazoyer C, De Almeida I, Trevers K et al (2015) Transcriptome analysis of chicken Es, blastodermal and germ cells reveals that chick Es cells are equivalent to mouse Es cells rather than Episc. Stem Cell Res 14(1):54–67Google Scholar
  15. Johnson PA, Giles JR (2006) Use of genetic strains of chickens in studies of ovarian cancer. Poult Sci 85(2):246–250Google Scholar
  16. Kress C, Montillet G, Jean C, Fuet A, Pain B (2016) Chicken embryonic stem cells and primordial germ cells display different heterochromatic histone marks than their mammalian counterparts. Epigenetics Chromatin 9:5Google Scholar
  17. Lavial F, Acloque H, Bachelard E, Nieto MA, Samarut J, Pain B (2009) Ectopic expression of Cvh (chicken vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate. Dev Biol 330(1):73–82Google Scholar
  18. Lillico SG, Sherman A, McGrew MJ, Robertson CD, Smith J, Haslam C, Barnard P, Radcliffe PA, Mitrophanous KA, Elliot EA, Sang HM (2007) Oviduct-specific expression of two therapeutic proteins in transgenic hens. Proc Natl Acad Sci U S A 104(6):1771–1776Google Scholar
  19. Lu Y, West FD, Jordan BJ, Jordan ET, West RC, Yu P, He Y, Barrios MA, Zhu Z, Petitte JN, Beckstead RB, Stice SL (2014) Induced pluripotency in chicken embryonic fibroblast results in a germ cell fate. Stem Cells Dev 23(15):1755–1764Google Scholar
  20. McGrew MJ, Sherman A, Ellard FM, Lillico SG, Gilhooley HJ, Kingsman AJ, Mitrophanous KA, Sang H (2004) Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Rep 5(7):728–733Google Scholar
  21. Naito M, Harumi T, Kuwana T (2015) Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens. Anim Reprod Sci 153:50–61Google Scholar
  22. Nakamura Y, Yamamoto Y, Usui F, Mushika T, Ono T, Setioko AR, Takeda K, Nirasawa K, Kagami H, Tagami T (2007) Migration and proliferation of primordial germ cells in the early chicken embryo. Poult Sci 86(10):2182–2193Google Scholar
  23. Oishi I, Yoshii K, Miyahara D, Kagami H, Tagami T (2016) Targeted mutagenesis in chicken using Crispr/Cas9 system. Sci Report 6:23980Google Scholar
  24. Park TS, Lee HG, Moon JK, Lee HJ, Yoon JW, Yun BN, Kang SC et al (2015) Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter. FASEB J 29(6):2386–2396Google Scholar
  25. Park TS, Lee HJ, Kim KH, Kim JS, Han JY (2014) Targeted gene knockout in chickens mediated by Talens. Proc Natl Acad Sci U S A 111(35):12716–12721Google Scholar
  26. Pesce M, Klinger FG, De Felici M (2002) Derivation in culture of primordial germ cells from cells of the mouse epiblast: phenotypic induction and growth control by Bmp4 signalling. Mech Dev 112(1–2):15–24Google Scholar
  27. Petitte JN, Clark ME, Liu G, Gibbins AMV, Etches RJ (1990) Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108(1):185–196Google Scholar
  28. Rengaraj D, Zheng YH, Kang KS, Park KJ, Lee BR, Lee SI, Choi JW, Han JY (2010) Conserved expression pattern of chicken Dazl in primordial germ cells and germ-line cells. Theriogenology 74(5):765–776Google Scholar
  29. Song YH, Duraisamy S, Ali J, Kizhakkayil J, Jacob VD, Mohammed MA, Eltigani MA, et al. (2014) Characteristics of long-term cultures of avian primordial germ cells and gonocytes. Biol Reprod 90, no. 1Google Scholar
  30. Tang X, Xu S, Zhang H, Chen Q, Li R, Wu W, Yu M, Liu H (2017) Retinoic acid promotes expression of germline-specific genes in chicken blastoderm cells by stimulating Smad1/5 phosphorylation in a feeder-free culture system. BMC Biotechnol 17(1):17Google Scholar
  31. Taylor L, Carlson DF, Nandi S, Sherman A, Fahrenkrug SC, McGrew MJ (2017) Efficient Talen-mediated gene targeting of chicken primordial germ cells. Development 144(5):928–934Google Scholar
  32. Thomson T, Lasko P (2005) Tudor and its domains: germ cell formation from a Tudor perspective. Cell Res 15(4):281–291Google Scholar
  33. Tsunekawa N, Naito M, Sakai Y, Nishida T, Noce T (2000) Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 127(12):2741–2750Google Scholar
  34. van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, Swanberg SE, Delany ME, Etches RJ (2006a) Germline transmission of genetically modified primordial germ cells. Nature 441(7094):766–769Google Scholar
  35. van de Lavoir MC, Mather-Love C, Leighton P, Diamond JH, Heyer BS, Roberts R, Zhu L, Winters-Digiacinto P, Kerchner A, Gessaro T, Swanberg S, Delany ME, Etches RJ (2006b) High-grade transgenic somatic chimeras from chicken embryonic stem cells. Mech Dev 123(1):31–41Google Scholar
  36. Wan Z, Rui L, Li Z (2014) Expression patterns of Prdm1 during chicken embryonic and germline development. Cell Tissue Res 356(2):341–356Google Scholar
  37. Wang Y, Chou BK, Dowey S, He C, Gerecht S, Cheng L (2013) Scalable expansion of human induced pluripotent stem cells in the defined Xeno-free E8 medium under adherent and suspension culture conditions. Stem Cell Res 11(3):1103–1116Google Scholar
  38. Zhou Q, Wang M, Yuan Y, Wang X, Fu R, Wan H, Xie M, Liu M, Guo X, Zheng Y, Feng G, Shi Q, Zhao XY, Sha J, Zhou Q (2016) Complete meiosis from embryonic stem cell-derived germ cells in vitro. Cell Stem Cell 18(3):330–340Google Scholar
  39. Zhu L, van de Lavoir MC, Albanese J, Beenhouwer DO, Cardarelli PM, Cuison S, Deng DF, Deshpande S, Diamond JH, Green L, Halk EL, Heyer BS, Kay RM, Kerchner A, Leighton PA, Mather CM, Morrison SL, Nikolov ZL, Passmore DB, Pradas-Monne A, Preston BT, Rangan VS, Shi M, Srinivasan M, White SG, Winters-Digiacinto P, Wong S, Zhou W, Etches RJ (2005) Production of human monoclonal antibody in eggs of chimeric chickens. Nat Biotechnol 23(9):1159–1169Google Scholar

Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Xiaochuan Tang
    • 1
  • Jun Shi
    • 1
  • Xiaolian Qin
    • 2
  • Ning Xiao
    • 2
  • Rongyang Li
    • 3
  • Hao Hu
    • 1
  • Fengshuo Yang
    • 1
  • Deshun Shi
    • 2
    Email author
  • Xiaoli Wang
    • 1
    Email author
  1. 1.College of Animal Science and TechnologyGuangxi UniversityNanningPeople’s Republic of China
  2. 2.State Key Laboratory for Conservation and Utilization of Subtropical Agro-BioresourcesNanningPeople’s Republic of China
  3. 3.College of Animal Science and TechnologyNanjing Agricultural UniversityNanjingPeople’s Republic of China

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