Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 2003 Jan;41(1):23–35. doi: 10.1023/A:1024283521966

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Yan Li 1, Douglas A Kniss 2, Larry C Lasky 3, Shang-Tian Yang 1
PMCID: PMC3449760  PMID: 19002959

Abstract

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.

Keywords: Differentiation, Embryonic stem cell, Perfusion bioreactor, Three-dimensional culture

Full Text

The Full Text of this article is available as a PDF (4.3 MB).

References

  1. Buttery L.D.K., Bourne S., Xynosn J.D., Wood H., Hughes F.J., Hughes S.P.F., et al. Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng. 2001;7:89–99. doi: 10.1089/107632700300003323. [DOI] [PubMed] [Google Scholar]
  2. Cukierman E., Pankov R., Stevens D.R., Yamada K.M. Taking cell-matrix adhesions to the third dimension. Science. 2001;294:1708–1712. doi: 10.1126/science.1064829. [DOI] [PubMed] [Google Scholar]
  3. Dang S.M., Kyba M., Perlingeiro R., Daley G.Q., Zandstra P.W. Efficiency of embryonic body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol Bioeng. 2002;78:442–453. doi: 10.1002/bit.10220. [DOI] [PubMed] [Google Scholar]
  4. Dani C. Embryonic stem cell-derived adipogenesis. Cells Tissues Organs. 1999;165:173–180. doi: 10.1159/000016697. [DOI] [PubMed] [Google Scholar]
  5. Draper J.S., Pigott C., Thomson J.A., Andrews P.W. Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat. 2002;200:249–258. doi: 10.1046/j.1469-7580.2002.00030.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Filippi M.-D., Porteu F., Pesteur F.L., Rameau P., Nogueira M.M., Debili N., et al. Embryonic stem cell differentiation to hematopoietic cells: A model to study the function of various regions of the intracytoplasmic domain of cytokine receptors in vitro. Exp Hematol. 2000;28:1363–1372. doi: 10.1016/s0301-472x(00)00549-x. [DOI] [PubMed] [Google Scholar]
  7. Guan K., Rohwedel J., Wobus A.M. Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro. Cytotechnology. 1999;30:211–226. doi: 10.1023/A:1008041420166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hole N. Embryonic stem cell-derived haematopoiesis. Cells Tissues Organs. 1999;165:181–189. doi: 10.1159/000016698. [DOI] [PubMed] [Google Scholar]
  9. Kaufman D.D., Hanson E.T., Lewis R.L., Auerbach R., Thomson J.A. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Nat Acad Sci USA. 2001;98:10716–10721. doi: 10.1073/pnas.191362598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Keller G., Kennedy M., Papayannopoulou T., Wiles M.V. Hematopoietic commitment during embryonic stem cells differentiation in culture. Mol Cell Biol. 1993;13:473–486. doi: 10.1128/mcb.13.1.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Li Y., Ma T., Kniss D.A., Yang S.T., Lasky L.C. Human cord cell hematopoiesis in three-dimensional nonwoven fibrous matrices: in vitro simulation of marrow environment. J Hematother Stem Cell Res. 2001;10:355–368. doi: 10.1089/152581601750288966. [DOI] [PubMed] [Google Scholar]
  12. Li Y., Ma T., Yang S.T., Kniss D.A. Thermal compression and characterization of three-dimensional nonwoven PET matrices as tissue engineering scaffolds. Biomaterials. 2001;22:609–618. doi: 10.1016/s0142-9612(00)00224-6. [DOI] [PubMed] [Google Scholar]
  13. Ling V., Neben S. In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies. J Cell Physiol. 1997;171:104–115. doi: 10.1002/(SICI)1097-4652(199704)171:1<104::AID-JCP12>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  14. Lumelsky N., Blondel O., Laeng P., Velasco I., Ravin R., Mckay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science. 2001;292:1389–1394. doi: 10.1126/science.1058866. [DOI] [PubMed] [Google Scholar]
  15. Ma T., Yang S.T., Kniss D.A. Development of an in vitro human placenta model by cultivation of human trophoblasts in a fiber-based bioreactor system. Tissue Eng. 1999;5:91–102. doi: 10.1089/ten.1999.5.91. [DOI] [PubMed] [Google Scholar]
  16. Ma T., Li Y., Yang S.T., Kniss D.A. Effects of pore size in 3-D PET fibrous matrix on human trophoblast tissue development. Biotechnol Bioeng. 2000;70:606–618. doi: 10.1002/1097-0290(20001220)70:6<606::aid-bit2>3.0.co;2-h. [DOI] [PubMed] [Google Scholar]
  17. McAdams T.A., Miller W.M., Papoutsakis E.T. Hematopoietic cell culture therapies (part I): cell culture considerations. Trends Biotechnol. 1996;14:341–349. doi: 10.1016/0167-7799(96)10047-0. [DOI] [PubMed] [Google Scholar]
  18. Manuilova E.S., Gordeeva O.F., Grivennikov I.A., Ozernyuk N.D. Embryonic stem cells: spontaneous and directed differentiation. Biol Bull. 2001;28:595–600. [PubMed] [Google Scholar]
  19. Martin I., Shastri V.P., Padera R.F., Yang J., Mackay A.J., Langer R., et al. Selective differentiation of mammalian bone marrow stromal cells cultured on three-dimensional polymer foams. J Biomed Mater Res. 2001;55:229–235. doi: 10.1002/1097-4636(200105)55:2<229::aid-jbm1009>3.0.co;2-q. [DOI] [PubMed] [Google Scholar]
  20. Nakano T. In vitro development of hematopoietic system from mouse embryonic stem cells: a new approach for embryonic hematopoiesis. Int J Hematol. 1996;65:1–8. doi: 10.1016/s0925-5710(96)00531-2. [DOI] [PubMed] [Google Scholar]
  21. Pardo B., Honegger P. Differentiation of rat striatal embryonic stem cells in vitro: monolayer culture vs. three-dimensional coculture with differentiated brain cells. J Neurosci Res. 2000;59:504–512. doi: 10.1002/(SICI)1097-4547(20000215)59:4<504::AID-JNR5>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  22. Pesce M., Anastassiadis K. and Scholer H.R. 1999. Oct-4: Lessons of totipotency from embryonic stem cells. Cells Tissues Organs 165: 144–152. [DOI] [PubMed]
  23. Robertson E., Bradley A., Kuehn M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature. 1986;323:445–448. doi: 10.1038/323445a0. [DOI] [PubMed] [Google Scholar]
  24. Schuldiner M., Yanuka O., Itskovitz-Eldor J., Melton D.A., Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Nat Acad Sci USA. 2000;97:11307–11312. doi: 10.1073/pnas.97.21.11307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shamblott M.J., Axelman J., Littlefield J.W., Blumenthal P.D., Huggins G.R., Cui Y., et al. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Nat Acad Sci USA. 2001;98:113–118. doi: 10.1073/pnas.021537998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith A.G. Embryo-derived stem cells: of mice and men. Ann Review Cell Dev Biol. 2001;17:435–462. doi: 10.1146/annurev.cellbio.17.1.435. [DOI] [PubMed] [Google Scholar]
  27. Thomson J.A., Itskovitz-Eldor J., Shapiro S.S., Waknitz M.A., Swiergiel J.J., Marshall V.S., et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. doi: 10.1126/science.282.5391.1145. [DOI] [PubMed] [Google Scholar]
  28. Thomson J.A., Odorico J.S. Human embryonic stem cell and embryonic germ cell lines. Trends Biotechnol. 2000;18:53–57. doi: 10.1016/s0167-7799(99)01410-9. [DOI] [PubMed] [Google Scholar]
  29. Wartenberg M., Gunther J., Hescheler J., Sauer H. The embryoid body as a novel in vitro assay system for antian giogenic agents. Lab Invest. 1998;78:1301–1314. [PubMed] [Google Scholar]
  30. Wiles M.V., Johansson B.M. Embryonic stem cell development in a chemically defined medium. Exp Cell Res. 1999;247:241–248. doi: 10.1006/excr.1998.4353. [DOI] [PubMed] [Google Scholar]
  31. Wobus A.M. Potential of embryonic stem cells. Mol Aspects Med. 2001;22:149–164. doi: 10.1016/s0098-2997(01)00006-1. [DOI] [PubMed] [Google Scholar]
  32. Xu C., Inokuma M.S., Denham J., Golds K., Kundu P., Gold J.D., et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001;19:971–974. doi: 10.1038/nbt1001-971. [DOI] [PubMed] [Google Scholar]
  33. Zandstra P.W., Nagy A. Stem cell bioengineering. Ann Rev Biomed Eng. 2001;3:275–305. doi: 10.1146/annurev.bioeng.3.1.275. [DOI] [PubMed] [Google Scholar]
  34. Zandstra P.W., Le H.-V., Daley G.Q., Griffith L.G., Lauffenburger D.A. Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. Biotechnol Bioeng. 2000;69:607–617. [PubMed] [Google Scholar]
  35. Zuk P.A., Zhu M., Mizuno H., Huang J., Futrell J.W., Katz A.J., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211–228. doi: 10.1089/107632701300062859. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES