In a study published last October in the journal Stem Cell Research, Chandrasekaran et al. compared the induction of neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) obtained by 2D and 3D culture methods. Neural differentiation of five hiPSC lines was analyzed at the protein level by immunocytochemistry and flow cytometry to reveal the expression of various markers : NPC, neuronal, cortical layer and glial markers.

NPCs derived from human PSCs are often induced using 3D aggregates, because these systems are considered as efficient differentiation tools to induce PSCs into the neural lineage. However, according to the authors, no publication had directly compared 3D neural induction (sphere) with 2D neural induction (monolayer) in terms of cell numbers, gene expression profiles, proliferation rates, differentiation potential, cell fate and functionality.

Representative confocal microscopic images of 3D spheres.

They found no significant difference between the two induction methods – or the different genetic background cell lines – regarding early differentiation events (neuroepithelial commitment and formation of neural rosettes). However, a higher number of NPCs positive for NESTIN and PAX6 was generated using 3D neural induction, compared to the 2D method. Interestingly, the analysis of neurite length revealed that the 3D-induced neurons exhibited longer neurites than 2D-induced cells.

This study, among others, shows that 3D culture methods might help to improve the differentiation of human cells in vitro, leading to more physiological phenotypes !