Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

AbstractPost-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes inaccessible to deep analysis in vivo. Mouse embryonic stem cells (mESCs) can form aggregates reflecting the post-occipital embryo (gastruloids), but lacking proper morphogenesis. Here we show that embedding of aggregates derived from mESCs in an extracellular matrix compound results in Trunk-Like-Structures (TLS) with a high level of organization comprising a neural tube and somites. Comparative single-cell RNA-seq analysis demonstrates that TLS execute gene-regulatory programs in an embryo-like order, and generate primordial germ cell like cells (PGCLCs). TLS lacking Tbx6 form ectopic neural tubes, mirroring the embryonic mutant phenotype. ESC-derived trunk-like structures thus constitute a novel powerful in vitro platform for investigating lineage decisions and morphogenetic processes shaping the post-implantation embryo.One sentence summaryA platform for generating trunk-like-structures with precursors of spinal cord, bone and muscle from stem cells in a dish

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Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Science ◽

10.1126/science.aba4937 ◽

2020 ◽

Vol 370 (6522) ◽

pp. eaba4937 ◽

Cited By ~ 3

Author(s):

Jesse V. Veenvliet ◽

Adriano Bolondi ◽

Helene Kretzmer ◽

Leah Haut ◽

Manuela Scholze-Wittler ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Neural Tube ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Sequencing Analysis ◽

Mouse Development ◽

Somite Formation ◽

Post Implantation

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized “trunk-like structures” (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish.

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Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

Molecular Medicine Reports ◽

10.3892/mmr.2015.4586 ◽

2015 ◽

Vol 13 (1) ◽

pp. 720-730 ◽

Cited By ~ 8

Author(s):

LIPING OU ◽

LIAOQIONG FANG ◽

HEJING TANG ◽

HAI QIAO ◽

XIAOMEI ZHANG ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells

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GABAergic Neurons from Mouse Embryonic Stem Cells Possess Functional Properties of Striatal Neurons In Vitro, and Develop into Striatal Neurons In Vivo in a Mouse Model of Huntington’s Disease

Stem Cell Reviews and Reports ◽

10.1007/s12015-011-9290-2 ◽

2011 ◽

Vol 8 (2) ◽

pp. 513-531 ◽

Cited By ~ 23

Author(s):

Eunju Shin ◽

Mary J. Palmer ◽

Meng Li ◽

Rosemary A. Fricker

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Mouse Model ◽

Functional Properties ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Gabaergic Neurons ◽

Striatal Neurons

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Functional Dissection of pri-miR-290~295 in Dgcr8 Knockout Mouse Embryonic Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20184345 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4345

Author(s):

Ming Shi ◽

Jing Hao ◽

Xi-Wen Wang ◽

Le-Qi Liao ◽

Huiqing Cao ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Mirna Cluster ◽

Neighboring Gene ◽

Region Gene ◽

Mesenchymal Transition

The DiGeorge syndrome critical region gene 8 (Dgcr8) knockout strategy has been widely used to study the function of canonical microRNAs (miRNAs) in vitro and in vivo. However, primary miRNA (pri-miRNA) transcripts are accumulated in Dgcr8 knockout cells due to interrupted processing. Whether abnormally accumulated pri-miRNAs have any function is unknown. Here, using clustered regularly interspaced short palindromic repeats system/CRISPR-associated protein 9 (CRISPR/Cas9), we successfully knocked out the primary microRNA-290~295 (pri-miR-290~295) cluster, the most highly expressed miRNA cluster in mouse embryonic stem cells (ESCs), in Dgcr8 knockout background. We found that the major defects associated with Dgcr8 knockout in mouse ESCs, including higher expression of epithelial-to-mesenchymal transition (EMT) markers, slower proliferation, G1 accumulation, and defects in silencing self-renewal, were not affected by the deletion of pri-miR-290~290 cluster. Interestingly, the transcription of neighboring gene nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 12(Nlrp12) was upregulated upon the deletion of the pri-miR-290~295 cluster. Together, our results suggested that the major defects in Dgcr8 knockout ESCs were not due to the accumulation of pri-miR-290~295, and the deletion of miRNA genes could affect the transcription of neighboring DNA elements.

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SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc- SMPDL3B

10.1101/2021.02.24.432815 ◽

2021 ◽

Author(s):

Wei Fan ◽

Shuang Tang ◽

Xiaojuan Fan ◽

Yi Fang ◽

Xiaojiang Xu ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Neural Differentiation ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Sphingolipid Metabolism

AbstractSphingolipids are important structural components of cell membranes and prominent signaling molecules controlling cell growth, differentiation, and apoptosis. Sphingolipids are particularly abundant in the brain, and defects in sphingolipid degradation are associated with several human neurodegenerative diseases. However, molecular mechanisms governing sphingolipid metabolism remain unclear. Here we report that sphingolipid degradation is under transcriptional control of SIRT1, a highly conserved mammalian NAD+-dependent protein deacetylase, in mouse embryonic stem cells (mESCs). Deletion of SIRT1 results in accumulation of sphingomyelin in mESCs, primarily due to reduction of SMPDL3B, a GPI-anchored plasma membrane bound sphingomyelin phosphodiesterase. Mechanistically, SIRT1 regulates transcription of Smpdl3b through c-Myc. Functionally, SIRT1 deficiency-induced accumulation of sphingomyelin increases membrane fluidity and impairs neural differentiation in vitro and in vivo. Our findings discover a key regulatory mechanism for sphingolipid homeostasis and neural differentiation, further imply that pharmacological manipulation of SIRT1-mediated sphingomyelin degradation might be beneficial for treatment of human neurological diseases.

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Glutamatergic Neuronal Differentiation of Mouse Embryonic Stem Cells after Transient Expression of Neurogenin 1 and Treatment with BDNF and GDNF: In Vitro and In Vivo Studies

Journal of Neuroscience ◽

10.1523/jneurosci.0563-08.2008 ◽

2008 ◽

Vol 28 (48) ◽

pp. 12622-12631 ◽

Cited By ~ 83

Author(s):

J. H. Reyes ◽

K. S. O'Shea ◽

N. L. Wys ◽

J. M. Velkey ◽

D. M. Prieskorn ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Neuronal Differentiation ◽

Transient Expression ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

In Vivo Studies

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SIRT1 regulates sphingolipid metabolism and neural differentiation of mouse embryonic stem cells through c-Myc- SMPDL3B

eLife ◽

10.7554/elife.67452 ◽

2021 ◽

Vol 10 ◽

Author(s):

Wei Fan ◽

Shuang Tang ◽

Xiaojuan Fan ◽

Yi Fang ◽

Xiaojiang Xu ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Neural Differentiation ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Sphingolipid Metabolism

Sphingolipids are important structural components of cell membranes and prominent signaling molecules controlling cell growth, differentiation, and apoptosis. Sphingolipids are particularly abundant in the brain, and defects in sphingolipid degradation are associated with several human neurodegenerative diseases. However, molecular mechanisms governing sphingolipid metabolism remain unclear. Here we report that sphingolipid degradation is under transcriptional control of SIRT1, a highly conserved mammalian NAD+-dependent protein deacetylase, in mouse embryonic stem cells (mESCs). Deletion of SIRT1 results in accumulation of sphingomyelin in mESCs, primarily due to reduction of SMPDL3B, a GPI-anchored plasma membrane bound sphingomyelin phosphodiesterase. Mechanistically, SIRT1 regulates transcription of Smpdl3b through c-Myc. Functionally, SIRT1 deficiency-induced accumulation of sphingomyelin increases membrane fluidity and impairs neural differentiation in vitro and in vivo. Our findings discover a key regulatory mechanism for sphingolipid homeostasis and neural differentiation, further imply that pharmacological manipulation of SIRT1-mediated sphingomyelin degradation might be beneficial for treatment of human neurological diseases.

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Generating gastruloids with somite-like structures from mouse embryonic stem cells

10.21203/rs.2.18203/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Vincent van Batenburg ◽

Susanne Carina van den Brink ◽

Marloes Blotenburg ◽

Anna Alemany ◽

Naomi Moris ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cultured Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Hands On ◽

Somite Formation ◽

Key Aspects ◽

Post Implantation

Abstract Gastruloids are aggregates of mouse embryonic stem cells that can be used to study key aspects of mammalian post-implantation development in vitro1–4. Gastruloids generated with previously published protocols do not generate somite-like structures4–6. Here, we describe a modified version of the gastruloids culture protocol5,6 that results in gastruloids that do generate somite-like structures in vitro (van den Brink et al., Nature, 2020)7. Under these conditions, about 50% of the gastruloids generated form structures with features that are characteristic of somites7.This protocol takes 6 days, with relatively little hands-on time. The protocol starts with the aggregation of the cultured cells. Then, the Wnt-agonist Chiron is added 2 days (48h) later. The medium of the aggregates is replaced 3 days (72h) after aggregation. To induce somite-formation, gastruloids are embedded in Matrigel 4 days (96h) after aggregation. After 5 days (120h) of culture, gastruloids resemble E8.5 mouse embryos. At this timepoint they can be fixed (fixative is added on day 5 and washed away on day 6 after overnight incubation in PFA) to prepare them for staining or microscopy experiments.

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