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Next Generation Sequencing Identifies the Heterogeneity of Human Pluripotent Stem Cell-Derived AGM-like Cells

ABSTRACT: Next-generation sequencing (NGS) has significantly advanced the elucidation of developmental signaling mechanisms that are important for different cell lineage formation from human pluripotent stem cells (hPSCs). We report here the application of single cell RNA-sequencing (scRNA-seq) technology for transcriptome profile of hPSC-derived aorta-gonad-mesonephros (AGM)-like endothelial and hematopoietic cells, and compare to those of primary AGM cells. Day 8 and day 18 samples were collected and performed in IIIumina NovaSeq6000. The resulting sequence reads (day 8: 90,000 reads/cells and day 18: 75,000 reads/cell) were mapped to human genome (hg19) using Cell Ranger, and the RefSeq transcript levels (RPKMs) were quantified using the Seurat R package version 3.2. Our scRNA-seq data confirmed the stable expression of key definitive hematopoietic cell markers including SOX17, RUNX1, LMO4 and CD44, and no detectable expression of primitive hematopoietic cell markers, such as GYPA (CD235a), were observed. This study represents the first detailed analysis of AGM-like cell transcriptomes generated by scRNA-seq technology, providing insight into the mechanisms underlying the differentiation of hPSCs into AGM-like cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE157944 | GEO | 2022/05/13

REPOSITORIES: GEO

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Project description:The nuclear LIM-only protein LMO4 is upregulated in breast cancer, especially estrogen receptor negative tumors, and its overexpression in mice leads to hyperplasia and tumor formation. Here, we show that deletion of LMO4 in the mammary glands of mice leads to impaired lobuloalveolar development due to decreased epithelial cell proliferation. With the goal of discovering potential LMO4-target genes, we also developed a conditional expression system in MCF-7 cells for both LMO4 and a dominant negative (DN) form of its co-regulator, Co-factor of LIM domains (Clim/Ldb/Nli). We then used DNA microarrays to identify genes responsive to LMO4 and DN-Clim upregulation. One of the genes common to both datasets was BMP7, whose expression is also significantly correlated with LMO4 transcript levels in a large dataset of human breast cancers, suggesting that BMP7 is a bona fide target gene of LMO4 in breast cancer. Inhibition of BMP7 partially blocks the effects of LMO4 on apoptosis, indicating that BMP7 mediates at least some functions of LMO4. Gene transfer studies show that LMO4 regulates the BMP7 promoter, and chromatin immunoprecipitation studies show that LMO4 and its co-factor Clim2 are recruited to the BMP7 promoter. Furthermore, we demonstrate that HDAC2 recruitment to the BMP7 promoter is inhibited by upregulation of LMO4 and that HDAC2 knockdown upregulates the promoter. These studies suggest a novel mechanism of action for LMO4: LMO4, Clim2 and HDAC2 are part of a transcriptional complex, and increased LMO4 levels can disrupt the complex, leading to decreased HDAC2 recruitment and increased promoter activity. Experiment Overall Design: We used the Tet-off system to establish several distinct MCF-7 cell clones, referred to as MCF7-LMO4-TetOff and MCF7-DN-Clim-TetOff cells, in which the expression of Myc-tagged LMO4 or DN-Clim was repressed by the presence of doxycycline in the medium. Total RNA was isolated from three distinct MCF7-LMO4-TetOff cell lines and three distinct MCF7-DN-Clim-TetOff cell lines in presence and absence of doxycycline after 6 days. Treatment with doxycycline To decrease variability, we pooled RNAs from three experiments for each of the three cell lines.

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Next Generation Sequencing Identifies the Heterogeneity of Human Pluripotent Stem Cell-Derived AGM-like Cells

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