Neuronal Differentiation from Induced Pluripotent Stem Cell-Derived Neurospheres by the Application of Oxidized Alginate-Gelatin-Laminin Hydrogels

Abstract

Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate-gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.

Keywords: bioprinting; human induced pluripotent stem cells (hiPSC); hydrogels; laminin; neurospheres; oxidized alginate; tissue engineering.

Figure 1

Schematic of ADA-GEL-LAM hydrogel. By oxidation of alginate, alginate di-aldehyde (ADA) is formed. The presence of aldehyde groups allows crosslinking with amine groups of proteins (gelatin, laminin) via nucleophilic attack of the amine on the aldehyde.

Figure 2

Material characterization. (A) Fourier transformed infrared spectroscopy (FTIR) analysis, absorbance spectra of ADA, GEL, ADA-GEL, ADA-GEL-LAM, and ADA-GEL hydrogels. (B) Scanning electron microscopy images of ADA-GEL and ADA-GEL-LAM hydrogels. Both hydrogels feature micro porosity (bottom row). (C) Nanoindentation of ADA-GEL and ADA-GEL-LAM showing the effective Young’s modulus (E_eff) of the hydrogels at 22 °C and 37 °C. Data are displayed as mean ± SD. (D) Qualitative load-indentation behavior of the hydrogels. Statistically significant differences of means analyzed using one-way ANOVA with * p < 0.05, ** p < 0.01, not significant (ns, p ≥ 0.05). Scale bar 1–10 µm.

Figure 3

Cell viability and toxicity of iPSC-derived NPCs. (A) Cell embedding scheme. Neurospheres were freshly chopped to 0.1 mm, mixed with ADA-GEL-X precursor at a density of 10,000 spheres/mL (corresponds to 1 × 10⁷ cells/mL), crosslinked, and maintained in differentiation medium. (B) Viability of neurospheres in the hydrogels measured via resazurin reduction to resorufin. Data shown as relative fluorescence units (RFU). 2D as positive and negative (2D lysis, TritonX-100 treated) controls. (C) Cytotoxicity assessment via LDH release after one day of incubation in differentiation medium, normalized to 2D TritonX-100 treated lysis control (n = 3 with 4 technical replicates; *** p < 0.001 compared to 2D control).

Figure 4

LIVE/DEAD staining of neurospheres in ADA-X hydrogels. Neurospheres were embedded in the indicated hydrogels and cultivated in differentiation medium. Samples were stained with Calcein-AM (LIVE, green) and Ethidium-homodimer (DEAD, red) and analyzed by light-microscopy in fluorescence and brightfield mode. Cell outgrowth was indicated in ADA-GEL-LAM (white arrows). Scale bar 100 µm.

Figure 5

Laminin supports neuronal migration out of neurospheres. (A) Confocal fluorescence microscopy of immunostainings for tubulin-β-III (red, Alexa546), filamentous actin (green, Phalloidin-Alexa488) and nuclei (blue, Hoechst) after 14 days of gel differentiation. The upper panel displays the tubulin-β-III positive neuronal outgrowth only. Representative maximum intensity projections are shown in the lower panel. (B) Quantification of the migration distance (black), measured from the edge of the sphere core to the distal end, normalized to the length of ADA-GEL. Number of spheres showing migration, normalized to ADA-GEL (red). Scale bar 200 µm.