FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Journal of animal science and biotechnology2013; 4(1); 40; doi: 10.1186/2049-1891-4-40

Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts.

Abstract: The use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA. Results: Relative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P < 0.001) and 5-bromo-2'-deoxyuridine (BrdU) incorporation (167 ± 6 and 120 ± 6%, respectively; P < 0.001). Treatment with DEX increased ALP activity compared with control (1,638 ± 38%; P  0.8). Runt-related transcription factor2 expression increased 3-fold (P < 0.001) by d 6 of culture. Osterix expression increased 9-fold (P < 0.05) by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 (P < 0.01); however expression was reduced 4-fold at d 18 (P < 0.01). Conclusions: Dexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.
Get Full Text
Publication Date: 2013-10-29 PubMed ID: 24169030PubMed Central: PMC3874597DOI: 10.1186/2049-1891-4-40Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study explored the optimal conditions for growing equine bone marrow mesenchymal stem cells (BMSC) and their transformation into osteoblasts, cells that aid bone regeneration. The research compared the use of autologous and commercial serum for cell growth, and the role of crucial transcription factors during the transformation process. The study concluded that dexamethasone catalysed the cells’ transformation into osteoblasts rather than a bone protein named BMP-2, with the expression levels of transcription factors Runx2 and osterix increasing and Tbx3 reducing during the transformation.

The Culture Conditions

  • The researchers used equine BMSCs taken from the sternum of three horses. The cell cultures were treated with either commercially purchased fetal bovine serum (FBS) or autologous horse serum (HS).
  • The proliferation of the cells in the two serum types was compared. Proliferation is the process by which cells divide and multiply.
  • The purpose was to identify which type of serum was better for the growth and differentiation (transformation) of equine BMSCs into osteoblasts.

Osteoblast Transformation Process

  • To initiate osteoblast differentiation, the cells were treated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate. They also received either human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or a combination of both.
  • They measured the Alkaline phosphatase (ALP) activity, a known marker of osteoblast differentiation, using an ELISA test.

Role of Transcription Factors

  • The level of transcription factors was tracked throughout the transformation process to monitor their role in the process. The transcription factors investigated were Runx2, Osterix (Osx), and T-box3 (Tbx3).
  • RNA was extracted from the cells at various stages of the differentiation process to determine the expression levels of these transcription factors.

Findings and Conclusion

  • The researchers found that both FBS and HS increased cell number and BrdU (a thymidine analog that gets incorporated into newly synthesized DNA molecules) incorporation significantly compared to the control.
  • DEX treatment increased ALP activity substantially compared to the control, but BMP-2 did not.
  • Expression of Runx2 increased three-fold by day six, Osx expression increased nine-fold by day 18, and Tbx3 expression initially increased 1.8-fold at day 3 but reduced four-fold by day 18.
  • The study concluded that dexamethasone was necessary for equine BMSC transformation into osteoblasts but not BMP-2.
  • Additionally, the expression of transcription factors Runx2 and osterix increased while Tbx3 decreased during the differentiation process.

Cite This Article

APA
Glynn ER, Londono AS, Zinn SA, Hoagland TA, Govoni KE. (2013). Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts. J Anim Sci Biotechnol, 4(1), 40. https://doi.org/10.1186/2049-1891-4-40

Publication

Journal of animal science and biotechnology
ISSN: 1674-9782
NlmUniqueID: 101581293
Country: England
Language: English
Volume: 4
Issue: 1
Pages: 40

Researcher Affiliations

Glynn, Elizabeth R A
    Londono, Alfredo Sanchez
      Zinn, Steven A
        Hoagland, Thomas A
          Govoni, Kristen E
          • Department of Animal Science, University of Connecticut, 3636 Horsebarn Road Ext,, Unit 4040, Storrs, CT 06269-4040, USA. kristen.govoni@uconn.edu.

          References

          This article includes 29 references
          1. Estberg L, Stover SM, Gardner IA, Johnson BJ, Case JT, Ardans A, Read DH, Anderson ML, Barr BC, Daft BM, Kinde H, Moore J, Stoltz J, Woods LW. Fatal musculoskeletal injuries incurred during racing and training in thoroughbreds.. J Am Vet Med Assoc 1996 Jan 1;208(1):92-6.
            pubmed: 8682713
          2. Lewis JM. New technology could help reduce bone fractures in horses.. DVM360.com July 1, 2008.
          3. Beyer A. Life, savings contribute to difficult calculation.. The Washington Post 2006. Sunday, May 28.
          4. Galuppo L. Equine Fractures: Emergency First Aid and Stabilization Techniques.. CEH The Horse Report pp. 1–4.
          5. Blanchette A. A second chance for Abby.. StarTribune 2008. p.  ..
          6. Perren SM. Fracture healing. The evolution of our understanding.. Acta Chir Orthop Traumatol Cech 2008 Aug;75(4):241-6.
            pubmed: 18760078
          7. Schindeler A, McDonald MM, Bokko P, Little DG. Bone remodeling during fracture repair: The cellular picture.. Semin Cell Dev Biol 2008 Oct;19(5):459-66.
            doi: 10.1016/j.semcdb.2008.07.004pubmed: 18692584google scholar: lookup
          8. Milner PI, Clegg PD, Stewart MC. Stem cell-based therapies for bone repair.. Vet Clin North Am Equine Pract 2011 Aug;27(2):299-314.
            doi: 10.1016/j.cveq.2011.05.002pubmed: 21872760google scholar: lookup
          9. Kraus KH, Kirker-Head C. Mesenchymal stem cells and bone regeneration.. Vet Surg 2006 Apr;35(3):232-42.
            doi: 10.1111/j.1532-950X.2006.00142.xpubmed: 16635002google scholar: lookup
          10. Koch TG, Berg LC, Betts DH. Concepts for the clinical use of stem cells in equine medicine.. Can Vet J 2008 Oct;49(10):1009-17.
            pmc: PMC2553494pubmed: 19119371
          11. Eslaminejad MB, Rouhi L, Arabnajafi M, Baharvand H. Rat marrow-derived mesenchymal stem cells developed in a medium supplemented with the autologous versus bovine serum.. Cell Biol Int 2009 May;33(5):607-16.
            doi: 10.1016/j.cellbi.2009.03.001pubmed: 19286467google scholar: lookup
          12. Dahl JA, Duggal S, Coulston N, Millar D, Melki J, Shahdadfar A, Brinchmann JE, Collas P. Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum.. Int J Dev Biol 2008;52(8):1033-42.
            doi: 10.1387/ijdb.082663jdpubmed: 18956336google scholar: lookup
          13. Toupadakis CA, Wong A, Genetos DC, Cheung WK, Borjesson DL, Ferraro GL, Galuppo LD, Leach JK, Owens SD, Yellowley CE. Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue.. Am J Vet Res 2010 Oct;71(10):1237-45.
            doi: 10.2460/ajvr.71.10.1237pubmed: 20919913google scholar: lookup
          14. Karsenty G. Minireview: transcriptional control of osteoblast differentiation.. Endocrinology 2001 Jul;142(7):2731-3.
            doi: 10.1210/en.142.7.2731pubmed: 11415989google scholar: lookup
          15. Govoni KE, Linares GR, Chen ST, Pourteymoor S, Mohan S. T-box 3 negatively regulates osteoblast differentiation by inhibiting expression of osterix and runx2.. J Cell Biochem 2009 Feb 15;106(3):482-90.
            doi: 10.1002/jcb.22035pmc: PMC2915761pubmed: 19115250google scholar: lookup
          16. Smith RK, Korda M, Blunn GW, Goodship AE. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment.. Equine Vet J 2003 Jan;35(1):99-102.
            pubmed: 12553472doi: 10.2746/042516403775467388google scholar: lookup
          17. D'Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow.. J Bone Miner Res 1999 Jul;14(7):1115-22.
            doi: 10.1359/jbmr.1999.14.7.1115pubmed: 10404011google scholar: lookup
          18. Murray SJ, Santangelo KS, Bertone AL. Evaluation of early cellular influences of bone morphogenetic proteins 12 and 2 on equine superficial digital flexor tenocytes and bone marrow-derived mesenchymal stem cells in vitro.. Am J Vet Res 2010 Jan;71(1):103-14.
            doi: 10.2460/ajvr.71.1.103pmc: PMC4246500pubmed: 20043789google scholar: lookup
          19. Carpenter RS, Goodrich LR, Frisbie DD, Kisiday JD, Carbone B, McIlwraith CW, Centeno CJ, Hidaka C. Osteoblastic differentiation of human and equine adult bone marrow-derived mesenchymal stem cells when BMP-2 or BMP-7 homodimer genetic modification is compared to BMP-2/7 heterodimer genetic modification in the presence and absence of dexamethasone.. J Orthop Res 2010 Oct;28(10):1330-7.
            doi: 10.1002/jor.21126pmc: PMC3200399pubmed: 20309952google scholar: lookup
          20. Govoni KE, Lee SK, Chadwick RB, Yu H, Kasukawa Y, Baylink DJ, Mohan S. Whole genome microarray analysis of growth hormone-induced gene expression in bone: T-box3, a novel transcription factor, regulates osteoblast proliferation.. Am J Physiol Endocrinol Metab 2006 Jul;291(1):E128-36.
            doi: 10.1152/ajpendo.00592.2005pmc: PMC3000614pubmed: 16464905google scholar: lookup
          21. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method.. Nat Protoc 2008;3(6):1101-8.
            doi: 10.1038/nprot.2008.73pubmed: 18546601google scholar: lookup
          22. Vidal MA, Kilroy GE, Johnson JR, Lopez MJ, Moore RM, Gimble JM. Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity.. Vet Surg 2006 Oct;35(7):601-10.
            doi: 10.1111/j.1532-950X.2006.00197.xpubmed: 17026544google scholar: lookup
          23. Arnhold SJ, Goletz I, Klein H, Stumpf G, Beluche LA, Rohde C, Addicks K, Litzke LF. Isolation and characterization of bone marrow-derived equine mesenchymal stem cells.. Am J Vet Res 2007 Oct;68(10):1095-105.
            doi: 10.2460/ajvr.68.10.1095pubmed: 17916017google scholar: lookup
          24. Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.. J Cell Biochem 1997 Feb;64(2):278-94.
            doi: 10.1002/(SICI)1097-4644(199702)64:2<278::AID-JCB11>3.0.CO;2-Fpubmed: 9027588google scholar: lookup
          25. Lissenberg-Thunnissen SN, de Gorter DJ, Sier CF, Schipper IB. Use and efficacy of bone morphogenetic proteins in fracture healing.. Int Orthop 2011 Sep;35(9):1271-80.
            doi: 10.1007/s00264-011-1301-zpmc: PMC3167450pubmed: 21698428google scholar: lookup
          26. Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R, Aro H, Atar D, Bishay M, Börner MG, Chiron P, Choong P, Cinats J, Courtenay B, Feibel R, Geulette B, Gravel C, Haas N, Raschke M, Hammacher E, van der Velde D, Hardy P, Holt M, Josten C, Ketterl RL, Lindeque B, Lob G, Mathevon H, McCoy G, Marsh D, Miller R, Munting E, Oevre S, Nordsletten L, Patel A, Pohl A, Rennie W, Reynders P, Rommens PM, Rondia J, Rossouw WC, Daneel PJ, Ruff S, Rüter A, Santavirta S, Schildhauer TA, Gekle C, Schnettler R, Segal D, Seiler H, Snowdowne RB, Stapert J, Taglang G, Verdonk R, Vogels L, Weckbach A, Wentzensen A, Wisniewski T. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients.. J Bone Joint Surg Am 2002 Dec;84(12):2123-34.
            pubmed: 12473698doi: 10.2106/00004623-200212000-00001google scholar: lookup
          27. Ter Brugge PJ, Jansen JA. In vitro osteogenic differentiation of rat bone marrow cells subcultured with and without dexamethasone.. Tissue Eng 2002 Apr;8(2):321-31.
            doi: 10.1089/107632702753725076pubmed: 12031120google scholar: lookup
          28. Heino TJ, Hentunen TA. Differentiation of osteoblasts and osteocytes from mesenchymal stem cells.. Curr Stem Cell Res Ther 2008 May;3(2):131-45.
            doi: 10.2174/157488808784223032pubmed: 18473879google scholar: lookup
          29. Franceschi RT, Ge C, Xiao G, Roca H, Jiang D. Transcriptional regulation of osteoblasts.. Cells Tissues Organs 2009;189(1-4):144-52.
            doi: 10.1159/000151747pmc: PMC3512205pubmed: 18728356google scholar: lookup

          Citations

          This article has been cited 19 times.
          Subscribe to our newsletter
          Join 99,658 horse owners like you who receive our email updates on horse health and nutrition.