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Single cell RNA-seq analysis of Tbx4+ and Tbx4- mouse lung mesenchymal cells

ABSTRACT: Single cell RNA-seq analysis of Tbx4+ and Tbx4- mouse lung mesenchymal cells

PROVIDER: PRJNA531191 | ENA |

REPOSITORIES: ENA

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Single cell RNA-seq analysis of Tbx4+ and Tbx4- mouse lung mesenchymal cells

Project description:Purpose: The goals of this study are to analyze Tbx4 lineage+ and Tbx4 lineage- cells sorted from Tbx4cre;RosaTm mouse lung. Methods: Tbx4 lineage+ and Tbx4 lineage- cells within epcam+ CD31- CD45- 7AAD- cells were sorted from Tbx4cre;RosaTm mouse lung, and run on the C1 chip (Fluidigm). Cell capture, lysis, reverse transcription and cDNA amplification was performed on the C1 IFC for mRNA-seq on Fluidigm C1 Single-Cell Auto Prep System following the manufacturer’s protocol. Results: C1 single cell RNA-seq platform captured and sequenced 54 Tbx4 lineage+ cells and 39 Tbx4 lineage - cells. Conclusions: Our study analyzed transcriptomes of Tbx4 lineage+ and Tbx4 lineage- lung mesenchymal cells. This study provides a framework for the application of transcriptome profiling towards characterization of differentiated genes Tbx4 lineage+ and Tbx4 lineage - cells.

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Single cell RNAseq of co-cultured mouse iPSC-derived lung mesenchyme and mouse iPSC-derived lung epithelial progenitors in distal and proximal differentiation media

Project description:Co-cultures of lung epithelium and mesenchyme are useful tools to study epithelial-mesenchymal crosstalk in lung development and disease. However, many previous attempts to generate such co-cultures have yielded poor juxtaposition between the epithelial and the mesenchymal lineage. In addition, induced pluripotent stem cell (iPSC)-derived co-cultures often contain generic mesenchyme that is not necessarily lung-specific. We sought to establish co-cultures of purified mouse iPSC-derived lung-specific mesenchyme and iPSC-derived lung epithelial progenitors. We used a mouse iPSC line carrying a lung mesenchyme-specific reporter/tracer (Tbx4-LERGFP) to generate lung mesenchymal progenitors by directed differentiation via a lateral plate mesodermal progenitor state (induced lung mesenchyme, iLM). In parallel we differentiated a mouse embryonic stem (ES) cell line carrying a Nkx2-1mCherry reporter into lung epithelial progenitor cells using our established directed differentiation protocol. We then combined the purified lung epithelial and mesenchymal progenitor cells and co-cultured them in distal or proximal differentiation media for 1 week on Matrigel. We find that cells self-organize into complex 3-dimensional organoids with closely juxtaposed epithelial and mesenchymal cells. Furthermore, co-culture affects the molecular phenotype of both lineages. Our iPSC-derived co-culture model can provide an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics.

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Investigation of the gene expression changes triggered by the lung mesenchymal Tsc2KO in each major cell subpopulation of the adult mouse lung

Project description:Tumor suppressor Tuberous sclerosis complex 2 (TSC2) is a key negative regulator of mammalian target of rapamycin (mTOR), a central controller of cell growth and metabolism in health and disease. Loss of TSC2 induces the constitutive activation of mTORC1 in rare lung disease pulmonary Lymphangioleiomyomatosis (LAM), which affects only women of childbearing age and characterized by lung destruction and progressive loss of pulmonary function. Little is known how TSC2 loss induces LAM and what is the LAM cell of origin. To determine cell-type specific effects of TSC2 loss and mTORC1 activation on lung homeostasis we generated new transgenic mice with targeted Tsc2 deletion in lung mesenchyme Tbx4-Cre+Tsc2flox/flox and lung epithelium Shh-Cre+Tsc2flox/flox. Both Tbx4-Cre+, Tsc2flox/flox and Shh-Cre+, Tsc2flox/flox mice were viable and fertile. Adult Tbx4-Cre+Tsc2flox/flox mice demonstrated mTORC1 activation in lung mesenchyme, progressive alveolar enlargement, female-specific pulmonary function decline and prgenancy-induced lesion growth. To investigate molecular mechanism underlying observed phenotypic changes in the Tsc2KO lungs we performed comparative bulk-RNAseq analysis of the gene expression changes in the major cell subpopulations of the adult female 8-week-old Tsc2KO mouse lungs compared to the age- and sex-matched WT controls.

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