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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.

No MeSH data available.


Induction of neurogenesis in human pluripotent stem cells by syn-5TFs mRNA cocktail.(a) Syn-5TFs-induces the efficient differentiation of human ES (SEES3) and iPS (201B7 and TkDA3-4) lines into neuronal cells. Representative images of TUBB3 staining at Day 5 are shown. The efficiency of TUBB3 expression in ES and iPS lines were as follows: TkDA3-4 human iPS cells, 86.2% ± 2.2 [mean ± SD] (two independent experiments, n = 2); 201B7 human iPS cells, 89.2% ± 9.6 [mean ± SD] (n = 2); and SEES3 human ES cells, 88.0% ± 12.1 [mean ± SD] (n = 2). Scale bar indicates 200 μm. (b) Small molecule modulators enhance the efficiency of neuronal differentiation induced by the Syn-5TFs cocktail in TkDA3-4 human iPS cells. Representative images of TUBB3 staining at Day 5 are shown. Scale bar indicates 200 μm. (c) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (red) and MAP2 (green) expressing cells at Day 7 are shown. Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm. The fraction of MAP2-positive cells among TUBB3-positive neurons (at Day 7) was 83.2% ± 0.6 [mean ± SD] (n = 2). (d) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (green) and NeuN (red) expressing cells at Day 10 are shown. The fraction of NeuN-positive cells among TUBB3-positive neurons (at Day 10) was 96.3% ± 0.4 [mean ± SD] (n = 2). Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm.
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f3: Induction of neurogenesis in human pluripotent stem cells by syn-5TFs mRNA cocktail.(a) Syn-5TFs-induces the efficient differentiation of human ES (SEES3) and iPS (201B7 and TkDA3-4) lines into neuronal cells. Representative images of TUBB3 staining at Day 5 are shown. The efficiency of TUBB3 expression in ES and iPS lines were as follows: TkDA3-4 human iPS cells, 86.2% ± 2.2 [mean ± SD] (two independent experiments, n = 2); 201B7 human iPS cells, 89.2% ± 9.6 [mean ± SD] (n = 2); and SEES3 human ES cells, 88.0% ± 12.1 [mean ± SD] (n = 2). Scale bar indicates 200 μm. (b) Small molecule modulators enhance the efficiency of neuronal differentiation induced by the Syn-5TFs cocktail in TkDA3-4 human iPS cells. Representative images of TUBB3 staining at Day 5 are shown. Scale bar indicates 200 μm. (c) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (red) and MAP2 (green) expressing cells at Day 7 are shown. Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm. The fraction of MAP2-positive cells among TUBB3-positive neurons (at Day 7) was 83.2% ± 0.6 [mean ± SD] (n = 2). (d) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (green) and NeuN (red) expressing cells at Day 10 are shown. The fraction of NeuN-positive cells among TUBB3-positive neurons (at Day 10) was 96.3% ± 0.4 [mean ± SD] (n = 2). Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm.

Mentions: When we transfected both ESCs (SEES3) and iPSCs (TkDA3-4 and 201B7) with the syn-5TFs cocktail, we found that rapid and highly efficient neural differentiation occurred by Day 5 after transfection (Fig. 3a). Efficiency of neural differentiation was similar between ESCs and iPSCs judged by the number of cells marked with neuron-specific TUBB3 in the total number of cells in the culture: 86.2% ± 2.2 [mean ± SD] (two independent experiments, n = 2) for TKDA3-4 human iPS cells, 89.2% ± 9.6 [mean ± SD] (n = 2) for 201B7 human iPS cells, and 88.0% ± 12.1 [mean ± SD] (n = 2) for SEES3 human ES cells. We did not notice any difference in the kinetics of differentiation between the cell lines (data not shown).


Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors
Induction of neurogenesis in human pluripotent stem cells by syn-5TFs mRNA cocktail.(a) Syn-5TFs-induces the efficient differentiation of human ES (SEES3) and iPS (201B7 and TkDA3-4) lines into neuronal cells. Representative images of TUBB3 staining at Day 5 are shown. The efficiency of TUBB3 expression in ES and iPS lines were as follows: TkDA3-4 human iPS cells, 86.2% ± 2.2 [mean ± SD] (two independent experiments, n = 2); 201B7 human iPS cells, 89.2% ± 9.6 [mean ± SD] (n = 2); and SEES3 human ES cells, 88.0% ± 12.1 [mean ± SD] (n = 2). Scale bar indicates 200 μm. (b) Small molecule modulators enhance the efficiency of neuronal differentiation induced by the Syn-5TFs cocktail in TkDA3-4 human iPS cells. Representative images of TUBB3 staining at Day 5 are shown. Scale bar indicates 200 μm. (c) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (red) and MAP2 (green) expressing cells at Day 7 are shown. Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm. The fraction of MAP2-positive cells among TUBB3-positive neurons (at Day 7) was 83.2% ± 0.6 [mean ± SD] (n = 2). (d) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (green) and NeuN (red) expressing cells at Day 10 are shown. The fraction of NeuN-positive cells among TUBB3-positive neurons (at Day 10) was 96.3% ± 0.4 [mean ± SD] (n = 2). Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm.
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f3: Induction of neurogenesis in human pluripotent stem cells by syn-5TFs mRNA cocktail.(a) Syn-5TFs-induces the efficient differentiation of human ES (SEES3) and iPS (201B7 and TkDA3-4) lines into neuronal cells. Representative images of TUBB3 staining at Day 5 are shown. The efficiency of TUBB3 expression in ES and iPS lines were as follows: TkDA3-4 human iPS cells, 86.2% ± 2.2 [mean ± SD] (two independent experiments, n = 2); 201B7 human iPS cells, 89.2% ± 9.6 [mean ± SD] (n = 2); and SEES3 human ES cells, 88.0% ± 12.1 [mean ± SD] (n = 2). Scale bar indicates 200 μm. (b) Small molecule modulators enhance the efficiency of neuronal differentiation induced by the Syn-5TFs cocktail in TkDA3-4 human iPS cells. Representative images of TUBB3 staining at Day 5 are shown. Scale bar indicates 200 μm. (c) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (red) and MAP2 (green) expressing cells at Day 7 are shown. Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm. The fraction of MAP2-positive cells among TUBB3-positive neurons (at Day 7) was 83.2% ± 0.6 [mean ± SD] (n = 2). (d) Highly efficient neuronal differentiation of TkDA3-4 human iPS by Syn-5TFs cocktail. Neuronal TUBB3 (green) and NeuN (red) expressing cells at Day 10 are shown. The fraction of NeuN-positive cells among TUBB3-positive neurons (at Day 10) was 96.3% ± 0.4 [mean ± SD] (n = 2). Upper row shows images of low magnification (10X). Scale bar indicates 200 μm. Lower row shows images of high magnification (40X). Scale bar indicates 50 μm.
Mentions: When we transfected both ESCs (SEES3) and iPSCs (TkDA3-4 and 201B7) with the syn-5TFs cocktail, we found that rapid and highly efficient neural differentiation occurred by Day 5 after transfection (Fig. 3a). Efficiency of neural differentiation was similar between ESCs and iPSCs judged by the number of cells marked with neuron-specific TUBB3 in the total number of cells in the culture: 86.2% ± 2.2 [mean ± SD] (two independent experiments, n = 2) for TKDA3-4 human iPS cells, 89.2% ± 9.6 [mean ± SD] (n = 2) for 201B7 human iPS cells, and 88.0% ± 12.1 [mean ± SD] (n = 2) for SEES3 human ES cells. We did not notice any difference in the kinetics of differentiation between the cell lines (data not shown).

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.