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Differentiation of PSC into lung organoids with endogenous functional alveolar macrophages and blood vessels

ABSTRACT: The goals of this study are to determine tissue composition of human lung organoids (hLOs) when maintained long-term (day 230). hLOs consist of alveolar epithelial cells, mesenchymal/endothelial cells, smooth muscle cells and immune cells. After dissociated hLOs, a target capture of 1x 104 cells was performed using the 10X Genomics Chromium Single Cell RNA sequencing. Briefly, single cell gel bead-in-emulsions(GEMs) are generated by passing cells with enzyme mix, partitioning oil, and barcoded gel beads by 10X Chromium chip. After GEM formation, the gel bead is dissolved and co-partitioned cell is lysed. Subsequently, reverse transcription (RT) occurs inside GEMs and barcoded full-length cDNA is generated. After RT, amplified cDNAs with barcode sequences (cell index and UMI) are pooled and single cell library is constructed using the Single Cell 3` Reagent Kit (v3 chemistry). The resulting library was sequenced on illumina HiseqX platform with 150bp paired end. Raw base calling files generated by illumine sequencing were demultiplexed based on the sample index read to generate FASTQ files using the 10X Genomics Cell Ranger (v3.0.2) pipeline. The raw reads were trimmed from the 3’ end to get the recommended number of cycle for read pairs (read1: 28bp; read2 : 91bp). The trimmed reads were mapped to the hg38 reference human genome with subsequent analysis, including filtering, barcode counting, and UMI counting. The resulting data were used to generate the multidimensional feature-barcode matrix using the CellRanger count with default parameters.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142838 | GEO | 2020/01/03

REPOSITORIES: GEO

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Project description:Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/−) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/− mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. To obtain a more comprehensive understanding of the changes within HSPCs at the molecular level, we employed single-cell RNA-sequencing (scRNAseq) using both WT and Son+/− bone marrow HSPCs isolated and sorted based on their Lin− cKit+ (LK) cell markers (24,355 total LK cells from 2 WT mice; 31,232 total LK cells from 2 Son+/− mice. The linage-marker-negative (Lin-) c-kit+ population containing hematopoietic stem and progenitor cells was sorted from dissociated mouse bone marrow cells by flow cytometry. Sorted single-cell suspension, 10x barcoded gel beads and oil (10xGenomics Chromium Next GEM Single Cell 3' Kit v3.1 PN 1000268) were loaded into 10x Chromium™ Single Cell Chip to capture single cells into nanoliter-scale oil droplets by 10xChromium X Controller and generate Gel Bead-In-EMulsions (GEMs). cDNA from single cells were synthesized and barcoded by incubation of the GEMs in a thermocycler machine and then purified from GEMs by DynaBeads (10xGenomics PN 2000048). cDNA was pre-amplified by PCR to generate sufficient mass for sequencing library construction. The single-cell cDNA libraries were constructed by following 10xGenomics 3’ NextGem 3.1 version kit instructions. The final constructed 3’-biased single-cell libraries were sequenced by the Illumina Novaseq6000 machine, targeting 50,000 read pairs/cell. We identified hematopoietic abnormalities in Son+/− mice, including leukocytopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing genes required for lymphoid and B-cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.

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Differentiation of PSC into lung organoids with endogenous functional alveolar macrophages and blood vessels

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