Embryonic Stem Cell Culture System Research Articles

MiR-3099 promotes neurogenesis and inhibits astrogliogenesis during murine neural development

MicroRNA-3099 is highly expressed during neuronal differentiation and development of the central nervous system. Here we characterised the role of miR-3099 during neural differentiation and embryonic brain development using a stable and regulatable mouse embryonic stem cell culture system for miR-3099 expression and in utero electroporation of miR-3099 expression construct into E15.5 embryonic mouse brains. In the in vitro system, miR-3099 overexpression upregulated gene related to neuronal markers such as Tuj1, NeuN, Gat1, vGluT1 and vGluT2. In contrast, gene related to astrocyte markers (Gfap, S100β and Slc1a3) were suppressed upon overexpression of miR-3099. Furthermore, miR-3099 overexpression between E15.5 and E18.5 mouse embryonic brains led to disorganised neuronal migration potentially due to significantly decreased Gfap+ cells. Collectively, our results indicated that miR-3099 plays a role in modulating and regulating expression of key markers involved in neuronal differentiation. In silico analysis was also performed to identify miR-3099 homologues in the human genome, and candidates were validated by stem-loop RT-qPCR. Analysis of the miR-3099 seed sequence AGGCUA against human transcriptomes revealed that a potential miRNA, mds21 (Chr21:39186698-39186677) (GenBank accession ID: MK521584), was 100% identical to the miR-3099 seed sequence. Mds21 expression was observed and validated in various human cell lines (293FT, human Wharton's jelly and dental pulp mesenchymal stem cells, and MCF-7, MDA-MB-231, C-Sert, SW780, RT112, 5637, EJ28 and SH-SY5Y cells), with the highest levels detected in human mesenchymal stem cell lines. The analysis validated mds21 as a novel miRNA and a novel homologue of miR-3099 in the human genome.

Inactivating sex chromosomes

Molecular Biology To compensate for sex chromosome dosage, XX females undergo epigenetic inactivation (XCI) of one X chromosome. Using a mouse embryonic stem cell culture system, Sousa et al. investigated this process in vitro. Surprisingly, they found that male cells also undergo XCI, albeit transiently, at the onset of stem cell differentiation. In addition, both X chromosomes are ephemerally inactivated in female cells before one X is randomly inactivated permanently. Although it remains to be shown in vivo, these results suggest gender-independent XCI initiation and additional, unknown female-specific mechanisms to maintain XCI. Cell Stem Cell 10.1016/j.stem.2018.05.001 (2018).

BMP4 acts as a dorsal telencephalic morphogen in a mouse embryonic stem cell culture system.

ABSTRACTThe concept of a morphogen – a molecule that specifies two or more cell fates in a concentration-dependent manner – is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate central nervous system (CNS), including the mouse dorsal telencephalic midline (DTM). Bone morphogenetic proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate – that of choroid plexus epithelial cells (CPECs) – in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal–Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2.

A mouse embryonic stem cell culture system with stable and regulatable expression miR-3099: an in vitro approach towards functional genomics study

Event Abstract Back to Event A mouse embryonic stem cell culture system with stable and regulatable expression miR-3099: an in vitro approach towards functional genomics study Shahidee Zainal Abidin1, 2, 3*, Jia-Wen Leong1, 2, 4, Norshariza Nordin2, 3, Syahril Abdullah2, 3, Pike-See Cheah1, 2, 4 and King-Hwa Ling1, 2, 3* 1 Universiti Putra Malaysia, NeuroBiology and Genetics Group, Faculty of Medicine and Health Sciences, Malaysia 2 Universiti Putra Malaysia, Genetics and Regenerative Medicine Research Center, Faculty of Medicine and Health Sciences, Malaysia 3 Universiti Putra Malaysia, Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Malaysia 4 Universiti Putra Malaysia, Department of Human Anatomy, Faculty of Medicine and Health Sciences, Malaysia miR-3099 is a small non-coding RNA that is highly expressed throughout embryogenesis and is localised in cortical plate between E13.5 and E17.5 in developing mouse brain. Majority of the cells in the brain at these time-points are committed to neuronal cell lineage. To understand more on the role of miR-3099 during neurogenesis, this study aims to characterise the role of miR-3099 via in vitro approach with respect to functional genomic study. The bioinformatics study was used to predict the downstream-targeted genes by using four independent prediction software (miRDB, miRanda, TargetScan and DIANA micro-T CDS). The target genes that were predicted by at least three different databases were subjected to DAVID bioinformatics analysis to understand their function in biological processes followed by Allen Brain Atlas expression profile analysis. Based on the analysis, it was suggested that miR-3099 was potentially involved in neural stem cell (NSC) fate determination pathway. This warrants further characterisation by generating mouse embryonic stem (mES) cell with regulatable expression miR-3099 to perform function study at molecular level. Interestingly, miR-3099 seed region was found to be 100% identical with chr21:40558602-40558624 in human genome. This indicates that, by extrapolating the finding from animal model will contribute to a more detailed knowledge of the molecular mechanisms of NSC fate determination toward specific lineages. Acknowledgements The study was supported by Malaysian Ministry of Higher Education's Fundamental Research Grant Scheme awarded to P.-S. Cheah (FRGS/1/2015/SKK08/UPM/02/1) and Malaysian Ministry of Science, Technology and Innovation's Sciencefund grant awarded to K.-H. Ling (02-01-04-SF2336). Keywords: Cerebral Cortex, Genomics, miRNA, in vitro, embryonic stem cell Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: O03: Postgraduate Travel Awardees Oral Session 3 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Zainal Abidin S, Leong J, Nordin N, Abdullah S, Cheah P and Ling K (2016). A mouse embryonic stem cell culture system with stable and regulatable expression miR-3099: an in vitro approach towards functional genomics study. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00091 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Mr. Shahidee Zainal Abidin, Universiti Putra Malaysia, NeuroBiology and Genetics Group, Faculty of Medicine and Health Sciences, Serdang, Selangor, Malaysia, shah.68000@gmail.com Dr. King-Hwa Ling, Universiti Putra Malaysia, NeuroBiology and Genetics Group, Faculty of Medicine and Health Sciences, Serdang, Selangor, Malaysia, lkh@upm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling Google Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling Google Scholar Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling PubMed Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

SWI/SNF Protein Component BAF250a Regulates Cardiac Progenitor Cell Differentiation by Modulating Chromatin Accessibility during Second Heart Field Development

ATP-dependent SWI/SNF chromatin remodeling complexes alter the structure of chromatin at specific loci and facilitate tissue-specific gene regulation during development. Several SWI/SNF subunits are required for cardiogenesis. However, the function and mechanisms of SWI/SNF in mediating cardiac progenitor cell (CPC) differentiation during cardiogenesis are not well understood. Our studies of the SWI/SNF chromatin remodeling complex identified that BAF250a, a regulatory subunit of the SWI/SNF, plays a key role in CPC differentiation. BAF250a ablation in mouse second heart field (SHF) led to trabeculation defects in the right ventricle, ventricular septal defect, persistent truncus arteriosus, reduced myocardial proliferation, and embryonic lethality around E13. Using an embryonic stem cell culture system that models the formation and differentiation of SHF CPCs in vivo, we have shown that BAF250a ablation in CPCs specifically inhibits cardiomyocyte formation. Moreover, BAF250a selectively regulates the expression of key cardiac factors Mef2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunoprecipitation and DNase I digestion assays indicate that BAF250a regulates gene expression by binding selectively to its target gene promoters and recruiting Brg1, the catalytic subunit of SWI/SNF, to modulate chromatin accessibility. Our results thus identify BAF250a-mediated chromatin remodeling as an essential epigenetic mechanism mediating CPC differentiation.

Bimodal effect of retinoic acid on melanocyte differentiation identified by time‐dependent analysis

Retinoic acid (RA) is considered to control melanocytes; however, its precise mechanism remains unclear because of a bimodal effect, which promotes or inhibits melanin synthesis depending on the cell type, culture condition of melanocytes and skin conditions. In this study, we examined the effects of RA throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell culture system to induce melanocytes. The results showed that RA has significantly different effects depending on the stage of differentiation of melanocytes. More specifically, RA promoted differentiation in earlier stages, wherein embryonic stem cells became melanoblasts via neural crest cells, and inhibited differentiation in later stages, wherein melanoblasts became melanocytes. It was revealed for the first time that melanocytes show markedly different reactions to RA depending on the stage of differentiation.

Axonal Growth Regulation of Fetal and Embryonic Stem Cell‐Derived Dopaminergic Neurons by Netrin‐1 and Slits

The physical restoration of dopamine circuits damaged or lost in Parkinson disease by implanting embryonic stem (ES)-derived cells may become a treatment. It is critical to understand responses of ES-derived dopamine (DA) neurons to guidance signals that determine axonal path and targeting. Using a collagen gel culture system, we examined effects of secreted molecules Netrin-1 and Slits on neurite outgrowth of fetal DA neurons and murine ES-differentiated DA neurons. We have previously shown that fetal DA neurons express DCC and Robo1/2 receptors and that Netrin-1 and Slit2 function as an attractant and a repellent for DA neurite outgrowth. In the present study, we observe that both Slit1 and Slit3 repel and inhibit neurite growth of fetal DA neurons. Here, we also demonstrate that ES-differentiated neurons including DA neurons express the Netrin receptor DCC and Slit receptor Robo proteins. In the gel culture system of ES cells, Netrin-1 promoted neurite outgrowth mediated by DCC receptor, and Slit1 and Slit3 were inhibitory for neurite outgrowth through Robo receptors. Slit2 appeared to exert inhibitory as well as repulsive effects in the coculture assay. However, unlike fetal DA neurites, no directed neurite outgrowth was observed in the cocultures of ES-derived DA neurons with Netrin-1-, Slit1-, and Slit3-producing cells. The findings suggest that ES-derived DA neurons generated by current protocols can respond to guidance cues in vitro in a similar manner to fetal cells but also exhibit distinct responses. This may result from developmental differences generated by present in vitro methods of cell patterning or conditioning during ES cell differentiation.

The Battlefield of Pluripotency

How embryonic stem cells maintain the potential to differentiate into multiple cell lineages is still unclear. In this issue of Cell, Niwa et al. (2005) show that a duel between the transcription factors Oct3/4 and Cdx2 can restrict embryonic stem cells to either embryonic or placental fate. The vulnerability of lineage potential to transcriptional perturbation may reflect an essential feature of pluripotency.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

AbstractAlthough the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

Developmental Analysis of Tropomyosin Gene Expression in Embryonic Stem Cells and Mouse Embryos

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.