Enhancement of neural functions through three dimensional neural differentiation

Enhancement of neural functions through three dimensional neural differentiation from human embryonic stem cells


Mi-Sun Choi, Jung-Hwa Oh, Se-Myo Park, Hyang-Ae Lee, Heeyoung Yang, Soojin Kim, Ki-Suk Kim, Chang-Woo Song and Seokjoo Yoon


Mi-Sun Choi


Korea Institute of Toxicology


The potential functionality of human embryonic stem cell(hESC)-derived neuronal cells is important to screen and prioritize the neurotoxic effects of test substances. However, hESC-derived neuronal cells have limitations to assess the functional neuronal activity on neurotoxicity due to the immaturity of cultured hESC-derived neuron. Here, we generated hESC-derived neuronal cells for measuring the electrical activity through three approaches: (1) generation of 3D neurosphere, (2) Astrocyte-conditioned media, and (3) co-cultures of 3D neurospheres with astrocyte strategies to enhance the neuronal network’s electrical functionality. Firstly, we attempted the induction of 3D neurospheres from hESC-derived neural precursor cells(hNPCs) using 3D Petri Dish®, which creates a natural 3D environment. Characterization of neurospheres was demonstrated by immunohistochemistry, showing the appearance of beta-tubulin III(Tuj1) and microtubule-associated protein 2 (MAP2) positive cells. Electrical activity was also observed for resting membrane potential, gain characteristic Na+, K+ currents and action potentials using patch clamp technique. Comparing to 2D differentiation system, 3D differentiated neuron enhanced electrophysiological activities. In addition, cocultures of hESC-derived neurospheres with astrocyte accelerated electrical functionality but did not alter action potential in astrocyte-condition medium(ACM). Here, we suggest the neural differentiation method to enhance the functional neural activities and this information could be used in drug and chemical safety screening for potential neurotoxic compounds. This work was supported by the General Research Program (NRF-2012M3A9C7050138) from the Ministry of Science, ICT.