Reprogramming strategies: comparing the effectiveness of monolayer and neurosphere techniques in mesenchymal stem cells dedifferentiation into neural stem cells

The remarkable plasticity of human dental pulp mesenchymal stem cells (hDPSCs) has paved the way for significant advancements in regenerative medicine, particularly in addressing challenges related to neural repair, neurodegenerative diseases, and glioblastoma (GBM) research [1]. GBM are highly aggressive brain tumors with limited treatment options and poor survival rates [2]. A major factor driving their progression and recurrence is the presence of glioblastoma stem cells (GSCs), which share molecular and functional traits with neural stem cells (NSCs) (3). These similarities complicate therapeutic strategies, as distinguishing between GSCs and normal NSCs is critical for developing precise and effective treatments. Additionally, NSCs interact with the tumor microenvironment, influencing GBM behavior and its response to therapies [3,4]. Despite their potential, sourcing NSCs for research and therapeutic purposes is often hindered by ethical and technical challenges [4,5]. To address this, researchers have turned to the dedifferentiation of hDPSCs into NSCs, offering a scalable, ethical, and reliable alternative [5]. These dedifferentiated hDPSC-derived NSCs are invaluable for refining glioblastoma models, enabling researchers to better understand the interactions between GSCs and the neural microenvironment [5,6]. The aim of this research is to identify the most effective method for dedifferentiating human dental pulp mesenchymal stem cells into neural stem cells that closely resemble commercial NSC lines. In total, five different cell lines derived from human dental pulp were previously validated as mesenchymal stem cells. For the induction of hDPSCs to dedifferentiate into NSCs were used DMEM/F12 conditioning media supplemented with B27, N2, and bFGF and EGF factors. Dedifferentiation was perfomed in two different ways: culturing the cells in neurospheres within low-adhesion dishes and by dedifferentiating them in a monolayer format. After 8 hours, 1 and 2 weeks of dedifferentiation, all cells were collected to assess changes in gene expression. RNA was extracted from the cell pellets using TRIzol reagent and then converted into complementary DNA (cDNA) with the High-Capacity RNA-to-cDNA kit. The expression levels of specific markers associated with neural stem cells were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) which revieled mRNA changes, compared to the control. Validated cells were dedifferentiated into neural stem cells, which after 8 hours, 1 and 2 weeks showed morphological changes in the cells and mRNA changes in the genes CDK5R1, MAP2 and MSI1. A comparative analysis of the two methods revealed that the method of dedifferentiating cells with neurospheres was more effective than dedifferentiation in monolayer. Nevertheless, the expression of these markers did not reach NSC values, which suggests that dedifferentiation occurred, but the efficiency was low.