Effect of 3D Spheroid Culturing on NF-κB Signaling Pathway and Neurogenic Potential in Human Amniotic Fluid Stem Cells

Author:

Valiulienė Giedrė1,Zentelytė Aistė1,Beržanskytė Elizabet1,Navakauskienė Rūta1ORCID

Affiliation:

1. Department of Molecular Cell Biology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio av. 7, LT-01257 Vilnius, Lithuania

Abstract

Human amniotic fluid stem cells (hAFSCs) are known for their advantageous properties when compared to somatic stem cells from other sources. Recently hAFSCs have gained attention for their neurogenic potential and secretory profile. However, hAFSCs in three-dimensional (3D) cultures remain poorly investigated. Therefore, we aimed to evaluate cellular properties, neural differentiation, and gene and protein expression in 3D spheroid cultures of hAFSCs in comparison to traditional two-dimensional (2D) monolayer cultures. For this purpose, hAFSCs were obtained from amniotic fluid of healthy pregnancies and cultivated in vitro, either in 2D, or 3D under untreated or neuro-differentiated conditions. We observed upregulated expression of pluripotency genes OCT4, NANOG, and MSI1 as well as augmentation in gene expression of NF-κB−TNFα pathway genes (NFKB2, RELA and TNFR2), associated miRNAs (miR103a-5p, miR199a-3p and miR223-3p), and NF-κB p65 protein levels in untreated hAFSC 3D cultures. Additionally, MS analysis of the 3D hAFSCs secretome revealed protein upregulation of IGFs signaling the cascade and downregulation of extracellular matrix proteins, whereas neural differentiation of hAFSC spheroids increased the expression of SOX2, miR223-3p, and MSI1. Summarizing, our study provides novel insights into how 3D culture affects neurogenic potential and signaling pathways of hAFSCs, especially NF-κB, although further studies are needed to elucidate the benefits of 3D cultures more thoroughly.

Funder

Vilnius University Science promotion fund

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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