Detection In Vitro and Quantitative Estimation of Artificial Microterritories Which Promote Osteogenic Differentiation and Maturation of Stromal Stem Cells

  • Igor A. Khlusov
  • Natalya M. Shevtsova
  • Marina Yu. Khlusova
Part of the Methods in Molecular Biology book series (MIMB, volume 1035)


Extracellular matrix can regulate multipotent mesenchymal stromal cells (MMSC) differentiation, with potential applications for tissue engineering. A relief of mineralized bone takes essential effect on MMSC fate. Nevertheless, delicate structure and a hierarchy of niches for stromal stem cells and its quantitative parameters are not practically known. Here, we describe the protocol to define the basic approach providing a guiding for in vitro identification of quantitative features of artificial calcium phosphate niches which promotes osteogenic differentiation and maturation of stromal stem cell.

Key words

Prenatal stromal cells of human lung Calcium phosphate surface Short-term culture Artificial osteogenic niches Alkaline phosphatase Acid phosphatase Osteocalcin Immunocytochemistry Light microscopy Scanning electron microscopy 



The authors are deeply indebted to: professor Yu.P. Sharkeev and E.V. Legostaeva Ph.D. (Institute of Strength Physics and Materials Science, SB of RAS, Tomsk, Russia) for designing and digital imaging of titanium specimens with calcium phosphate coating; K.V. Zaitsev Ph.D. (Stem Cells Bank Ltd., Tomsk, Russia) for cell culture provision; Joint Use Center for Materials Science (Tomsk State University, Tomsk, Russia) for microscopic equipment use.

This work was supported by the Federal Goal Program of Russian Federation (grant No 8036).


  1. 1.
    Schofield R (1978) The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 4:7–25PubMedGoogle Scholar
  2. 2.
    Li L, Xie T (2005) Stem cell niche: structure and function. Annu Rev Cell Dev Biol 21:605–631PubMedCrossRefGoogle Scholar
  3. 3.
    Dellatore SM, Garsia AS, Miller WM (2008) Mimicking stem cell niches to increase stem cell expansion. Curr Opin Biotechnol 19:534–540PubMedCrossRefGoogle Scholar
  4. 4.
    Calvi LM, Adams GB, Weibrect KW et al (2003) Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425:841–846PubMedCrossRefGoogle Scholar
  5. 5.
    Yin T, Li L (2006) The stem cell niches in bone. JCI 116:1195–1201PubMedCrossRefGoogle Scholar
  6. 6.
    Jing D, Fonseca A-V, Alakel N et al (2010) Hematopoietic stem cells in co-culture with mesenchymal stromal cells—modeling the niche compartments in vitro. Haematologica 95:542–550PubMedCrossRefGoogle Scholar
  7. 7.
    Curtis AS, Varde M (1964) Control of cell behavior: topological factors. J Natl Cancer Inst 33:15–26PubMedGoogle Scholar
  8. 8.
    Datta N, Holtorf HL, Sikavitsas VI et al (2005) Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells. Biomaterials 26:971–977PubMedCrossRefGoogle Scholar
  9. 9.
    Kolf CM, Cho E, Tuan RS (2007) Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Res Ther 9:204–219PubMedCrossRefGoogle Scholar
  10. 10.
    Sniadecki NJ, Desai RA, Ruiz SA, Chen CS (2006) Nanotechnology for cell-substrate interactions. Ann Biomed Eng 34:59–74PubMedCrossRefGoogle Scholar
  11. 11.
    Lutolf MP, Gilbert PM, Blau HM (2009) Designing materials to direct stem-cell fate. Nature 462:433–441PubMedCrossRefGoogle Scholar
  12. 12.
    Lutolf MP, Doyonnas R, Havenstrite K et al (2009) Perturbation of single hematopoietic stem cell fates in artificial niches. Integr Biol (Camb) 1:59–69CrossRefGoogle Scholar
  13. 13.
    Khlusov IA, Karlov AV, Sharkeev Yu P et al (2005) Osteogenic potential of mesenchymal stem cells from bone marrow in situ: role of physicochemical properties of artificial surfaces. Bull Exp Biol Med 140:144–152PubMedCrossRefGoogle Scholar
  14. 14.
    Khlusov IA, Khlusova M Yu, Zaitsev KV et al (2011) Pilot in vitro study of the parameters of artificial niche for osteogenic differentiation of human stromal stem cell pool. Bull Exp Biol Med 150:535–542Google Scholar
  15. 15.
    Burtis CA, Ashwood ER (eds) (2001) Tietz fundamentals of clinical chemistry, 5th edn. W.B. Saunders Company, PhiladelphiaGoogle Scholar
  16. 16.
    Sharkeev Yu P, Legostaeva EV, Eroshenko A Yu et al (2009) The structure and physical and mechanical properties of a novel biocomposite material, nanostructured titanium-calcium-phosphate coating. Compos Interfac 16:535–546Google Scholar
  17. 17.
    Aerts F, Wagemaker G (2006) Mesenchymal stem cell engineering and transplantation. In: Nolta JA (ed) Genetic engineering of mesenchymal stem cells. Springer, Dordrecht, pp 1–44CrossRefGoogle Scholar
  18. 18.
    Lama VN, Smith L, Badri L et al (2007) Evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts. JCI 117:989–996PubMedCrossRefGoogle Scholar
  19. 19.
    da Silva Meirelles L, Chagastelles PC, Nardi NB (2006) Mesenchymal stem cells reside in virtually all post-natal organs and tissues. JCS 119:2204–2213Google Scholar
  20. 20.
    de Bruijn JD, van den Brink I, Mendes S et al (1999) Bone induction by implants coated with cultured osteogenic bone marrow cells. Adv Dent Res 13:74–81PubMedCrossRefGoogle Scholar
  21. 21.
    Riggs BL, Melton LJ III (1996) Osteoporosis. Etiology, diagnosis, and management, 2nd edn. Lippincott-Raven, Philadelphia, New YorkGoogle Scholar
  22. 22.
    Scadden DT (2007) The stem cell niche in health and leukemic disease. Best Pract Res Clin Haematol 20:19–27PubMedCrossRefGoogle Scholar
  23. 23.
    Khlusov IA, Dekhtyar Yu, Khlusova M Yu et al (2013) Novel concepts of “Niche-Relief” and “Niche-Voltage” for stem cells as a base of bone and hematopoietic tissues biomimetic engineering. IFMBE Proc 38:99–102. doi: 10.1007/978-3-642-34197-7_26

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Igor A. Khlusov
    • 1
  • Natalya M. Shevtsova
    • 1
  • Marina Yu. Khlusova
    • 1
  1. 1.Scientific Educational Center, Biocompatible Materials and BioengineeringSiberian State Medical UniversityTomskRussia

Personalised recommendations