Abstract
Neural stem cells-based therapies have shown great potential for central nervous system regeneration, with three-dimensional (3D) culture systems representing a key technique for tissue engineering applications, as well as disease modeling and drug screenings. Self-assembling peptides (SAPs), providing biomimetic synthetic micro-environments regulating cellular functionality and tissue repair, constitute a suitable tool for the production of complex tissue-like structures in vitro. However, one of the most important drawbacks in 3D cultures, obtained via animal-derived substrates and serum-rich media, is the reproducibility and tunability of a standardized methodology capable to coax neural differentiation of different human cell lines. In this work we cultured four distinct human neural stem cell (hNSC) lines in 3D synthetic multifunctionalized hydrogel (named HYDROSAP) for up to 6 weeks. Three-dimensional cultures of differentiating hNSCs exhibited a progressive differentiation and maturation over time. All hNSCs-derived neurons in 3D culture system exhibited randomly organized entangled networks with increasing expression of GABAergic and glutamatergic phenotypes and presence of cholinergic ones. Oligodendrocytes formed insulating myelin sheaths positive for myelin basic protein (MBP). In summary, results demonstrated a successfully standardized and reproducible 3D cell culture system for hNSC differentiation and maturation in serum-free conditions useful for future therapies.
Highlights
Conventional in vitro neuronal models rely on cell growth in 2D platform that are a poor representation of cell behavior in vivo
A schematic representation of experimental protocol is portrayed in Figure 1 and largely described in section “Materials and Methods.” human neural stem cells (hNSCs) at high concentration (4.5 × 104 cells/μl) were encapsulated in pureHYDROSAP, composed by multifunctionalized self-assembling peptide (SAP) and branched SAPs, supplemented with sucrose and sodium hydroxide (NaOH) solutions
To deeply characterize the difference among four independent hNSC lines in vitro, results were divided in two parts: (1) comparisons among hNSC lines at each timepoint (1DIV, 1WIV and 6WIV); (2) time-tracking of marker expression for each hNSC line
Summary
Conventional in vitro neuronal models rely on cell growth in 2D platform that are a poor representation of cell behavior in vivo. Hydrogel-based biomaterials are an excellent platform for 3D cultures because of their biocompatibility, elasticity, adjustable mechanical properties, and the ability to mimic native extracellular matrix (ECM). They have been widely used in biomedical researches, ranging from tissue engineering (Christman, 2019) and regenerative medicine (Cembran et al, 2020) to drug delivery (Narayanaswamy and Torchilin, 2019) and microfluidic devices (Alessandri et al, 2016; Natividad-Diaz et al, 2019). In vitro 3D hydrogel cultures provide new tools to direct stem cell differentiation into defined phenotypes prior transplantation (Lai et al, 2018)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
