Skip to main content
SpringerLink
Log in

Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells

  • Published:
Biomedical Microdevices Aims and scope Submit manuscript

Abstract

Access to unlimited numbers of live human neurons derived from stem cells offers unique opportunities for in vitro modeling of neural development, disease-related cellular phenotypes, and drug testing and discovery. However, to develop informative cellular in vitro assays, it is important to consider the relevant in vivo environment of neural tissues. Biomimetic 3D scaffolds are tools to culture human neurons under defined mechanical and physico-chemical properties providing an interconnected porous structure that may potentially enable a higher or more complex organization than traditional two-dimensional monolayer conditions. It is known that even minor variations in the internal geometry and mechanical properties of 3D scaffolds can impact cell behavior including survival, growth, and cell fate choice. In this report, we describe the design and engineering of 3D synthetic polyethylene glycol (PEG)-based and biodegradable gelatin-based scaffolds generated by a free form fabrication technique with precise internal geometry and elastic stiffnesses. We show that human neurons, derived from human embryonic stem (hESC) cells, are able to adhere to these scaffolds and form organoid structures that extend in three dimensions as demonstrated by confocal and electron microscopy. Future refinements of scaffold structure, size and surface chemistries may facilitate long term experiments and designing clinically applicable bioassays.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • T.B. Bini, S. Gao, S. Wang, S. Ramakrishna, Poly(l-lactide-co-glycolide) biodegradable microfibers and electrospun nanofibers for nerve tissue engineering: an in vitro study. J. Mater. Sci. 41(19), 6453–6459 (2006)

    Article  Google Scholar 

  • M.Z.A. Björn Carlberg, Nannmark Ulf, Liu Johan, H. Georg Kuhn, Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells. Biomed. Mater. 4(4), 045004 (2009)

    Article  Google Scholar 

  • S.M. Chambers, C.A. Fasano, E.P. Papapetrou, M. Tomishima, M. Sadelain, L. Studer, Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27(3), 275–280 (2009)

    Article  Google Scholar 

  • P.L. Chandran, V. Barocas, Affine versus non-affine fibril kinematics in collagen networks: theoretical studies of network behavior. J. Biomech. Eng. 128(2), 259–270 (2006)

    Article  Google Scholar 

  • L.P. Deleyrolle, B.A. Reynolds, Isolation, expansion, and differentiation of adult Mammalian neural stem and progenitor cells using the neurosphere assay. Methods Mol. Biol. 549, 91–101 (2009)

    Article  Google Scholar 

  • D.B. Edelman, E.W. Keefer, A cultural renaissance: in vitro cell biology embraces three-dimensional context. Exp. Neurol. 192(1), 1–6 (2005)

    Article  Google Scholar 

  • Y. Elkabetz, G. Panagiotakos, G. Al Shamy, N.D. Socci, V. Tabar, L. Studer, Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 22(2), 152–165 (2008)

    Article  Google Scholar 

  • D.Y. Fozdar, P. Soman, W. LeeJin, L.-H. Han, S. Chen, Three-dimensional polymer constructs exhibiting a tunable negative poisson’s ratio. Adv. Funct. Mater. (2011) In Press

  • R. Gauvin, Y.-C. Chen, J.W. Lee, P. Soman, P. Zorlutuna, J.W. Nichol et al., Microfabrication of complex porous tissue engineering scaffolds using 3D projection stereolithography. Biomaterials 33(15), 3824–3834 (2012)

    Article  Google Scholar 

  • K. Gelse, E. Pöschl, T. Aigner, Collagens–structure, function, and biosynthesis. Adv. Drug Deliv. Rev. 55(12), 1531–1546 (2003)

    Article  Google Scholar 

  • L.H. Han, G. Mapili, S. Chen, K. Roy, Projection microfabrication of three-dimensional scaffolds for tissue engineering. J Manuf Sci E–T ASME 2008 Apr;130(2) (2008)

  • J.B. Jensen, M. Parmar, Strengths and limitations of the neurosphere culture system. Mol. Neurobiol. 34(3), 153–161 (2006)

    Article  Google Scholar 

  • A. Kunze, M. Giugliano, A. Valero, P. Renaud, Micropatterning neural cell cultures in 3D with a multi-layered scaffold. Biomaterials 32(8), 2088–2098 (2011)

    Article  Google Scholar 

  • H.J. Lam, S. Patel, A. Wang, J. Chu, S. Li, In vitro regulation of neural differentiation and axon growth by growth factors and bioactive nanofibers. Tissue Eng. Part A 16(8), 2641–2648 (2011)

    Article  Google Scholar 

  • H. Lee, G.A. Shamy, Y. Elkabetz, C.M. Schofield, N.L. Harrsion, G. Panagiotakos et al., Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells 25(8), 1931–1939 (2007)

    Article  Google Scholar 

  • J. Lee, M.J. Cuddihy, N.A. Kotov, Three-dimensional cell culture matrices: state of the art. Tissue Eng. Part B Rev. 14(1), 61–86 (2008)

    Article  Google Scholar 

  • S. Levenberg, N.F. Huang, E. Lavik, A.B. Rogers, J. Itskovitz-Eldor, R. Langer, Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc. Natl. Acad. Sci. 100(22), 12741–12746 (2003)

    Article  Google Scholar 

  • W. Li, W. Sun, Y. Zhang, W. Wei, R. Ambasudhan, P. Xia et al., Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc. Natl. Acad. Sci. U. S. A. 108(20), 8299–8304 (2011)

    Article  Google Scholar 

  • H. Liu, S.C. Zhang, Specification of neuronal and glial subtypes from human pluripotent stem cells. Cell Mol. Life Sci. 68(24), 3995–4008 (2011)

    Article  Google Scholar 

  • W. Ma, W. Fitzgerald, Q.Y. Liu, T.J. O’Shaughnessy, D. Maric, H.J. Lin et al., CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels. Exp. Neurol. 190(2), 276–288 (2004)

    Article  Google Scholar 

  • M.C. Manzini, C.A. Walsh, What disorders of cortical development tell us about the cortex: one plus one does not always make two. Curr. Opin. Genet. Dev. 21(3), 333–339 (2011)

    Article  Google Scholar 

  • G.P. Marshall 2nd, B.A. Reynolds, E.D. Laywell, Using the neurosphere assay to quantify neural stem cells in vivo. Curr. Pharm. Biotechnol. 8(3), 141–145 (2007)

    Article  Google Scholar 

  • A. Pathak, S. Kumar, Biophysical regulation of tumor cell invasion: moving beyond matrix stiffness. Integr. Biol. 3(4), 267–278 (2011)

    Article  MathSciNet  Google Scholar 

  • J. Pedersen, M. Swartz, Mechanobiology in the third dimension. Ann. Biomed. Eng. 33(11), 1469–1490 (2005)

    Article  Google Scholar 

  • A.L. Perrier, V. Tabar, T. Barberi, M.E. Rubio, J. Bruses, N. Topf et al., Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 101(34), 12543–12548 (2004)

    Article  Google Scholar 

  • D. Ren, J.D. Miller, Primary cell culture of suprachiasmatic nucleus. Brain Res. Bull. 61(5), 547–553 (2003)

    Article  Google Scholar 

  • K. Sango, H. Saito, M. Takano, A. Tokashiki, S. Inoue, H. Horie, Cultured adult animal neurons and schwann cells give us new insights into diabetic neuropathy. Curr. Diabetes Rev. 2(2), 169–183 (2006)

    Article  Google Scholar 

  • E. Shahbazi, S. Kiani, H. Gourabi, H. Baharvand., Electrospun nanofibrillar surfaces promote neuronal differentiation and function from human embryonic stem cells. Tissue Eng. Part A 2011/08/17 (2011)

  • R.F. Silva, A.S. Falcao, A. Fernandes, A.C. Gordo, M.A. Brito, D. Brites, Dissociated primary nerve cell cultures as models for assessment of neurotoxicity. Toxicol. Lett. 163(1), 1–9 (2006)

    Article  Google Scholar 

  • I. Singec, R. Knoth, R.P. Meyer, J. Maciaczyk, B. Volk, G. Nikkhah et al., Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nature methods 3(10), 801–806 (2006)

    Article  Google Scholar 

  • P. Soman, J.W. Lee, A. Phadke, S. Varghese, S. Chen. Spatial tuning of negative and positive Poisson’s ratio in a multi-layer scaffold. Acta Biomaterialia (0) (2012)

  • C. Storm, J.J. Pastore, F.C. MacKintosh, T.C. Lubensky, P.A. Janmey, Nonlinear elasticity in biological gels. Nature 435(7039), 191–194 (2005)

    Article  Google Scholar 

  • G. Vunjak-Novakovic, D.T. Scadden, Biomimetic platforms for human stem cell research. Cell Stem Cell 8(3), 252–261 (2011)

    Article  Google Scholar 

  • S.M. Willerth, K.J. Arendas, D.I. Gottlieb, S.E. Sakiyama-Elbert, Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells. Biomaterials 27(36), 5990–6003 (2006)

    Article  Google Scholar 

  • J.P. Winer, S. Oake, P.A. Janmey, Non-linear elasticity of extracellular matrices enables contractile cells to communicate local position and orientation. PLoS One 4(7), e6382 (2009)

    Article  Google Scholar 

  • F. Yang, R. Murugan, S. Wang, S. Ramakrishna, Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 26(15), 2603–2610 (2005)

    Article  Google Scholar 

  • S.C. Zhang, Neural subtype specification from embryonic stem cells. Brain Pathol. 16(2), 132–142 (2006)

    Article  Google Scholar 

Download references

Acknowledgements

The project described was supported in part by Award Number R01EB012597 from the National Institute of Biomedical Imaging And Bioengineering and grants (CMMI-1130894, CMMI-1120795) from the National Science Foundation. Thanks to Joseph Russo of Sanford-Burnham Imaging Core Facility for technical expertise. BT is funded by UCSD Dept. of Psychiatry NIH T32.

Author information

Authors and Affiliations

  1. Department of NanoEngineering, University of California, San Diego, 9500 Gilman Drive, Atkinson Hall, MC-0448, La Jolla, CA, 92093, USA

    Pranav Soman, Jin Woo Lee, Kenneth S. Vecchio & Shaochen Chen

  2. Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA, 92037, USA

    Brian T. D. Tobe, Alicia A. M. Winquist, Ilyas Singec, Evan Y. Snyder & Shaochen Chen

  3. Department of Psychiatry, University of California-San Diego, La Jolla, CA, USA

    Brian T. D. Tobe & Shaochen Chen

Authors
  1. Pranav Soman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brian T. D. Tobe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jin Woo Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alicia A. M. Winquist
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ilyas Singec
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kenneth S. Vecchio
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Evan Y. Snyder
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shaochen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Evan Y. Snyder or Shaochen Chen.

Additional information

Pranav Soman and Brian T. D. Tobe contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Soman, P., Tobe, B.T.D., Lee, J.W. et al. Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells. Biomed Microdevices 14, 829–838 (2012). https://doi.org/10.1007/s10544-012-9662-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10544-012-9662-7

Keywords

Search

Navigation