Skip to main content
Log in

Three-dimensional biomimetic scaffolds for hepatic differentiation of size-controlled embryoid bodies

  • Article
  • Published:
Journal of Materials Research Aims and scope Submit manuscript

Abstract

Three-dimensional (3D) biomimetic scaffolds are critical for tissue engineering to support stem cell culture and organoid formation. Embryonic stem (ES) cells hold promising potential for tissue regeneration and ES cell-derived specific lineages are expected to be strongly influenced by the size of embryoid bodies (EBs). However, the fundamental knowledge needed to achieve the goal of highly reproducible, efficient, and scalable differentiation of how EB size affects differentiation is missing. Here, we used 3D biomimetic scaffolds with highly uniform porous structure to regulate size of EBs and differentiated them toward hepatic fate. The results showed EBs formed within the scaffolds were precisely controlled by pore sizes of the scaffolds. We found that EBs equals to or larger than 180 ± 27 µm maintained the ability to differentiate to hepatic lineage. The 3D biomimetic scaffold provides the effective tools toward accurate regulation of EB sizes for tissue engineering.

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

Access this article

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

Instant access to the full article PDF.

Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Figure 6:
Figure 7:

Similar content being viewed by others

References

  1. C.E. Murry and G. Keller: Differentiation of embryonic stem cells to clinically relevant populations: Lessons from embryonic development. Cell 132, 661–680 (2008).

    Article  CAS  Google Scholar 

  2. A.M. Wobus and K.R. Boheler: Embryonic stem cells: Prospects for developmental biology and cell therapy. Physiol. Rev. 85, 635–678 (2005).

    Article  CAS  Google Scholar 

  3. P.W. Burridge, D. Anderson, H. Priddle, M.D. Barbadillo Muñoz, S. Chamberlain, C. Allegrucci, L.E. Young, and C. Denning: Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells 25, 929–938 (2007).

    Article  CAS  Google Scholar 

  4. J.M. Messana, N.S. Hwang, J. Coburn, J.H. Elisseeff, and Z. Zhang: Size of the embryoid body influences chondrogenesis of mouse embryonic stem cells. J. Tissue Eng. Regener. Med. 2, 499–506 (2008).

    Article  CAS  Google Scholar 

  5. E.S. Ng, R. Davis, E.G. Stanley, and A.G. Elefanty: A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat. Protoc. 3, 768–776 (2008).

    Article  CAS  Google Scholar 

  6. C-C. Huang, C-K. Liao, M-J. Yang, C-H. Chen, S-M. Hwang, Y-W. Hung, Y. Chang, and H-W. Sung: A strategy for fabrication of a three-dimensional tissue construct containing uniformly distributed embryoid body-derived cells as a cardiac patch. Biomaterials 31, 6218–6227 (2010).

    Article  CAS  Google Scholar 

  7. E.S. Ng, R.P. Davis, L. Azzola, E.G. Stanley, and A.G. Elefanty: Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 106, 1601–1603 (2005).

    Article  CAS  Google Scholar 

  8. S-H. Moon, J. Ju, S-J. Park, D. Bae, H-M. Chung, and S-H. Lee: Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies. Biomaterials 35, 5987–5997 (2014).

    Article  CAS  Google Scholar 

  9. J.M. Cha, H. Bae, N. Sadr, S. Manoucheri, F. Edalat, K. Kim, S.B. Kim, I.K. Kwon, Y-S. Hwang, and A. Khademhosseini: Embryoid body size-mediated differential endodermal and mesodermal differentiation using polyethylene glycol (PEG) microwell array. Macromol. Res. 23, 245–255 (2015).

    Article  CAS  Google Scholar 

  10. D. Sasaki, T. Shimizu, S. Masuda, J. Kobayashi, K. Itoga, Y. Tsuda, J.K. Yamashita, M. Yamato, and T. Okano: Mass preparation of size-controlled mouse embryonic stem cell aggregates and induction of cardiac differentiation by cell patterning method. Biomaterials 30, 4384–4389 (2009).

    Article  CAS  Google Scholar 

  11. A.D. Dias, A.M. Unser, Y. Xie, D.B. Chrisey, and D.T. Corr: Generating size-controlled embryoid bodies using laser direct-write. Biofabrication 6, 025007 (2014).

    Article  CAS  Google Scholar 

  12. J.C. Mohr, J. Zhang, S.M. Azarin, A.G. Soerens, J.J. de Pablo, J.A. Thomson, G.E. Lyons, S.P. Palecek, and T.J. Kamp: The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells. Biomaterials 31, 1885–1893 (2010).

    Article  CAS  Google Scholar 

  13. Y.Y. Choi, B.G. Chung, D.H. Lee, A. Khademhosseini, J-H. Kim, and S-H. Lee: Controlled-size embryoid body formation in concave microwell arrays. Biomaterials 31, 4296–4303 (2010).

    Article  CAS  Google Scholar 

  14. Y. Sakai, Y. Yoshiura, and K. Nakazawa: Embryoid body culture of mouse embryonic stem cells using microwell and micropatterned chips. J. Biosci. Bioeng. 111, 85–91 (2011).

    Article  CAS  Google Scholar 

  15. S. Vijayavenkataraman, S. Zhang, W.F. Lu, and J.Y.H. Fuh: Electrohydrodynamic-jetting (EHD-Jet) 3D-printed functionally graded scaffolds for tissue engineering applications. J. Mater. Res. 33, 1999–2011 (2018).

    Article  CAS  Google Scholar 

  16. A. Bruyas, F. Lou, A.M. Stahl, M. Gardner, W. Maloney, S. Goodman, and Y.P. Yang: Systematic characterization of 3D-printed PCL/β-TCP scaffolds for biomedical devices and bone tissue engineering: Influence of composition and porosity. J. Mater. Res. 33, 1948–1959 (2018).

    Article  CAS  Google Scholar 

  17. Y-S. Hwang, B.G. Chung, D. Ortmann, N. Hattori, H-C. Moeller, and A. Khademhosseini: Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11. Proc. Natl. Acad. Sci. U. S. A. 106, 16978–16983 (2009).

    Article  CAS  Google Scholar 

  18. G.K. Michalopoulos: Liver regeneration: Alternative epithelial pathways. Int. J. Biochem. Cell Biol. 43, 173–179 (2011).

    Article  CAS  Google Scholar 

  19. Y. Wang, J.H. Bahng, Q. Che, J. Han, and N.A. Kotov: Anomalously fast diffusion of targeted carbon nanotubes in cellular spheroids. ACS Nano 9, 8231–8238 (2015).

    Article  CAS  Google Scholar 

  20. J. Lee, M.J. Cuddihy, G.M. Cater, and N.A. Kotov: Engineering liver tissue spheroids with inverted colloidal crystal scaffolds. Biomaterials 30, 4687–4694 (2009).

    Article  CAS  Google Scholar 

  21. R.A. Carpenter, J-G. Kwak, S.R. Peyton, and J. Lee: Implantable pre-metastatic niches for the study of the microenvironmental regulation of disseminated human tumour cells. Nat. Biomed. Eng. 2, 915–929 (2018).

    Article  CAS  Google Scholar 

  22. L.G. Griffith and M.A. Swartz: Capturing complex 3D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 7, 211–224 (2006).

    Article  CAS  Google Scholar 

  23. A.R.C. Duarte, J.F. Mano, and R.L. Reis: Thermosensitive polymeric matrices for three-dimensional cell culture strategies. Acta Biomater. 7, 526–529 (2011).

    Article  CAS  Google Scholar 

  24. V.N. Goral, Y-C. Hsieh, O.N. Petzold, J.S. Clark, P.K. Yuen, and R.A. Faris: Perfusion-based microfluidic device for three-dimensional dynamic primary human hepatocyte cell culture in the absence of biological or synthetic matrices or coagulants. Lab Chip 10, 3380 (2010).

    Article  CAS  Google Scholar 

  25. Y. Wang, E. Jan, M. Cuddihy, J.H. Bahng, and N. Kotov: Layered biomimetic nanocomposites replicate bone surface in three-dimensional cell cultures. Nanocomposites, 4, 155–156 (2019).

    Google Scholar 

  26. P. Podsiadlo, A.K. Kaushik, B.S. Shim, A. Agarwal, Z. Tang, A.M. Waas, E.M. Arruda, and N.A. Kotov: Can nature’s design Be improved upon? High strength, transparent nacre-like nanocomposites with double network of sacrificial cross links. J. Phys. Chem. B 112, 14359–14363 (2008).

    Article  CAS  Google Scholar 

  27. E. Fineout-Overholt, R.F. Levin, and B.M. Melnyk: Strategies for advancing evidence-based practice in clinical settings. J. N. Y. State Nurses. Assoc. 35, 28–32 (2004).

    Google Scholar 

  28. D. Solter and B.B. Knowles: Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc. Natl. Acad. Sci. U. S. A. 75, 5565–5569 (1978).

    Article  CAS  Google Scholar 

  29. R. Matoba, H. Niwa, S. Masui, S. Ohtsuka, M.G. Carter, A.A. Sharov, and M.S.H. Ko: Dissecting Oct3/4-regulated gene networks in embryonic stem cells by expression profiling. PLoS One 1, e26 (2006).

    Article  Google Scholar 

  30. S. Vassilieva, K. Guan, U. Pich, and A.M. Wobus: Establishment of SSEA-1- and Oct-4-expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth. Exp. Cell Res. 258, 361–373 (2000).

    Article  CAS  Google Scholar 

  31. E. Marani, J.W. van Oers, P.A. Tetteroo, R.E. Poelmann, J. van der Veeken, and M.G. Deenen: Stage specific embryonic carbohydrate surface antigens of primordial germ cells in mouse embryos: FAL (S.S.E.A.-1) and globoside (S.S.E.A.-3). Acta Morphol. Neerl.-Scand. 24, 103–110 (1986).

    CAS  Google Scholar 

  32. H. Niwa, J. Miyazaki, and A.G. Smith: Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

    Article  CAS  Google Scholar 

  33. R.E. Schwartz, J.L. Linehan, M.S. Painschab, W-S. Hu, C.M. Verfaillie, and D.S. Kaufman: Defined conditions for development of functional hepatic cells from human embryonic stem cells. Stem Cells Dev. 14, 643–655 (2005).

    Article  CAS  Google Scholar 

  34. C.D. Capo-chichi, M.E. Rula, J.L. Smedberg, L. Vanderveer, M.S. Parmacek, E.E. Morrisey, A.K. Godwin, and X-X. Xu: Perception of differentiation cues by GATA factors in primitive endoderm lineage determination of mouse embryonic stem cells. Dev. Biol. 286, 574–586 (2005).

    Article  CAS  Google Scholar 

  35. C. Lange, P. Bassler, M.V. Lioznov, H. Bruns, D. Kluth, A.R. Zander, and H.C. Fiegel: Hepatocytic gene expression in cultured rat mesenchymal stem cells. Transplant. Proc. 37, 276–279 (2005).

    Article  CAS  Google Scholar 

  36. A. Ben-Ze’ev, G.S. Robinson, N.L. Bucher, and S.R. Farmer: Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. Proc. Natl. Acad. Sci. U. S. A. 85, 2161–2165 (1988).

    Article  Google Scholar 

  37. S. Zhu, M. Rezvani, J. Harbell, A.N. Mattis, A.R. Wolfe, L.Z. Benet, H. Willenbring, and S. Ding: Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93–97 (2014).

    Article  CAS  Google Scholar 

  38. Y-C. Lee, C-J. Chang, D. Bali, Y-T. Chen, and Y-T. Yan: Glycogen-branching enzyme deficiency leads to abnormal cardiac development: Novel insights into glycogen storage disease IV. Hum. Mol. Genet. 20, 455–465 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The central part of this work was supported by the MURI project from the Department of-Army W911NF-10-1-0518 Reconfigurable Matter from Programmable Colloids, assistance with electron microscopy, and for the NSF grant #DMR-9871177 for funding of the FEI Nova 200 analytical electron microscope used in this work.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yichun Wang.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, Y., Bahng, J.H. & Kotov, N.A. Three-dimensional biomimetic scaffolds for hepatic differentiation of size-controlled embryoid bodies. Journal of Materials Research 34, 1371–1380 (2019). https://doi.org/10.1557/jmr.2019.80

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1557/jmr.2019.80

Navigation