Supramolecular hydrogels induce differentiation of stem cell into neural lineages

Peptide-based supramolecular hydrogels have been proposed as supports for tissue engineering. [1] They are generated by the supramolecular organization of bioactive peptide amphiphiles (i.e., building blocks) into nanofibres, that are maintained together through several non-covalent interactions, e.g., p-p staking, and H-bonding. These nanofibers can further form hydrogels by interactions with ions from the cellular milieu. The obtained hydrogels are dynamic and sensitive to different stimulus: changes in temperature, pH or ionic strength can trigger their assembly/disassembly or gelation/solubilization. [2] The peptide can be functionalized with carbohydrates to copycat bioactive glycoproteins present in the extracellular matrix (ECM). Herein, we hypothesized that short self-assembling glycodipeptides can be used as building blocks of supramolecular gels that induce stem cell differentiation. [2]

Our molecular design is based on the Fmoc-diphenylalanine (Fmoc-FF) known to generate supramolecular hydrogels under physiological conditions. Fmoc-FF was functionalized with glucosamine-6-sulfate (GlcN6S) - a structural element of several glycosaminoglycans known for their ability to modulate cellular behavior via interactions with numerous proteins. [3] The gelation of Fmoc-FF-GlcN6S in response to temperature change (T, i.e., a heating-cooling cycle) and solvent-switch (S, dilution of a DMSO solution with water) was studied. Both methods generated hydrogels that were stable for at least 21 days under cell culture conditions. Importantly, the preparation method influenced the stiffness of the hydrogels: at the same concentration of Fmoc-FF-GlcN6S (10 mM), gels obtained by method T had a Young’s modulus of 2.1 kPa, while S method gels had a modulus of 0.5 kPa (in the range of neural tissues, i.e., between 0.5-1.9kPa). CD, fluorescence and AFM data showed that this difference is due to different molecular packing and nanofiber morphology.

Both types of hydrogels were cytocompatible with adherent adipose-derived stem cells (ADSC), that overexpressed neural genetic markers, such as GFAP (glial fibrillary acidic protein) and Nestin (neuronal stem cell maker) after three days of culture. Longer cultures (nine days) overexpressed the microtubule associated proteins MAP2 and βIII-tubulin. These qPCR results were also confirmed by immunofluorescence.

In conclusion, the developed Fmoc-FF-GlcN6S hydrogels induce stem cell differentiation into neural cell lineages and present the conditions to be tested for the regeneration of neural tissues.

References:

1.Alakpa, E. V., et al.,Chem, 2016. 1(3).

2.Lampel, A., Chem, 2020.

3.Gama, C.I., et al., Nat Chem Biol, 2006. 2(9): p. 467-73.