Similarly derived and cultured hESC lines show variation in their developmental potential towards neuronal cells in long-term culture.

BACKGROUND: Human embryonic stem cells (hESCs) can differentiate into any human cell type, including CNS cells, and thus have high potential in regenerative medicine. Several protocols exist for neuronal differentiation of hESCs, which do not necessarily work for all hESC lines. MATERIALS & METHODS: We tested the differentiation capacity of four similarly derived and cultured hESC lines (HS181, HS360, HS362 and HS401) in suspension culture in relatively simple neural differentiation medium for up to 20 weeks. RESULTS: All the hESC lines differentiated into neuronal cells, but in a line-dependent manner. Using our method, the HS181- and HS360-derived neurospheres differentiated in vitro into pure neuronal cell populations within 6 weeks, whereas HS362 and HS401 reached their peak of differentiation in 12 weeks, but never produced pure neuronal cell populations using the present method. The withdrawal of FGF from suspension culture increased the in vitro differentiation potential. The hESC-derived neurospheres formed functional neuronal networks when replated on a microelectrode array and responded as expected to pharmacologic modulation. CONCLUSION: Simple neurosphere culture is a suitable method for producing hESC-derived neuronal cells that can form functional neuronal networks from a number of hESC lines. The variation in the differentiation potential of hESC lines into neuronal cells must be carefully considered by those comparing various differentiation methods and designing transplantation therapies for neuronal disorders.

Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.