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Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors

View Article: PubMed Central - PubMed

ABSTRACT

Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.

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Syn-5TFs-induced neurons are functional.(a–f) Electric stimulation-evoked calcium transients in neurons induced by syn-5TFs on Day 7. Fluo-4 loaded cells before electric stimulation in DIC microscopy (a) and fluorescence microscopy (b). Cells after electric stimulation with 40 Hz pulses for 5 secs (c). Cells pre-treated with tetrodotoxin (TTX) (0.5 μM) for two min to block sodium channels, followed by electric stimulation (d). (e) Time line of representative trace of cell, indicated by an arrowhead in (a). The timings of image capture are indicated as (b–d). (f) Time line trace shows the recovery of cells to evoke calcium transients after TTX wash-off. (g) Repetitive multiple action potentials induced by current injections in the current clamp mode in syn-5TFs-induced neurons on Day 10. Application of TTX blocked the firing. (h) In a voltage clamp mode, syn-5TFs-induced neurons exhibited fast activating and inactivating sodium currents, which were blocked by TTX. The square pulses shown in the bottom panels indicate 20 pA current steps in (g) or 10 mV voltage steps in (h) applied to the syn-5TFs-induced neurons.
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f5: Syn-5TFs-induced neurons are functional.(a–f) Electric stimulation-evoked calcium transients in neurons induced by syn-5TFs on Day 7. Fluo-4 loaded cells before electric stimulation in DIC microscopy (a) and fluorescence microscopy (b). Cells after electric stimulation with 40 Hz pulses for 5 secs (c). Cells pre-treated with tetrodotoxin (TTX) (0.5 μM) for two min to block sodium channels, followed by electric stimulation (d). (e) Time line of representative trace of cell, indicated by an arrowhead in (a). The timings of image capture are indicated as (b–d). (f) Time line trace shows the recovery of cells to evoke calcium transients after TTX wash-off. (g) Repetitive multiple action potentials induced by current injections in the current clamp mode in syn-5TFs-induced neurons on Day 10. Application of TTX blocked the firing. (h) In a voltage clamp mode, syn-5TFs-induced neurons exhibited fast activating and inactivating sodium currents, which were blocked by TTX. The square pulses shown in the bottom panels indicate 20 pA current steps in (g) or 10 mV voltage steps in (h) applied to the syn-5TFs-induced neurons.

Mentions: We then functionally characterized the syn-5TFs-derived neurons by Fluo-4-based Ca2+ imaging. Electric field stimulation at 40 Hz for 5 seconds (secs) induced Ca2+ increase in most syn-5TFs-derived neurons from TkDA3-4 iPS cells by Day 7 (94%, n = 89; Fig. 5a–d and Supplementary Movie M1). Addition of tetrodotoxin (TTX), a neuronal voltage-gated sodium channel blocker, to the extracellular solution reversibly inhibited the electric stimulation-induced Ca2+ increase (Fig. 5e,f). These data indicate that these Ca2+ transients were mediated by voltage-gated sodium channels, followed by Ca2+ influx through voltage-gated Ca2+ channels.


Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors
Syn-5TFs-induced neurons are functional.(a–f) Electric stimulation-evoked calcium transients in neurons induced by syn-5TFs on Day 7. Fluo-4 loaded cells before electric stimulation in DIC microscopy (a) and fluorescence microscopy (b). Cells after electric stimulation with 40 Hz pulses for 5 secs (c). Cells pre-treated with tetrodotoxin (TTX) (0.5 μM) for two min to block sodium channels, followed by electric stimulation (d). (e) Time line of representative trace of cell, indicated by an arrowhead in (a). The timings of image capture are indicated as (b–d). (f) Time line trace shows the recovery of cells to evoke calcium transients after TTX wash-off. (g) Repetitive multiple action potentials induced by current injections in the current clamp mode in syn-5TFs-induced neurons on Day 10. Application of TTX blocked the firing. (h) In a voltage clamp mode, syn-5TFs-induced neurons exhibited fast activating and inactivating sodium currents, which were blocked by TTX. The square pulses shown in the bottom panels indicate 20 pA current steps in (g) or 10 mV voltage steps in (h) applied to the syn-5TFs-induced neurons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5304326&req=5

f5: Syn-5TFs-induced neurons are functional.(a–f) Electric stimulation-evoked calcium transients in neurons induced by syn-5TFs on Day 7. Fluo-4 loaded cells before electric stimulation in DIC microscopy (a) and fluorescence microscopy (b). Cells after electric stimulation with 40 Hz pulses for 5 secs (c). Cells pre-treated with tetrodotoxin (TTX) (0.5 μM) for two min to block sodium channels, followed by electric stimulation (d). (e) Time line of representative trace of cell, indicated by an arrowhead in (a). The timings of image capture are indicated as (b–d). (f) Time line trace shows the recovery of cells to evoke calcium transients after TTX wash-off. (g) Repetitive multiple action potentials induced by current injections in the current clamp mode in syn-5TFs-induced neurons on Day 10. Application of TTX blocked the firing. (h) In a voltage clamp mode, syn-5TFs-induced neurons exhibited fast activating and inactivating sodium currents, which were blocked by TTX. The square pulses shown in the bottom panels indicate 20 pA current steps in (g) or 10 mV voltage steps in (h) applied to the syn-5TFs-induced neurons.
Mentions: We then functionally characterized the syn-5TFs-derived neurons by Fluo-4-based Ca2+ imaging. Electric field stimulation at 40 Hz for 5 seconds (secs) induced Ca2+ increase in most syn-5TFs-derived neurons from TkDA3-4 iPS cells by Day 7 (94%, n = 89; Fig. 5a–d and Supplementary Movie M1). Addition of tetrodotoxin (TTX), a neuronal voltage-gated sodium channel blocker, to the extracellular solution reversibly inhibited the electric stimulation-induced Ca2+ increase (Fig. 5e,f). These data indicate that these Ca2+ transients were mediated by voltage-gated sodium channels, followed by Ca2+ influx through voltage-gated Ca2+ channels.

View Article: PubMed Central - PubMed

ABSTRACT

Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs.

No MeSH data available.


Related in: MedlinePlus