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RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex [3D cerebral organoid]

ABSTRACT: RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex [3D cerebral organoid]

PROVIDER: PRJNA662380 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12611964_1.fastq.gz	Fastqsanger.gz
	SRR12611964_2.fastq.gz	Fastqsanger.gz
	SRR12611965_1.fastq.gz	Fastqsanger.gz
	SRR12611965_2.fastq.gz	Fastqsanger.gz
	SRR12611966_1.fastq.gz	Fastqsanger.gz

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Project description:Cerebral organoids â three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells â have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. 734 single-cell transcriptomes from human fetal neocortex or human cerebral organoids from multiple time points were analyzed in this study. All single cell samples were processed on the microfluidic Fluidigm C1 platform and contain 92 external RNA spike-ins. Fetal neocortex data were generated at 12 weeks post conception (chip 1: 81 cells; chip 2: 83 cells) and 13 weeks post conception (62 cells). Cerebral organoid data were generated from dissociated whole organoids derived from induced pluripotent stem cell line 409B2 (iPSC 409B2) at 33 days (40 cells), 35 days (68 cells), 37 days (71 cells), 41 days (74 cells), and 65 days (80 cells) after the start of embryoid body culture. Cerebral organoid data were also generated from microdissected cortical-like regions from H9 embryonic stem cell derived organoids at 53 days (region 1, 48 cells; region 2, 48 cells) or from iPSC 409B2 organoids at 58 days (region 3, 43 cells; region 4, 36 cells).

Project description:Background: More than 90% mortality of triple negative breast cancer (TNBC) patients are attributed to cancer metastasis with organotropism. Lung is the frequent site of TNBC metastasis. However, the precise mechanism of lung-specific metastasis of TNBC, particularly the driving factors, is not well understood. Methods: RNA sequencing was performed to identify patterns of gene expression associated with lung metastatic behavior using 4T1-LM3, 231-LM3 and their parental cells (4T1-P, 231-P). Expressions of RGCC, encoding the completion 32 protein response protein, were detected in TNBC cells and tissues by qRT-PCR, western blotting and IHC. Kinase activity assay were performed to evaluated PLK1 kinase activity and the amount of phosphorylated AMP-activated protein kinase α2 (AMPKα2). RGCC-mediated metabolism was determined by UHPLC system. Oxidative phosphorylation was evaluated by JC-1 staining and oxygen consumption rate (OCR) assay. Fatty acid oxidation assay, NADPH and ROS was applied to measure the status of RGCC-mediated fatty acid oxidation. The chemical sensitivity of cells was evaluated by CCK8 assay. Results: RGCC is aberrantly upregulated in pulmonary metastatic cells. High level of RGCC is significantly related with lung metastasis rather than other organ metastases. RGCC can effectively promote the kinase activity of PLK1, and activated PLK1 phosphorylates AMPKα2 to facilitate TNBC lung metastasis. Mechanistically, Survival and colonization of TNBC cells in the lungs by upregulating oxidative phosphorylation of mitochondria to obtain more energy and promoting fatty acid oxidation to produce abundant NADPH and sustains redox homeostasis, thus prevents excessive accumulation of potentially detrimental ROS in metastatic tumor cells, ultimately establishing metastases. Importantly, targeting RGCC in combination with paclitaxel/carboplatin effectively suppresses pulmonary metastases of TNBC in a mouse model. Conclusions: The enhanced RGCC is closely associated with lung-specific metastasis of TNBC. RGCC leads to activation of AMPKα2 and downstream signaling through RGCC-driven PLK1 activity to facilitate TNBC lung metastasis, which may determine a potential value of RGCC-driven OXPHOS and fatty acid oxidation as an important therapeutic target.

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RGCC balances self-renewal and neuronal differentiation of neural stem cells in the developing mammalian neocortex [3D cerebral organoid]

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