An optimized method for neuronal differentiation of embryonic stem cells in vitro

Li, Yuan; Mao, Xiang; Zhou, Xianju; Su, Yu-Ting; Zhou, Xiangyu; Shi, Kaituo; Zhao, Shasha

doi:10.1016/j.jneumeth.2019.108486

Cited by 10 publications

(4 citation statements)

References 33 publications

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“…The reintroduction of exogenous Hoxa1 was needed for 5 μM ATRA-induced ES cell neuronal differentiation ( Martinez-Ceballos and Gudas, 2008 ). Other investigators reported that 1 μM ATRA enhanced the efficiency of differentiation of ES cells into neural precursor cells ( Li et al, 2019 ) and that this conversion required both fibroblast growth factor and the elimination of signals for other fates ( Ying et al, 2003 ). Neural precursor cells have been efficiently derived from ES cells without the addition of ATRA by using a three-dimensional culture system followed by two-dimensional derivation.…”

Section: Findings From Studies Of the Development Of Embryonic Cellsmentioning

confidence: 99%

Retinoic acid receptor regulation of decision-making for cell differentiation

Brown

2023

Front. Cell Dev. Biol.

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All-trans retinoic acid (ATRA) activation of retinoic acid receptors (RARs) is crucial to an organism’s proper development as established by findings for mouse foetuses from dams fed a vitamin A-deficient diet. ATRA influences decision-making by embryonic stem (ES) cells for differentiation including lineage fate. From studies of knockout mice, RARα and RARγ regulate haematopoiesis whereby active RARα modulates the frequency of decision-making for myeloid differentiation, but is not essential for myelopoiesis, and active RARγ supports stem cell self-renewal and maintenance. From studies of zebrafish embryo development, active RARγ plays a negative role in stem cell decision-making for differentiation whereby, in the absence of exogenous ATRA, selective agonism of RARγ disrupted stem cell decision-making for differentiation patterning for development. From transactivation studies, 0.24 nM ATRA transactivated RARγ and 19.3 nM (80-fold more) was needed to transactivate RARα. Therefore, the dose of ATRA that cells are exposed to in vivo, from gradients created by cells that synthesize and metabolize, is important to RARγ versus RARα and RARγ activation and balancing of the involvements in modulating stem cell maintenance versus decision-making for differentiation. RARγ activation favours stemness whereas concomitant or temporal activation of RARγ and RARα favours differentiation. Crosstalk with signalling events that are provoked by membrane receptors is also important.

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Section: Findings From Studies Of the Development Of Embryonic Cellsmentioning

confidence: 99%

Retinoic acid receptor regulation of decision-making for cell differentiation

Brown

2023

Front. Cell Dev. Biol.

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“…On Day 4, Day 6 and Day 8, wash the cells with DPBS change the medium using basal differentiation medium containing 1 µM RA. After 8-day culture in these conditions, mESCs was differentiated into the neural progenitor cells (NPCs) 41,53 .…”

Section: Induced Neural Differentiation Of Mescs Into Npcs In Vitromentioning

confidence: 99%

Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq

Lyu,

Gao,

et al. 2024

Preprint

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Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation network and dynamics during cellular processes. In this study, we developed Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to provide quantitative insights into transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences in genomes. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNA transcription and specific transcription factors (TFs) binding sites that determine cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes; KAS-ATAC-seq signals on SSTEs exhibit more robust correlation with enhancer activities when compared with ATAC-seq data and active histone mark profiles. Our analysis of promoters and SSTEs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. We further discovered that ETS TFs and YY1 are critical in initiating early neural differentiation from mESCs to NPCs. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes and biomedical applications.

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“…iPSCs are induced to become NPCs through neuralization, either by EB formation [ 59 , 60 , 61 , 62 ], or by dual SMAD inhibition in monolayers [ 49 , 63 , 64 ]. EB formation is the most widely used method for neuralization [ 65 , 66 , 67 , 68 , 69 ]. In this process, cells grow in suspension and spontaneously arrange in three-dimensional aggregates upon withdrawal of factors promoting pluripotency.…”

Section: Generation Of Hipsc-derived Mnsmentioning

confidence: 99%

Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons

Akter

Ding

2022

Cells

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Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN differentiation and their applications in the generation of MNs from hiPSCs, with a particular focus on two approaches: induction by small molecules and induction by lentiviral delivery of transcription factors. At each induction stage, different culture media and supplements, typical growth conditions and cellular morphology, and specific markers for validation of cell identity and quality control are specifically discussed. Both approaches can generate functional MNs. Currently, the major challenges in modeling neurological diseases using iPSC-derived neurons are: obtaining neurons with high purity and yield; long-term neuron culture to reach full maturation; and how to culture neurons more physiologically to maximize relevance to in vivo conditions.

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An optimized method for neuronal differentiation of embryonic stem cells in vitro

Cited by 10 publications

References 33 publications

Retinoic acid receptor regulation of decision-making for cell differentiation

Retinoic acid receptor regulation of decision-making for cell differentiation

Quantitative analysis of cis-regulatory elements in transcription with KAS-ATAC-seq

Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons

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