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

Goparaju, Sravan K.; Kohda, Kazuhisa; Ibata, Keiji; Soma, Atsumi; Nakatake, Yukhi; Akiyama, Tasuku; Wakabayashi, Shokichi; Matsushita, Maiko; Sakota, Miki; Kimura, Hiromi; Yuzaki, Michisuke; Ko, Shigeru B. H.; Ko, Minoru S.H.

doi:10.1038/srep42367

Cited by 74 publications

(85 citation statements)

References 32 publications

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“…A recent study has reported a cocktail of synthetic transcription factors to initiate neural differentiation of human pluripotent stem cells in which a set of small molecules consisting of forskolin, SB431542, dorsomorphin, and retinoic acid included in the B27 supplement are added to the differentiation medium. This study demonstrated that the extracellular differentiation cues from small molecules can significantly increase the efficacy of pluripotent stem cells for differentiation into functional neurons (Goparaju et al, ).…”

Section: Discussionmentioning

confidence: 95%

Down‐regulation of the non‐coding RNA H19 and its derived miR‐675 is concomitant with up‐regulation of insulin‐like growth factor receptor type 1 during neural‐like differentiation of human bone marrow mesenchymal stem cells

Farzi-Molan

Babashah

Bakhshinejad

et al. 2018

Cell Biology International

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The differentiation of human bone marrow mesenchymal stem cells (BMSCs) into specific lineages offers new opportunities to use the therapeutic efficiency of these pluripotent cells in regenerative medicine. Multiple lines of evidence have revealed that non-coding RNAs play major roles in the differentiation of BMSCs into neural cells. Here, we applied a cocktail of neural inducing factors (NIFs) to differentiate BMSCs into neural-like cells. Our data demonstrated that during neurogenic induction, BMSCs obtained a neuron-like morphology. Also, the results of gene expression analysis by qRT-PCR showed progressively increasing expression levels of neuron-specific enolase (NSE) as well as microtubule-associated protein 2 (MAP-2) and immunocytochemical staining detected the expression of these neuron-specific markers along differentiated BMSC bodies and cytoplasmic processes, confirming the differentiation of BMSCs into neuronal lineages. We also compared differences in the expression levels of the long non-coding RNA (lncRNA) H19 and H19-derived miR-675 between undifferentiated and neurally differentiated BMSCs and found that during neural differentiation down-regulation of the lncRNA H19/miR-675 axis is concomitant with up-regulation of insulin-like growth factor type-1 (IGF-1R), a well-established target of miR-675 involved in neurogenesis. The findings of the current study provide support for the hypothesis that miR-675 may confer functionality to H19, suggesting a key role for this miRNA in the neural differentiation of BSMCs. However, further investigation is required to gain deeper insights into the biological roles of this miRNA in the complex process of neurogenesis.

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Section: Discussionmentioning

confidence: 95%

Down‐regulation of the non‐coding RNA H19 and its derived miR‐675 is concomitant with up‐regulation of insulin‐like growth factor receptor type 1 during neural‐like differentiation of human bone marrow mesenchymal stem cells

Farzi-Molan

Babashah

Bakhshinejad

et al. 2018

Cell Biology International

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“…hMSC derived neurons (hMd-Neurons) displayed spontaneous activity and showed response in miliseconds to electrical stimulation as a typical maturating neuron [32]. [34][35][36]. This is a phenomenon that can be discussed since spontaneous activity is not an ability of all primary neurons.…”

Section: Discussionmentioning

confidence: 99%

Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity

Karakaş

Bay

Türkel

et al. 2020

Preprint

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ABSTRACTMesenchymal stem cells are one of the promising tissue specific stem cell source for neural tissue regeneration applications. Previous studies on human mesenchymal stem cell (hMSC) derived neurons have been limited and not statisfactory in terms of neuronal activity. In this study, we analysed the functionality of bone marrow hMSCs differentiated into neural protein expressing cells by a single step cytokine based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (≥75%) as demonstrated by Ca++ imaging. Furthermore, when electrically stimulated, hMSC induced neurons (hMd-Neuron) matched the response of a typical neuron in the process of maturation. Our results reveal that enriched neurothrophic factors enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. hMd-Neurons have the potential to be used as a tool for disease modelling of neuropathologies and neural differentiation studies.

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“…The result from Ngn2 mRNA also supports this conclusion. Recently, Goparaju et al reported a novel mRNA‐based strategy using the cotransfection of five TFs every 8 hours to generate iPSC‐derived motor neurons . It is possible that using the same TFs with phosphosite modification may make this protocol more efficient by reducing mRNA amount and/or transfection intervals.…”

Section: Discussionmentioning

confidence: 99%

“…mRNA‐based reprogramming kits are widely used for generating integration‐free iPSCs. In comparison, mRNA‐driven iPSC differentiation is also feasible but more challenging , because an optimal iPSC differentiation protocol requires significantly higher efficiency (>80%) than that of cell reprogramming (0.01%–2%). Here, we focus on developing highly efficient strategies for generating mRNA‐induced mDA neurons, hereby referred to as miDA neurons.…”

Section: Introductionmentioning

confidence: 99%

Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

Xue

Zhan

Sun

et al. 2018

Stem Cells Translational Medicine

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Proneural transcription factors (TFs) drive highly efficient differentiation of pluripotent stem cells to lineage‐specific neurons. However, current strategies mainly rely on genome‐integrating viruses. Here, we used synthetic mRNAs coding two proneural TFs (Atoh1 and Ngn2) to differentiate induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons. mRNAs coding Atoh1 and Ngn2 with defined phosphosite modifications led to higher and more stable protein expression, and induced more efficient neuron conversion, as compared to mRNAs coding wild‐type proteins. Using these two modified mRNAs with morphogens, we established a 5‐day protocol that can rapidly generate mDA neurons with >90% purity from normal and Parkinson's disease iPSCs. After in vitro maturation, these mRNA‐induced mDA (miDA) neurons recapitulate key biochemical and electrophysiological features of primary mDA neurons and can provide high‐content neuron cultures for drug discovery. Proteomic analysis of Atoh1‐binding proteins identified the nonmuscle myosin II (NM‐II) complex as a new binding partner of nuclear Atoh1. The NM‐II complex, commonly known as an ATP‐dependent molecular motor, binds more strongly to phosphosite‐modified Atoh1 than the wild type. Blebbistatin, an NM‐II complex antagonist, and bradykinin, an NM‐II complex agonist, inhibited and promoted, respectively, the transcriptional activity of Atoh1 and the efficiency of miDA neuron generation. These findings established the first mRNA‐driven strategy for efficient iPSC differentiation to mDA neurons. We further identified the NM‐II complex as a positive modulator of Atoh1‐driven neuron differentiation. The methodology described here will facilitate the development of mRNA‐driven differentiation strategies for generating iPSC‐derived progenies widely applicable to disease modeling and cell replacement therapy. stem cells translational medicine 2019;8:112&12

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

Cited by 74 publications

References 32 publications

Down‐regulation of the non‐coding RNA H19 and its derived miR‐675 is concomitant with up‐regulation of insulin‐like growth factor receptor type 1 during neural‐like differentiation of human bone marrow mesenchymal stem cells

Down‐regulation of the non‐coding RNA H19 and its derived miR‐675 is concomitant with up‐regulation of insulin‐like growth factor receptor type 1 during neural‐like differentiation of human bone marrow mesenchymal stem cells

Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity

Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons

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