Skip to main content

Advertisement

SpringerLink
Log in

Epidermal growth factor can optimize a serum-free culture system for bone marrow stem cell proliferation in a miniature pig model

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

Bone marrow-derived mesenchymal stem cells have become an attractive cell source for periodontal ligament regeneration treatment because of their potential to engraft to several tissue types after injury. Most researchers have focused on the transplantation process, but few have paid attention to cell safety concerns and rapid proliferation before transplantation. Using serum-free medium to culture stem cells may be an effective method to avoid problems associated with exogenous serum and the addition of growth factors to promote cell proliferation. Here, we randomly divided our serum-free cultures and treated them with different levels of epidermal growth factor (EGF). We then evaluated changes in rates of cell adhesion, proliferation, apoptosis, and cell cycle ratio as well as their differentiation potential. The data showed that all of these parameters were significantly different when comparing serum-free cultures with and without 10 nM/L EGF (p < 0.05/0.01); however, cells with 10 nM/L EGF did not respond differently than cells grown in standard serum-containing media without EGF (p > 0.05). In summary, our results demonstrate that 10 nM/L EGF was the optimal dose for serum-free culture, which can replace traditional standard serum medium for in vitro expansion of miniature pig bone marrow-derived mesenchymal stem cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

Similar content being viewed by others

References

  • Bosch P.; Pratt S. L.; Stice S. L. Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells. Biol. Reprod. 74: 46–57; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Bose M.; Olivan B.; Teixeira J.; Pi-Sunyer F. X.; Laferrere B. Do incretins play a role in the remission of type 2 diabetes after gastric bypass surgery: what are the evidence? Obes. Surg. 19: 217–229; 2009.

    Article  PubMed  Google Scholar 

  • Cao L.; Yao Y.; Lee V.; Kiani C.; Spaner D.; Lin Z.; Zhang Y.; Adams M. E.; Yang B. B. Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion. J. Cell. Biochem. 77: 569–583; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Chase L. G.; Lakshmipathy U.; Solchaga L. A.; Rao M. S.; Vemuri M. C. A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Res. Ther. 1: 8; 2010.

    Article  PubMed  Google Scholar 

  • Chen H. F.; Kuo H. C.; Chien C. L.; Shun C. T.; Yao Y. L.; Ip P. L.; Chuang C. Y.; Wang C. C.; Yang Y. S.; Ho H. N. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum. Reprod. 22: 567–577; 2007.

    Article  PubMed  Google Scholar 

  • Clabaut A.; Delplace S.; Chauveau C.; Hardouin P.; Broux O. Human osteogenics derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipogenics. Differentiation 80: 40–45; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Conforti E.; Arrigoni E.; Piccoli M.; Lopa S.; de Girolamo L.; Ibatici A.; Di Matteo A.; Tettamanti G.; Brini A. T.; Anastasia L. Reversine increases multipotent human mesenchymal cells differentiation potential. J. Biol. Regul. Homeost. Agents 25: S25–S33; 2011.

    PubMed  CAS  Google Scholar 

  • Coxam V.; Miller M. A.; Bowman M. B.; Miller S. C. Ontogenesis of IGF regulation of longitudinal bone growth in rat metatarsal rudiments cultured in serum-free medium. Arch. Physiol. Biochem. 104: 173–179; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Deasy B. M.; Qu-Peterson Z.; Greenberger J. S.; Huard J. Mechanisms of muscle stem cell expansion with cytokines. Stem Cells 20: 50–60; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Ding G.; Liu Y.; Wang W.; Wei F.; Liu D.; Fan Z.; An Y.; Zhang C.; Wang S. Allogeneic periodontal ligament stem cell therapy for periodontitis in swine. Stem Cells 28: 1829–1838; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Ethier C.; Raymond V. A.; Musallam L.; Houle R.; Bilodeau M. Antiapoptotic effect of EGF on mouse hepatocytes associated with downregulation of proapoptotic Bid protein. Am. J. Physiol. Gastrointest. Liver Physiol. 285: G298–G308; 2003.

    PubMed  CAS  Google Scholar 

  • Hasegawa N.; Kawaguchi H.; Hirachi A.; Takeda K.; Mizuno N.; Nishimura M.; Koike C.; Tsuji K.; Iba H.; Kato Y.; Kurihara H. Behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects. J. Periodontol. 77: 1003–1007; 2006.

    Article  PubMed  Google Scholar 

  • Hofer E. L.; La Russa V.; Honegger A. E.; Bullorsky E. O.; Bordenave R. H.; Chasseing N. A. Alteration on the expression of IL-1, PDGF, TGF-beta, EGF, and FGF receptors and c-Fos and c-Myc proteins in bone marrow mesenchymal stroma cells from advanced untreated lung and breast cancer patients. Stem Cells Dev. 14: 587–594; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Ingram J. L.; Bonner J. C. EGF and PDGF receptor tyrosine kinases as therapeutic targets for chronic lung diseases. Curr. Mol. Med. 6: 409–421; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Iwata K.; Asawa Y.; Nishizawa S.; Mori Y.; Nagata S.; Takato T.; Hoshi K. The development of a serum-free medium utilizing the interaction between growth factors and biomaterials. Biomaterials 33: 444–454; 2012.

    Article  PubMed  CAS  Google Scholar 

  • Kawaguchi H.; Hirachi A.; Hasegawa N.; Iwata T.; Hamaguchi H.; Shiba H.; Takata T.; Kato Y.; Kurihara H. Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells. J. Periodontol. 75: 1281–1287; 2004.

    Article  PubMed  Google Scholar 

  • Kramer P. R.; Nares S.; Kramer S. F.; Grogan D.; Kaiser M. Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro. J. Dent. Res. 83: 27–34; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Krampera M.; Pasini A.; Rigo A.; Scupoli M. T.; Tecchio C.; Malpeli G.; Scarpa A.; Dazzi F.; Pizzolo G.; Vinante F. HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells: inducing cell expansion and reversibly preventing multilineage differentiation. Blood 106: 59–66; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Kratchmarova I.; Blagoev B.; Haack-Sorensen M.; Kassem M.; Mann M. Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science 308: 1472–1477; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Lange C.; Cakiroglu F.; Spiess A. N.; Cappallo-Obermann H.; Dierlamm J.; Zander A. R. Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J. Cell. Physiol. 213: 18–26; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Mandl E. W.; van der Veen S. W.; Verhaar J. A.; van Osch G. J. Serum-free medium supplemented with high-concentration FGF2 for cell expansion culture of human ear chondrocytes promotes redifferentiation capacity. Tissue Eng. 8: 573–580; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Mareddy S.; Dhaliwal N.; Crawford R.; Xiao Y. Stem cell-related gene expression in clonal populations of mesenchymal stromal cells from bone marrow. Tissue Eng. Part A 16: 749–758; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Marei M. K.; Saad M. M.; El-Ashwah A. M.; Ei-Backly R. M.; Al-Khodary M. A. Experimental formation of periodontal structure around titanium implants utilizing bone marrow mesenchymal stem cells: a pilot study. J. Oral Implantol. 35: 106–129; 2009.

    Article  PubMed  Google Scholar 

  • Martino G.; Bacigaluppi M.; Peruzzotti-Jametti L. Therapeutic stem cell plasticity orchestrates tissue plasticity. Brain 134: 1585–1587; 2011.

    Article  PubMed  Google Scholar 

  • McCarty R. C.; Gronthos S.; Zannettino A. C.; Foster B. K.; Xian C. J. Characterisation and developmental potential of ovine bone marrow derived mesenchymal stem cells. J. Cell. Physiol. 219: 324–333; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Meuleman N.; Tondreau T.; Delforge A.; Dejeneffe M.; Massy M.; Libertalis M.; Bron D.; Lagneaux L. Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-MEM medium. Eur. J. Haematol. 76: 309–316; 2006.

    Article  PubMed  Google Scholar 

  • Modino S. A.; Sharpe P. T. Tissue engineering of teeth using adult stem cells. Arch. Oral Biol. 50: 255–258; 2005.

    Article  PubMed  Google Scholar 

  • Pasquinelli G.; Tazzari P.; Ricci F.; Vaselli C.; Buzzi M.; Conte R.; Orrico C.; Foroni L.; Stella A.; Alviano F.; Bagnara G. P.; Lucarelli E. Ultrastructural characteristics of human mesenchymal stromal (stem) cells derived from bone marrow and term placenta. Ultrastruct. Pathol. 31: 23–31; 2007.

    Article  PubMed  Google Scholar 

  • Platt M. O.; Roman A. J.; Wells A.; Lauffenburger D. A.; Griffith L. G. Sustained epidermal growth factor receptor levels and activation by tethered ligand binding enhances osteogenic differentiation of multi-potent marrow stromal cells. J. Cell. Physiol. 221: 306–317; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Pytlik R.; Stehlik D.; Soukup T.; Kalbacova M.; Rypacek F.; Trc T.; Mulinkova K.; Michnova P.; Kideryova L.; Zivny J.; Klener Jr. P.; Vesela R.; Trneny M.; Klener P. The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering. Biomaterials 30: 3415–3427; 2009.

    Article  PubMed  CAS  Google Scholar 

  • Rottmar M.; Hakanson M.; Smith M.; Maniura-Weber K. Stem cell plasticity, osteogenic differentiation and the third dimension. J. Mater. Sci. Mater. Med. 21: 999–1004; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Sadler G. P.; Jones D. L.; Woodhead J. S.; Horgan K.; Wheeler M. H. Effect of growth factors on growth of bovine parathyroid cells in serum-free medium. World J. Surg. 20: 822–828; 1996. discussion 828–829.

    Article  PubMed  CAS  Google Scholar 

  • Scott W. J.; Mann P. Substrate effects on endothelial cell adherence rates. ASAIO Trans. 36: M739–M741; 1990.

    PubMed  CAS  Google Scholar 

  • Sonoyama W.; Liu Y.; Fang D.; Yamaza T.; Seo B. M.; Zhang C.; Liu H.; Gronthos S.; Wang C. Y.; Wang S.; Shi S. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One 1: e79; 2006.

    Article  PubMed  Google Scholar 

  • Tamama K.; Fan V. H.; Griffith L. G.; Blair H. C.; Wells A. Epidermal growth factor as a candidate for ex vivo expansion of bone marrow-derived mesenchymal stem cells. Stem Cells 24: 686–695; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Tarle S. A.; Shi S.; Kaigler D. Development of a serum-free system to expand dental-derived stem cells: PDLSCs and SHEDs. J. Cell. Physiol. 226: 66–73; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Wang C. Y.; Chen L. L.; Kuo P. Y.; Chang J. L.; Wang Y. J.; Hung S. C. Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements. Apoptosis 15: 439–449; 2010.

    Article  PubMed  Google Scholar 

  • Wei N.; Gong P.; Liao D.; Yang X.; Li X.; Liu Y.; Yuan Q.; Tan Z. Auto-transplanted mesenchymal stromal cell fate in periodontal tissue of beagle dogs. Cytotherapy 12: 514–521; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Wu W.; Zhou Y.; Tan W. In vitro culture of bone marrow-derived mesenchymal stem cells in a chemically-defined serum-free medium. Sheng Wu Gong Cheng Xue Bao 25: 121–128; 2009.

    PubMed  CAS  Google Scholar 

  • Xiang M.; Wang J.; Kaplan E.; Oettgen P.; Lipsitz L.; Morgan J. P.; Min J. Y. Antiapoptotic effect of implanted embryonic stem cell-derived early-differentiated cells in aging rats after myocardial infarction. J. Gerontol. A Biol. Sci. Med. Sci. 61: 1219–1227; 2006.

    Article  PubMed  Google Scholar 

  • Xu S.; De Becker A.; Van Camp B.; Vanderkerken K.; Van Riet I. An improved harvest and in vitro expansion protocol for murine bone marrow-derived mesenchymal stem cells. J. Biomed. Biotechnol. 2010: 105940; 2010.

    PubMed  Google Scholar 

  • Yang Y.; Rossi F. M.; Putnins E. E. Periodontal regeneration using engineered bone marrow mesenchymal stromal cells. Biomaterials 31: 8574–8582; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Zhao Q.; Gong P.; Tan Z.; Yang X. Differentiation control of transplanted mesenchymal stem cells (MSCs): a new possible strategy to promote periodontal regeneration. Med. Hypotheses 70: 944–947; 2008.

    Article  PubMed  Google Scholar 

  • Zhu H.; Miosge N.; Schulz J.; Schliephake H. Regulation of multilineage gene expression and apoptosis during in vitro expansion of human bone marrow stromal cells with different cell culture media. Cells Tissues Organs 192: 211–220; 2010.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful to Yan Ma and Xiaojuan Bi for guidance in stem cell culture and Shutao Zheng and Ousheng Liu for molecular biology assistance. This work was funded by Youth Fund of Natural Science Foundation of Xinjiang Uygur Autonomous Region, nos.: 2013211B58.

Competing interests

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South University, No. 110 Xiangya Road, Changsha, Hunan, 410078, China

    Xuan Wang, Ousheng Liu, Yuehong Wang & Zhangui Tang

  2. Department of Pediatric Dentistry and Oral Prevention and Health Care, 1st Affiliated Hospital Xinjiang Medical University, No. 1, Liyushan South Road, Urumqi, Xinjiang Uygur Autonomous Region, 830011, China

    Xuan Wang & Yishan Liu

  3. Department of Cardiovascular Surgery, 1st Affiliated Hospital of Xinjiang Medical University, No. 1, Liyushan South Road, Urumqi, 830011, Xinjiang Uygur Autonomous Region, China

    Feng Zheng

  4. Clinical Medical Research Institute, State Key Lab Incubation Base of Xinjiang Major Diseases Research, First Affiliated Hospital of Xinjiang Medical University, No. 1, Liyushan South Road, Urumqi, 830011, Xinjiang Uygur Autonomous Region, China

    Shutao Zheng

  5. Department of Dental, Affiliated Hospital of Hangzhou Normal University, No. 126, Wenzhou Road, Gong-shu District, Hangzhou, Zhejiang, 310011, China

    Liangjun Zhong

Authors
  1. Xuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ousheng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shutao Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yishan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuehong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhangui Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Liangjun Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Zhangui Tang or Liangjun Zhong.

Additional information

Editor: T. Okamoto

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, X., Zheng, F., Liu, O. et al. Epidermal growth factor can optimize a serum-free culture system for bone marrow stem cell proliferation in a miniature pig model. In Vitro Cell.Dev.Biol.-Animal 49, 815–825 (2013). https://doi.org/10.1007/s11626-013-9665-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-013-9665-6

Keyword

Search

Navigation