Adipose-Derived Stem Cells Spontaneously Express Neural Markers When Grown in a PEG-Based 3D Matrix

Abstract

Neurological diseases are among the leading causes of disability and death worldwide and remain difficult to treat. Tissue engineering offers avenues to test potential treatments; however, the development of biologically accurate models of brain tissues remains challenging. Given their neurogenic potential and availability, adipose-derived stem cells (ADSCs) are of interest for creating neural models. While progress has been made in differentiating ADSCs into neural cells, their differentiation in 3D environments, which are more representative of the in vivo physiological conditions of the nervous system, is crucial. This can be achieved by modulating the 3D matrix composition and stiffness. Human ADSCs were cultured for 14 days in a 1.1 kPa polyethylene glycol-based 3D hydrogel matrix to assess effects on cell morphology, cell viability, proteome changes and spontaneous neural differentiation. Results showed that cells continued to proliferate over the 14-day period and presented a different morphology to 2D cultures, with the cells elongating and aligning with one another. The proteome analysis revealed 439 proteins changed in abundance by >1.5 fold. Cyclic nucleotide 3′-phosphodiesterase (CNPase) markers were identified using immunocytochemistry and confirmed with proteomics. Findings indicate that ADSCs spontaneously increase neural marker expression when grown in an environment with similar mechanical properties to the central nervous system.

Neural Marker Expression in Adipose-Derived Stem Cells Grown in PEG-Based 3D Matrix Is Enhanced in the Presence of B27 and CultureOne Supplements

“…Similarly, to our previous work [30], the cells grown in PEG-based 3D matrices did not experience a statistically significant decrease in viability or proliferation. In addition, treatment with either B27, C1, or N2 had no statistically significant effect on the viability and proliferation of the 3D-grown cells over the treatment course.…”

“…It is known that as stem cells start to differentiate, proliferation decreases, and as neural cells mature, they reach the G 0 cell cycle stage, where division stops [32]. When looking at DMEM 3D control cells, these have shown a significant increase in confluence over time (Figure 2e,f), similar to that previously reported [30]. While the treated cells' proliferation may not have significantly decreased from when treatment started, no increase in proliferation is a sign of metabolic activity slowing down.…”

“…Our previous work and the literature more broadly demonstrate that ADSCs grown in traditional 2D culture with neural culture supplements exhibit changes consistent with neural differentiation [31]. Similarly, ADSCs grown in a 3D culture system have shown a degree of differentiation toward neural cell subtypes with the expression of neural markers [30]. Thus, it was hypothesised that the supplements would further enhance neural differentiation of the ADSCs grown in 3D and there would be biological differences and changes in the expressed proteome indicative of this.…”

“…Of particular interest, human-derived mesenchymal stem cells (hMSCs) have been shown to differentiate towards a neural lineage when grown in scaffolds resembling a brain stiffness of 1 kPa [28]. Additionally, our previous work showed that hADSCs spontaneously express neural markers when grown in PEG-based hydrogels of 1.1 kPa stiffness [30], further highlighting the importance of growth matrices and cell growth environment when conducting neural differentiation of hADSCs.…”

“…This study builds upon our previous work, where we used the readily available media supplements B27, C1, and N2 for neural induction of ADSCs in 2D matrices [31], and on our most recent investigation of the effects of mechanical properties of 3D polyethylene glycol (PEG)-based hydrogels containing RGD and YIGSR peptides to initiate neural differentiation of hADSCs [30]. This work merges both concepts and explores the effects of treatment and matrix combined using cost-effective and standardised supplements together with the reproducibility of commercially available system rastrum bioprinting (Inventia, Sydney, Australia).…”