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Substrate stiffness affects the morphology and gene expression of epidermal neural crest stem cells in a short term culture.

Biotechnology and bioengineering (2019-10-28)
Sareh Pandamooz, Arman Jafari, Mohammad S Salehi, Benjamin Jurek, Abolhassan Ahmadiani, Anahid Safari, Shadi Hassanajili, Afshin Borhani-Haghighi, Mehdi Dianatpour, Hassan Niknejad, Negar Azarpira, Leila Dargahi
ABSTRACT

According to the intrinsic plasticity of stem cells, controlling their fate is a critical issue in cell-based therapies. Recently, a growing body of evidence has suggested that substrate stiffness can affect the fate decisions of various stem cells. Epidermal neural crest stem cells as one of the main neural crest cell derivatives hold great promise for cell therapies due to presenting a high level of plasticity. This study was conducted to define the influence of substrate stiffness on the lineage commitment of these cells. Here, four different polyacrylamide hydrogels with elastic modulus in the range of 0.7-30 kPa were synthesized and coated with collagen and stem cells were seeded on them for 24 hr. The obtained data showed that cells can attach faster to hydrogels compared with culture plate and cells on <1 kPa stiffness show more neuronal-like morphology as they presented several branches and extended longer neurites over time. Moreover, the transcription of actin downregulated on all hydrogels, while the expression of Nestin, Tubulin, and PDGFR-α increased on all of them and SOX-10 and doublecortin gene expression were higher only on <1 kPa. Also, it was revealed that soft hydrogels can enhance the expression of glial cell line-derived neurotrophic factor, neurotrophin-3, and vascular endothelial growth factor in these stem cells. On the basis of the results, these cells can respond to the substrate stiffness in the short term culture and soft hydrogels can alter their morphology and gene expression. These findings suggested that employing proper substrate stiffness might result in cells with more natural profiles similar to the nervous system and superior usefulness in therapeutic applications.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, BioXtra
Sigma-Aldrich
Fluorescein diacetate, used as cell viability stain
Supelco
Sodium hydroxide, pellets EMPLURA®
Sigma-Aldrich
DAPI, dilactate, ≥98% (HPLC)
Sigma-Aldrich
Minimum Essential Medium Eagle, Alpha Modification, with L-glutamine, ribonucleosides and deoxyribonucleosides, without sodium bicarbonate, powder, suitable for cell culture
Sigma-Aldrich
Paraformaldehyde, for synthesis
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule), F(ab′)2 fragment–FITC antibody produced in goat, affinity isolated antibody, buffered aqueous solution