Effect of various origins conditioned media on the migration of neural cells in vitro

Abstract

An important direction in the development of the latest technologies for the restoration of damaged central nervous system is the use of stem/progenitor cells (SPCs), mainly neurogenic SPCs (NSPCs) and mesenchymal multipotent stromal cells (MMSCs). One of the main mechanisms of SPCs action is indirect paracrine effects due to the ability to produce a wide range of biologically active signaling molecules (secretome). The study of regenerative effects of conditioned media (CM) of NSPCs and MMSCs as a source of their secretome seems to be actual and potentially beneficial. The aim of the study is to compare the impact of CM from 24-h cultures of fetal neurogenic cells (NCs (E14), as a source of NSPCs) and adiposederived mononuclear cells (AMCs as a source of MMSCs) on migration capacity of rat neural cells in vitro. AMCs-CM were obtained from 24-h cultures with prevalence of CD105+ cells and ability upon further cultivation to form “spheroids” and potency to differentiate into different cell types. NCs-CM were obtained from 24-h cultures with prevalence of Nestin+ cells and ability upon further cultivation to form “neurospheres” and potency to differentiate into astrocytes (GFAP+) and neurons (β-Tubulin III+). Rat fetal neural cells (E14) were cultured to achieve a confluent monolayer with basic cellular elements of nervous tissue (5-7th day), which was dissected with forming a transection site and DMEM with 10% fetal calf serum (control) or 0.1-0.3 mg/ml (by total protein amount) of NCs-CM or AMCs-CM were added. In control cultures of rat neural cells partial overgrowth of the dissected area of the monolayer was observed due to the migration of cells, formation of a network of processes and intercellular contacts; reaching 13.2% (4th day) – 23.2% (8th day) of its full length. The overgrown area increased after addition of CM: NCsCM – 3 times (0.1-0.2 mg/ml) and 3-4 times (0.3 mg/ml, 4th-8th day), reaching 70.5% of full length of the transection site; AMCs-CM – 1.5 times (0.1-0.2 mg/ml) and 4-7 times (0.3 mg/ml, 4th-8th day), reaching 97.4-100% of full length of the transection site. The addition of NCs CM and AMCs CM resulted in β-catenin translocation into nucleus of cells in rat neural cell cultures, which correlated with the overgrowth of the transection zone. NCs-CM as well as AMCs-CM in dose-dependent manner stimulate migration processes in culture of rat neural cells, obviously, involving β-catenin signaling pathway, contributing to overgrowing of the dissected area (reparation of a mechanical defect). NCs-CM and AMCs-CM are a source of signaling molecules that modulate the microenvironment and activate endogenous repair mechanisms in culture (in vitro model of nerve tissue regeneration).