ScRNA-seq of pluripotent stem cell derived human intestinal organoids, adult intestine, and human fetal endoderm derived organs
ABSTRACT: Here, we used single cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human definitive endoderm and intestinal organoids (HIOs) at several timepoints of in vitro growth (7, 14, and 28 days) and after in vivo growth beneath the kidney capsule of a murine host (4 and 8 wks post-transplant). Additionally, we profiled HIOs grown in a non-adhesive alginate hydrogel and also CDX2 knockout HIOs. In order to benchmark the organoid cultures, we used scRNA-seq to profile primary human fetal esophagus (14.3 pcw, 16.7 pcw), stomach (6.7, 14.3, and 16.7 pcw), liver (14.4 pcw), small intestine ( 11.4 and 14.4 pcw) and colon (11.4, 14.4, and 18.9 pcw). Diverse cell lineages were captured across all tissues profiled, including: epithelium, mesenchyme, neurons, endothelium, and immune lineages.
Project description:Here, we used single cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human intestinal organoids (HIOs) grown in matrigel or a non-adhesive alginate hydrogel after 28 days of in vitro growth. Additionally, we used scRNA-seq to profile HIOs derived in the presence of Neuregulin 1 (NRG1) and/or EGF after 40 days of in vitro growth.
Project description:Here, we used single-cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human intestinal organoids (HIOs) grown in an alginate matrix after 3, 7, and 14 days of in vitro growth. Samples were grown in minigut media supplemented with either ENR or EGF.
Project description:The development of human pluripotent stem cell (hPSC)- derived small intestinal organoids (HIOs) has led to an improved understanding of human intestinal development and physiology. HIOs generated using directed differentiation lack some cellular populations found in the native organ, including vasculature. We performed single cell RNA sequencing (scRNA-seq) on approximately 13,000 cells at various timepoints (0, 3, 7, and 14 days) across HIO in vitro development and observed a transient population of endothelial-like cells (ECs) present within HIOs early during differentiation. Our data demonstrate that EC-like cells fail to be robustly maintained in long term culture. Here, we have developed a new directed differentiation approach to enhance co-differentiation and maintenance of ECs within HIOs, leading to the development of vascularized HIOs (vHIOs). scRNAseq was used to compare vHIOs to control HIOs after 59d months in culture.
Project description:Here, we performed single cell RNA sequencing (scRNA-seq) of human fetal kidney tissue samples from 2 individual biological specimens (13.7 and 15.4 weeks gestation). The data set is composed of approximately 10,000 cells from diverse renal lineages. Lineages captured include nephron progenitors, epithelium, stroma, immune, and endothelium.
Project description:Here, we performed single cell RNA sequencing (scRNA-seq) of human fetal ileum tissue samples from 3 individual biological specimens ages 11.4, 14.4, and 18.9 weeks post conception. The data set is composed of over 16,000 cells from diverse intestinal lineages.
Project description:Underdeveloped lungs are a primary cause of morbidity and mortality in premature infants, but our ability to help these patients by speeding up lung development are hindered by a lack of understanding of human lung developmental biology. Here, we performed single cell RNA sequencing of the human fetal lung from samples spanning from 11.5 weeks gestation to 21 weeks gestation from the distal lung, middle airways, and the tracheal epithelium. The primary goal of this experiment was to define fetal cell states to serve as a gold standard for pluripotent stem cell-derived lung cells and tissues, and to identify potential signaling pathways that drive differentiation of lung progenitor cells to mature cell types. Additionally, we generated bud tip progenitor organoids from 12 week human fetal lung bud tip progenitors. We show that treatment of bud tip progenitor organoids with a short pulse of dual SMAD activation (BMP4+TGFb1) led to the upregulation of lung basal cell markers, a cell type that serves as a critical stem cell for the adult airway, and that further treatment with dual SMAD inhibition leads to the generation of airway-like organoids containing differentiated cell types of the adult airway, including basal stem cells.
Project description:Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. Therapeutic targeting of the CSF1R pathway is a promising strategy in many human diseases, including neurological disorders or cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish csf1ra and csf1rb in adult myelopoiesis using single-cell RNA sequencing. Our analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors and that the expression of csf1ra and csf1rb is non-overlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.
Project description:Photo-damage represents an important aspect of cutaneous carcinogenesis. The photo-damaged microenvironment significantly facilitates progression of malignant melanoma. Therefore human primary dermal fibroblasts from photo-damaged adult skin and juvenile sun-protected skin were isolated and expanded as described by Dvorankova et al. (PMID: 29675784). Low passage fibroblasts were mixed in suspension (in ratio 1:1) with G361 malignant melanoma cell line (CVCL_1220). Using the hanging drop technique (PMID: 29786551), the melanoma/fibroblast spheres containing 50,000 cells per sphere were formed during the next 72 hours. The organoids were consequently maintained for another 2 days in non-adhesive culture wells in complete DMEM culture medium. At this time, heterogeneous spheres were harvested for immunohistochemical analysis or for further invasion studies. Single-cell suspensions from heterogeneous spheres were analysed by single-cell RNA sequencing (10x Chromium V3) to reveal the transcriptional heterogeneity of model cell populations under given conditions with emphasis on the features of dermal fibroblasts exposed to the influence of malignant melanoma.
Project description:Bulk RNA-sequencing experiments were performed to analyze the transcriptomic effects of such integrations into two newly established genomic safe harbor sites. Jurkat and HEK293T cells were edited to integrate CMV-mRuby expressing cassette into Rogi2 genomic safe harbor site using Cas9 RNP
Project description:Here, we used single-cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human intestinal organoids (HIOs) grown in suspension culture after 28 days of in vitro growth. Grown in minigut media supplemented with EGF.