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Differentiation of mouse embryonic stem cells into a defined neuronal lineage

Abstract

Although it has long been known that cultured embryonic stem cells can generate neurons, the lineage relationships with their immediate precursors remain unclear. We report here that selection of highly proliferative stem cells followed by treatment with retinoic acid generated essentially pure precursors that markers identified as Pax-6-positive radial glial cells. As they do in vivo, these cells went on to generate neurons with remarkably uniform biochemical and electrophysiological characteristics.

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Figure 1: Schematic representation of the neuronal differentiation procedure.
Figure 2: ES cells differentiate into a homogeneous population of radial glial cells.
Figure 3: Pax-6 is initially expressed by most cells but rapidly disappears.
Figure 4: Neuronal differentiation after 4 d in culture.
Figure 5: Morphology of GFP-positive neurons.
Figure 6: Western blots of extracts of in vitro differentiated neurons prepared at different time points.
Figure 7: Electrophysiological properties of stem cell-derived neurons (see also Supplementary Fig. 1 online).

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Acknowledgements

We thank A. Smith and M. Li for allowing M.B. to learn techniques of ES cell differentiation in their laboratory and for valuable suggestions, and D. Gerosa for technical assistance.

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Correspondence to Yves-Alain Barde.

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Supplementary information

Supplementary Fig. 1

Detailed electrophysiological characteristics of stem cell-derived neurons. (a) Method to calculate cell properties from Table 2. Tau was calculated as the time needed to reach 63% of the membrane voltage at the end of the hyperpolarizing pulse. (b) Increase of spontaneous activity by application of 50 μM bicuculline (current-clamp recording) clearly shows the existence of GABAergic input to the recorded cell that was 20-d old. (c) Cell response to a 2 s hyper- and depolarizing current pulse. (Left cell 14 DIV, right cell 20 DIV). From 20 cells tested 13 could follow the depolarization pulse with action potentials during the whole time, whereas seven cells only could follow approx. one-third of the time. (d) To further distinguish between EPSCs and IPSCs the cell (20 DIV) was clamped to -30 mV which is above the reversal potential of IPSCs. Left column: holding potential -60 mV, right column: holding potential -30 mV. In normal ACSF, EPSCs and IPSCs are present at -30 mV (top row). During the application of NBQX/AP5 only IPSCs could be detected (second row), whereas all responses could be blocked by further application of bicuculline (third row). The bottom row shows responses after washout of the drugs. (GIF 207 kb)

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Bibel, M., Richter, J., Schrenk, K. et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nat Neurosci 7, 1003–1009 (2004). https://doi.org/10.1038/nn1301

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