Neuronal Differentiation of Human Embryonic Stem Cell Derived Otic Neural Progenitors Using a Nanofibrillar Cellulose and Hydrogel Scaffold and Polyhedrin Delivery System

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Kyle S. Coots1, Shun Kobayashi1, Duncan M. Chadly1, Kevin T. Nella1, Charles A. Miller1,

 Andrew Oleksijew1, John Kessler2, Akihiro J. Matsuoka1,3,4

1Department of Otolaryngology-Head and Neck Surgery, 2Department of Neurology, 3Department of Communication Sciences and Disorders, 4Knowles Hearing Center, Northwestern University,

Chicago and Evanston, IL, U.S.A.


Stem cell therapy is a promising method to alleviate sensorineural hearing loss, but its potential is currently hindered by low cell survival post transplantation (1-4). The harsh, nutrient deprived environment of the cochlea is thought to be a leading factor in poor cell survival (5,6). To alleviate this problem, we have proposed to use GrowDex®-T as an artificial extracellular matrix that is capable of both providing scaffolding for cells to attach and as a way to keep slow releasing brain derived neurotrophic factor (BDNF) PODSTM -crystals in place.


  • GrowDex-T 1% (Cat No. 200 103 005, UPM)
  • EZSPHERE™ (Nacalai)
  • BrainPhys™ Neuronal Medium (Cat No. 05790, Stem Cell Technologies)
  • BDNF-PODS™ (Cat No. PPH1-250, Cell Guidance Systems)
  • DMEM/F-12 supplemented with 1% N2 supplement, 2% B27 supplement, 2 mM glutamine, 100 μM beta-mercaptoethanol (all reagents were obtained from Life Technologies)


Human embryonic stem cell (hESC) derived late-stage otic neural progenitors (ONPs), as previously described and characterized (7), were single dissociated from a traditional monolayer plate and transferred to a micro-fabricated three dimensional cell culturing device (EZSPHERE, Nacalai) that consists of uniformly sized microwells coated with a low-binding affinity polymer (2-methacryloylxyethyl phosphorycholine). Cells were cultured in an ONP maintenance media (ONPMM) (7) at 37°C, 5% CO2 for two days and allowed to form spheroids with diameters of roughly 250 𝜇m.

1.GrowDex-T stock was diluted to 0.25% (v/v) with PBS (-/-). 

2.100 𝜇l of 0.25% GrowDex-T was plated into a 48-well plate.

3.Spheroids were transferred into the GrowDex-T using a P200 micropipette tip and 500 𝜇l  of BrainPhys was gently added on top of the mixture.

4.75,000 BDNF-PODS (equal to 10 ng/ml) were added to the GrowDex-T. 

5.Spheroids were cultured for 7 days at 37°C, 5% CO2 with no media changes.

6.Following 7 days, the media was removed and the cells and GrowDex-T were fixed with 4% PFA and fluorescently stained for Peripherin, VGLUT2, and DAPI.


Immunocytochemical (ICC) analysis shows that the hESC-derived ONP spheroids stained positive for DAPI, Peripherin (peripheral neuron marker) and VGLUT2 (glutamate transporter marker) after one week in differentiation media. Z-stack (n=18) photomicrograph (Fig. 1) obtained using a Nikon A1(C) confocal microscope. Image shown from the ninth plane. Note that the 3D culture condition produces multiple image planes; differing apparent fluorescence intensities are caused by out-of-focus cells within spheres.



Figure 1. A Photomicrograph of ICC image on an immature glutamatergic neuronal spheroid stained with DAPI, Peripherin, and VGLUT2. Scale bar: 100 𝜇m.


Our results indicate that 0.25% GrowDex-T allows for robust differentiation of LONPs into immature glutamatergic peripheral neurons when supplemented with 75,000 BDNF-PODS. In conjunction with slow-releasing BDNF with PODS crystals, GrowDex-T can be a suitable artificial extracellular matrix that is potentially capable of facilitating survival and neurite growth of hESC-derived neuronal progenitors in the future.


1.Chen W, Jongkamonwiwat N, Abbas L, Eshtan SJ, Johnson SL, Kuhn S, et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature. Nature Publishing Group; 2012;490(7419):278–82.

2.Matsuoka AJ, Kondo T, Miyamoto RT, Hashino E. In vivo and in vitro characterization of bone marrow- derived stem cells in the cochlea. Laryngoscope. 2006;116(August):1363–7.

3.Matsuoka AJ, Kondo T, Miyamoto RT, Hashino E. Enhanced survival of bone-marrow-derived pluripotent stem cells in an animal model of auditory neuropathy. Laryngoscope. 2007;117(9):1629–35.

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6.Reichardt LF. Neurotrophin-regulated signalling pathways. Philos Trans R Soc B Biol Sci. 2006;361(1473):1545–64.

7.Matsuoka AJ, Morrissey ZD, Zhang C, Homma K, Belmadani A, Miller CA, et al. Directed Differentiation of Human Embryonic Stem Cells Toward Placode-Derived Spiral Ganglion-Like Sensory Neurons. Stem Cells Transl Med. 2017 Mar;6(3):923–36.