Cell and Tissue Research

, Volume 364, Issue 2, pp 429–441 | Cite as

bFGF signaling-mediated reprogramming of porcine primordial germ cells

  • Yu Zhang
  • Jing Ma
  • Hai Li
  • Jiawei Lv
  • Renyue Wei
  • Yimei Cong
  • Zhonghua LiuEmail author
Regular Article


Primordial germ cells (PGCs) have the ability to be reprogrammed into embryonic germ cells (EGCs) in vitro and are an alternative source of embryonic stem cells. Other than for the mouse, the systematic characterization of mammalian PGCs is still lacking, especially the process by which PGCs convert to pluripotency. This hampers the understanding of germ cell development and the derivation of authenticated EGCs from other species. We observed the morphological development of the genital ridge from Bama miniature pigs and found primary sexual differentiation in the E28 porcine embryo, coinciding with Blimp1 nuclear exclusion in PGCs. To explore molecular events involved in porcine PGC reprogramming, transcriptome data of porcine EGCs and fetal fibroblasts (FFs) were assembled and 1169 differentially expressed genes were used for Gene Ontology analysis. These genes were significantly enriched in cell-surface receptor-linked signal transduction, in agreement with the activation of LIF/Stat3 signaling and FGF signaling during the derivation of porcine EG-like cells. Using a growth-factor-defined culture system, we explored the effects of bFGF on the process and found that bFGF not only functioned at the very beginning of PGC dedifferentiation by impeding Blimp1 nuclear expression via a PI3K/AKT-dependent pathway but also maintained the viability of cultured PGCs thereafter. These results provide further insights into the development of germ cells from livestock and the mechanism of porcine PGC reprogramming.


Primordial germ cells Embryonic germ cells Differentiation bFGF Bama miniature pig 



We thank Dr. Yu Gao from the University of Wisconsin (Madison) for his valuable discussion during the manuscript preparation.

Supplementary material

441_2015_2326_Fig8_ESM.gif (154 kb)
Fig S1

Expression of Blimp1 in gonadal PGCs from male embryos. Genital ridge sections were stained with anti-Stella antibody (red) and anti-Blimp1 antibody (green) at the indicated embryonic stage. Cell nuclei were highlighted with Hoechst 33342 (blue). Bar 50 μm. (GIF 153 kb)

441_2015_2326_MOESM1_ESM.tif (4.5 mb)
High resolution image (TIF 4648 kb)
441_2015_2326_Fig9_ESM.gif (7 kb)
Fig S2

RPKM of mouse ESC-enriched target genes shared by Oct4, Sox2, Klf4 and c-Myc in porcine EGCs and porcine FFs. Red line indicates fold change > 3. (GIF 7 kb)

441_2015_2326_MOESM2_ESM.tif (235 kb)
High resolution image (TIF 234 kb)
441_2015_2326_Fig10_ESM.gif (26 kb)
Fig S3

Gene ontology analysis of the 788 putative target genes of Oct4, Sox2, Klf4 and c-Myc with P-value < 0.05 (GIF 26 kb)

441_2015_2326_MOESM3_ESM.tif (1.1 mb)
High resolution image (TIF 1121 kb)
441_2015_2326_Fig11_ESM.gif (19 kb)
Fig S4

Effects of various combinations of growth factors on formation of EG-like colonies. a-c After 5 days of cultivation, AP staining was performed to identify AP-positive colonies of each group. a LIF/SCF/bFGF. b LIF/SCF. c Control. (GIF 18 kb)

441_2015_2326_MOESM4_ESM.tif (915 kb)
High resolution image (TIF 915 kb)
441_2015_2326_Fig12_ESM.gif (27 kb)
Fig S5

Effects of various combinations of growth factors and inhibitors on formation of EG-like colonies. a-e After 5 days of cultivation as indicated top in Fig. 7c, AP staining was performed to identify AP-positive colonies of each group. a + bFGF. b -bFGF. c -bFGF/+FGFRi. d + bFGF/+LY294002. e + bFGF/+PD0325901. (GIF 26 kb)

441_2015_2326_MOESM5_ESM.tif (1.3 mb)
High resolution image (TIF 1320 kb)
441_2015_2326_MOESM6_ESM.pdf (124 kb)
ESM S1 (PDF 123 kb)
441_2015_2326_MOESM7_ESM.pdf (274 kb)
ESM S2 (PDF 273 kb)
441_2015_2326_MOESM8_ESM.pdf (173 kb)
ESM S3 (PDF 173 kb)
441_2015_2326_MOESM9_ESM.pdf (179 kb)
ESM S4 (PDF 178 kb)
441_2015_2326_MOESM10_ESM.pdf (342 kb)
ESM S5 (PDF 342 kb)


  1. Ancelin K, Lange UC, Hajkova P, Schneider R, Bannister AJ, Kouzarides T, Surani MA (2006) Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells. Nat Cell Biol 8:623–630CrossRefPubMedGoogle Scholar
  2. Brevini T, Pennarossa G, Maffei S, Gandolfi F (2012) Pluripotency network in porcine embryos and derived cell lines. Reprod Domest Anim S4:86–91CrossRefGoogle Scholar
  3. Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH (2008) Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133:1106–1117CrossRefPubMedGoogle Scholar
  4. Cheng L, Xiao L (2009) Pig induced pluripotent stem cells: a new resource for generating genetically modified pigs. Regen Med 4:787–789CrossRefPubMedGoogle Scholar
  5. Choi JW, Kim S, Kim TM, Kim YM, Seo HW, Park TS, Jeong JW, Song G, Han JY (2010) Basic fibroblast growth factor activates MEK/ERK cell signaling pathway and stimulates the proliferation of chicken primordial germ cells. PLoS One 5:e12968CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cong Y, Ma J, Sun R, Wang J, Xue B, Wang J, Xie B, Wang J, Hu K, Liu Z (2013) Derivation of putative porcine embryonic germ cells and analysis of their multi-lineage differentiation potential. J Genet Genomics 40:453–464CrossRefPubMedGoogle Scholar
  7. Durcova-Hills G, Prelle K, Müller S, Stojkovic M, Motlik J, Wolf E, Brem G (1998) Primary culture of porcine PGCs requires LIF and porcine membrane-bound stem cell factor. Zygote 6:271–275CrossRefPubMedGoogle Scholar
  8. Durcova-Hills G, Adams IR, Barton SC, Surani MA, McLaren A (2006) The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem Cells 24:1441–1449CrossRefPubMedGoogle Scholar
  9. Durcova-Hills G, Tang F, Doody G, Tooze R, Surani MA (2008) Reprogramming primordial germ cells into pluripotent stem cells. PLoS One 3:e3531CrossRefPubMedPubMedCentralGoogle Scholar
  10. Esteban MA, Xu J, Yang J, Peng M, Qin D, Li W, Jiang Z, Chen J, Deng K, Zhong M, Cai J, Lai L, Pei D (2009) Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem 284:17634–17640CrossRefPubMedPubMedCentralGoogle Scholar
  11. Hajkova P, Erhardt S, Lane N, Haaf T, El-Maarri O, Reik W, Walter J, Surani MA (2002) Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 117:15–23CrossRefPubMedGoogle Scholar
  12. Hyldig SM, Ostrup O, Vejlsted M, Thomsen PD (2011) Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos. Biol Reprod 84:1087–1093CrossRefPubMedGoogle Scholar
  13. Kim S, Günesdogan U, Zylicz JJ, Hackett JA, Cougot D, Bao S, Lee C, Dietmann S, Allen GE, Sengupta R, Surani MA (2014) PRMT5 protects genomic integrity during global DNA demethylation in primordial germ cells and preimplantation embryos. Mol Cell 56:564–579CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kimura T, Nakano T (2011) Induction of pluripotency in primordial germ cells. Histol Histopathol 26:643–650PubMedGoogle Scholar
  15. Kimura T, Tomooka M, Yamano N, Murayama K, Matoba S, Umehara H, Kanai Y, Nakano T (2008) AKT signaling promotes derivation of embryonic germ cells from primordial germ cells. Development 135:869–879CrossRefPubMedGoogle Scholar
  16. Kuijk EW, Colenbrander B, Roelen BA (2009) The effects of growth factors on in vitro-cultured porcine testicular cells. Reproduction 138:721–731CrossRefPubMedGoogle Scholar
  17. Lee CK, Piedrahita JA (2000) Effects of growth factors and feeder cells on porcine primordial germ cells in vitro. Cloning 2:197–205CrossRefPubMedGoogle Scholar
  18. Leitch HG, Nichols J, Humphreys P, Mulas C, Martello G, Lee C, Jones K, Surani MA, Smith A (2013) Rebuilding pluripotency from primordial germ cells. Stem Cell Rep 1:66–78CrossRefGoogle Scholar
  19. Lin IY, Chiu FL, Yeang CH, Chen HF, Chuang CY, Yang SY, Hou PS, Sintupisut N, Ho HN, Kuo HC, Lin KI (2014) Suppression of the SOX2 neural effector gene by PRDM1 promotes human germ cell fate in embryonic stem cells. Stem Cell Rep 2:189–204CrossRefGoogle Scholar
  20. Linher K, Cheung Q, Baker P, Bedecarrats G, Shiota K, Li J (2009) An epigenetic mechanism regulates germ cell-specific expression of the porcine deleted in azoospermia-like (DAZL) gene. Differentiation 77:335–349CrossRefPubMedGoogle Scholar
  21. Matsui Y, Zsebo K, Hogan B (1992) Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70:841–847CrossRefPubMedGoogle Scholar
  22. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, Lee W, Mendenhall E, O’Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448:553–560CrossRefPubMedPubMedCentralGoogle Scholar
  23. Mueller S, Prelle K, Rieger N, Petznek H, Lassnig C, Luksch U, Aigner B, Baetscher M, Wolf E, Mueller M, Brem G (1999) Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells. Mol Reprod Dev 54:244–254CrossRefPubMedGoogle Scholar
  24. Nagamatsu G, Kosaka T, Saito S, Honda H, Takubo K, Kinoshita T, Akiyama H, Sudo T, Horimoto K, Oya M, Suda T (2013) Induction of pluripotent stem cells from primordial germ cells by single reprogramming factors. Stem Cells 31:479–487CrossRefPubMedGoogle Scholar
  25. Perrett RM, Turnpenny L, Eckert JJ, O’Shea M, Sonne SB, Cameron IT, Wilson DI, Rajpert-De Meyts E, Hanley NA (2008) The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture. Biol Reprod 78:852–858CrossRefPubMedGoogle Scholar
  26. Petkov SG, Reh WA, Anderson GB (2009) Methylation changes in porcine primordial germ cells. Mol Reprod Dev 76:22–30CrossRefPubMedGoogle Scholar
  27. Petkov SG, Marks H, Klein T, Garcia RS, Gao Y, Stunnenberg H, Hyttel P (2011) In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and Yucatan mini pig embryos at days 20-24 of gestation. Stem Cell Res 6:226–237CrossRefPubMedGoogle Scholar
  28. Piedrahita JA, Anderson GB, Bondurant RH (1990) Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines. Theriogenology 34:865–877CrossRefPubMedGoogle Scholar
  29. Piedrahita JA, Moore K, Oetama B, Lee CK, Scales N, Ramsoondar J, Bazer FW, Ott T (1998) Generation of transgenic porcine chimeras using primordial germ cell-derived colonies. Biol Reprod 58:1321–1329CrossRefPubMedGoogle Scholar
  30. Resnick J, Bixler L, Cheng L, Donovan P (1992) Long-term proliferation of mouse primordial germ cells in culture. Nature 359:550–551CrossRefPubMedGoogle Scholar
  31. Rodríguez A, Allegrucci C, Alberio R (2012) Modulation of pluripotency in the porcine embryo and iPS cells. PLoS One 7:e49079CrossRefPubMedPubMedCentralGoogle Scholar
  32. Sette C, Dolci S, Geremia R, Rossi P (2000) The role of stem cell factor and of alternative c-kit gene products in the establishment, maintenance and function of germ cells. Int J Dev Biol 44:599–608PubMedGoogle Scholar
  33. Shim H, Anderson GB (1998) In vitro survival and proliferation of porcine primordial germ cells. Theriogenology 49:521–528CrossRefPubMedGoogle Scholar
  34. Soufi A, Donahue G, Zaret KS (2012) Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell 151:994–1004CrossRefPubMedPubMedCentralGoogle Scholar
  35. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676CrossRefPubMedGoogle Scholar
  36. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872CrossRefPubMedGoogle Scholar
  37. Tsung HC, Du ZW, Rui R, Li XL, Bao LP, Wu J, Bao SM, Yao Z (2003) The culture and establishment of embryonic germ (EG) cell lines from Chinese mini swine. Cell Res 13:195–202CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Yu Zhang
    • 1
  • Jing Ma
    • 1
  • Hai Li
    • 2
  • Jiawei Lv
    • 1
  • Renyue Wei
    • 1
  • Yimei Cong
    • 1
    • 3
  • Zhonghua Liu
    • 1
    Email author
  1. 1.College of Life ScienceNortheast Agricultural UniversityHarbinPeople’s Republic of China
  2. 2.State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbinPeople’s Republic of China
  3. 3.College of Veterinary MedicineNortheast Agricultural UniversityHarbinPeople’s Republic of China

Personalised recommendations