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A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

ABSTRACT: Empirical optimization of stem cell differentiation protocols is time consuming, is labor intensive, and typi- cally does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet sin- gle-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA ‘‘barcodelets’’ to identify them on the basis of the treatments they receive. We apply barRNA-seq to simultaneously manipulate up to seven developmental pathways and study effects on embryonic stem cell (ESC) germ layer specification and mesodermal specification, uncovering combina- torial effects of signaling pathway activation on gene expression. We further develop a data-driven frame- work for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNA-seq gastrulation atlas, and use this approach to guide ESC differen- tiation into a notochord-like population. We expect that barRNA-seq will have broad utility for investigating Q2 and understanding how cooperative signaling pathways drive cell fate acquisition.

ORGANISM(S): Mus musculus

PROVIDER: GSE122009 | GEO | 2020/05/26

REPOSITORIES: GEO

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A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

Project description:A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

| PRJNA503119 | ENA

Transcriptional profiling of Hepa1-6-bearing mouse model

Project description:Hepa1-6-bearing C57BL/6 mouse model was established to evaluate the therapeutic efficacy of Ganoderma lucidum extract (GLE) in HCC. The GLE treated and untreated mice were used for transcriptioanl profiles detection, and 126 differentially expressed lncRNAs and 558 DE mRNAs were identified, respectively. The bioinformatics analysis shows that the GLE could suppress the growth and proliferation of HCC by PI3K/Akt/mTOR and MAPK signaling pathway, and also could induce apoptosis of tumor cell by mitochondrial and death receptor pathway.

2018-08-02 | GSE117503 | GEO

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

Project description:Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMPK, a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK-/- EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs over-expressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. 2 WT and 2 AMPK DKO ESC lines were differentiated into embryoid bodies (EBs) for various lengths of time (2, 4, 8, and 12 days) in high and low glucose conditions. Both ESC and EB samples were profiled by mRNA-seq to examine how global gene expression changes associated with ESC differentiation are affected by AMPK deletion.

2016-03-09 | E-GEOD-77705 | biostudies-arrayexpress

Correlation with Apoptosis Process through RNA-Seq Data Analysis of Hep3B Hepatocellular Carcinoma Cells Treated with Glehnia littoralis Extract (GLE)

Project description:Glehnia littoralis is a perennial herb found in coastal sand dunes throughout East Asia. This herb has been reported to have hepatoprotective, immunomodulatory, antioxidant, antibacterial, antifungal, anti-inflammatory, and anticancer activities. It may be effective against hepatocellular carcinoma (HCC). However, whether this has been proven through gene-level RNA-seq analysis is still being determined. Therefore, we are attempting to identify target genes for the cell death process by analyzing the transcriptome of Hep3B cells among HCC treated with GLE (Glehnia littoralis extract) using RNA-seq. Hep3B was used for the GLE treatment, and the MTT test was performed. Hep3B was then treated with GLE at a set concentration of 300 μg/mL and stored for 24 h, followed by RNA isolation and sequencing. We then used the data to create a plot. As a result of the MTT analysis, cell death was observed when Hep3B cells were treated with GLE, and the IC50 was about 300 μg/mL. As a result of making plots using the RNA-seq data of Hep3B treated with 300 μg/mL GLE, a tendency for the apoptotic process was found. Flow cytometry and annexin V/propidium iodide (PI) staining verified the apoptosis of HEP3B cells treated with GLE. Therefore, an increase or decrease in the DEGs involved in the apoptosis process was confirmed. The top five genes increased were GADD45B, DDIT3, GADD45G, CHAC1, and PPP1R15A. The bottom five genes decreased were SGK1, CX3CL1, ZC3H12A, IER3, and HNF1A. In summary, we investigated the RNA-seq dataset of GLE to identify potential targets that may be involved in the apoptotic process in HCC. These goals may aid in the identification and management of HCC.

2024-09-04 | GSE276192 | GEO

Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1

Project description:A major challenge in adoptive cancer immunotherapy is the effective balance between the proliferation and cytotoxic capacity of the effector CD8+ T-cells. In this study we explored the molecular mechanisms whereby a “synthetic immune niche” (SIN), composed of immobilized CCL21 and ICAM1, modulates the interplay between these two cellular properties. Temporal phenotypic profiling of OVA-specific murine T-cells revealed major differences in the response to the SIN stimulation following antigen-specific and non-specific activation. Thus, SIN treatment enhanced both the expansion and cytotoxicity of cells activated by OVA-loaded DCs, at day 4, while, similar treatment of cells activated by anti CD3/CD28-conjugated beads dramatically enhanced the cells’ expansion, but unexpectedly blocked their cytotoxicity, which fully recovered only on day 7. Molecular profiling suggest that upregulation of cytotoxic gene signatures drive the SIN effect following DC activation, while down-regulation of exhaustion and pro-apoptotic genes optimize the proliferation-cytotoxicity balance in the beads-activated cells.

2024-06-28 | GSE254335 | GEO

A new identified suppressor of Cdc7p/SepH kinase, PomA, regulates fungal asexual reproduction via affecting phosphorylation of MAPK-HogA

Project description:The septation initiation network (SIN), composed of a conserved SepH (Cdc7p) kinase cascade, plays an essential role in fungal cytokinesis/septation and conidiation for asexual reproduction, while the mitogen-activated protein kinase (MAPK) pathway depends on successive signaling cascade phosphorylation to sense and respond to stress and environmental factors. In this study, a SepH suppressor–PomA in the filamentous fungus A. nidulans was identified as a negative regulator of septation and conidiation such that the pomA mutant was able to cure defects of sepH in septation and conidiation and overexpression of pomA remarkably suppressed septation. Under the normal cultural condition, SepH positively regulates the phosphorylation of MAPK-HogA, while PomA reversely affects this process and the phosphorylated HogA in MAPK could be a downstream product of the activated SIN. However, under the osmostress condition, the induced phosphorylated HogA is capable of bypassing the requirement of SepH to fulfill septation and conidiation. Findings demonstrate that crosstalk exists between the SIN and MAPK pathways.

2019-06-17 | PXD012514 | Pride

REST knock-out ESCs: a role for REST in embryonic stem cells' cardiac lineage specification

Project description:During development, lineage specification is controlled by several signaling pathways involving various transcription factors (TFs). Here, we studied the RE1-silencing transcription factor (REST) and identified an important role of this TF in cardiac differentiation. Using mouse embryonic stem cells (ESC) to model development, we analyzed the effect of REST knock-out on the ability to these cells to differentiate into the cardiac lineage. Detailed analysis of specific lineage markers expression showed selective down-regulation of endoderm markers in REST-null cells, thus contributing to a loss of cardiogenic signals. We propose here a new role for REST in cell fate specification besides its well-known repressive role of neuronal differentiation.

2016-02-01 | GSE76580 | GEO

miRNA profiling of Hepa1-6-bearing mouse model

Project description:the Hepa1-6-bearing C57 BL/6 mouse model was utilized to explore the therapeutic efficacy of Ganoderma lucidum extract (GLE), documenting that it could effectively inhibit tumor growth. Furthermore, the microRNA (miRNA) profiles of GLE-treated and untreated mice were detected, and 25 differential expressed (DE) miRNAs were determined, including 24 up-expressed and one down-expressed miRNAs., documenting that it could effectively inhibit tumor growth. Furthermore, the microRNA (miRNA profiles of GLE-treated and untreated mice were detected, and 25 differential expressed (DE miRNAs were determined, including 24 up-expressed and one down-expressed miRNAs.

2019-12-29 | GSE142692 | GEO

Generating human neural diversity with a multiplexed morphogen screen in organoids

Project description:Morphogens choreograph the generation of remarkable cellular diversity in the developing nervous system. Differen-tiation of stem cells toward particular neural cell fates in vitro often relies upon combinatorial modulation of these signaling pathways. However, the lack of a systematic approach to understand morphogen-directed differentiation has precluded the generation of many neural cell populations, and knowledge of the general principles of regional specification remain incomplete. Here, we developed an arrayed screen of 14 morphogen modulators in human neu-ral organoids cultured for over 70 days. Leveraging advances in multiplexed RNA sequencing technology and anno-tated single cell references of the human fetal brain we discovered that this screening approach generated consid-erable regional and cell type diversity across the neural axis. By deconvoluting morphogen-cell type relationships, we extracted design principles of brain region specification, including critical morphogen timing windows and combinatorics yielding an array of neurons with distinct neurotransmitter identities. Tuning GABAergic neural sub-type diversity unexpectedly led to the derivation of primate-specific interneurons. Taken together, this serves as a platform towards an in vitro morphogen atlas of human neural cell differentiation that will bring insights into hu-man development, evolution, and disease.

2023-05-30 | GSE233574 | GEO

Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia

Project description:Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.

2022-04-01 | GSE199352 | GEO

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