Project description:Here, we used a lentiviral-based barcoding method (CellTagging) to lineage trace progenitor cells in a human lung organoid model. 21-day cultures were transduced for 7 days, fluorescence activated cell sorted for GFP+ expression, and regrown as sparsely plated cells for additional 30 days before sequencing. Cultures were grown in serum-free basal media supplemented with FGF-10, A-8301, NOGGIN, and Y-27632 (‘DSI media’).
Project description:Underdeveloped lungs are the primary cause of death in premature infants, however, little is known about stem and progenitor cell maintenance during human lung development. In this study, we have identified that FGF7, Retinoic Acid and CHIR-99021, a small molecule that inhibits GSK3 to activate Wnt signaling, support in vitro maintenance of primary human fetal lung bud tip progenitor cells in a progenitor state. Furthermore, these factors are sufficient to derive a population of human bud tip-like progenitor cells in 3D organoid structures from human pluripotent stem cells (hPSC). Functional studies showed that hPSC-derived bud tip progenitor organoids do not contain any mesenchymal cell types, maintain multilineage potential in vitro and are able to engraft into the airways of injured mice and respond to systemic factors. We performed RNA-sequencing to assess the degree of similarity in global gene expression profiles between the full human fetal lung (59-127 days gestation), isolated human fetal bud tip progenitors, organoids grown from primary fetal bud tip progenitors, and hPSC-derived bud tip organoids. Results showed that hPSC-derived organoids have molecular profiles similar to organoids generated from primary human fetal lung tissue. Gene expression differences between hPSC-derived bud tip organoids and fetal progenitor organoids may be related to the presence of contaminating mesenchymal cells in primary cultures. hPSC-derived bud tip organoids are generated from a well-defined human cell sources, offering a distinct advantage over rare primary tissue as a means to study human specific lung development, homeostasis and disease.<br>Sample Nomenclature - Description<br> -------------------------------------------------------------------------<br> Peripheral fetal lung the distal/peripheral portion of the fetal lung (i.e., distal 0.5 cm) was excised from the rest of the lung using a scalpel. This includes all components of the lung (e.g., epithelial, mesenchymal, vascular). <br>Isolated fetal bud tip the bud peripheral portion of the fetal lung was excised with a scalpel and subjected to enzymatic digestion and microdissection. The epithelium was dissected and separated from the mesenchyme, but a small amount of associated mesenchyme likely remained. <br>Fetal progenitor organoid 3D organoid structures that arose from culturing isolated fetal epithelial bud tips. <br>Foregut spheroid 3D foregut endoderm structure as described in Dye et al. (2015). Gives rise to patterned lung organoid (PLO) when grown in 3F medium. <br> Patterned lung organoid (PLO) lung organoids that were generated by differentiating hPSCs, as described throughout the manuscript. <br> Bud tip organoid organoids derived from PLOs, enriched for SOX2/SOX9 co-expressing cells, and grown/passaged in 3F medium.
Project description:The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically-defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia.
Project description:The biggest problem with lung cancer organoid (LCO) is that most lung cancer organoids are derived from surgical specimens of early-stage lung cancer patients. In fact, patients who initially need chemotherapy are patients with advanced lung cancer who cannot undergo a surgery. So, making lung organoids from biopsy specimens successfully is an urgent task. The success rate of culturing LCO from biopsy tissues is very low because conventional lung biopsies such as transthoracic needle biopsy and forcep biopsy only get a small amount of lung tissue. The possibility of critical complications such as bleeding and pneumothorax makes it difficult to obtain enough specimens. Overgrowth of normal lung cells in late passages is also a critical problem in LCO and optimized culture conditions for LCO remain to be identified. To overcome the hurdles of lung cancer organoid, we made LCOs from cryobiopsy specimens of all stages of lung cancer patients. Transbronchial cryobiopsy can obtain over 10 times greater volume of tissue compared to bronchoscopic forceps biopsy and it can be applied to even peripheral lesions with radial endobronchial ultrasonography. The success rate of LCO culture was also markedly improved and recapitulated the characteristics of primary tumors. LCOs derived from cryobiopsy specimens can overcome the critical limitations of present lung cancer organoids. We expect that cryobiopsy will be a breakthrough strategy of clinical application of LCO in all stages of lung cancer.
Project description:Lung organoids made from pluripotent stem cells have the potential to enhance our understanding of disease mechanisms in pediatric lung disorders. As proof of concept, we have established a reproducible ex vivo model of lung organoid development derived from human induced pluripotent stem cells generated from fetuses and infants with Bockdalek congenital diaphragmatic hernia (CDH), a polygenic disorder associated with fetal lung compression and often lethal pulmonary hypoplasia at birth. We used microarrays to compare transcriptomes among the different cell types focusing on genes associated with lung development and extracellular matrix. We seek to identity anomalous gene expression during lung development, using lung organoids generated from hiPS of patients with congenital diafragmatic hernia.
Project description:Human fetal lung tip cells were purifed from developing human lungs at pseudoglandular and canalicular stages and in vitro cultured as organoids. Whole transcriptomic data for each organoid line at different stages were profiled by RNA seq.
Project description:Organoid cultures derived from menstrual flow faithfully replicate secretory phase endometrial glands and provide a novel, non-invasive technique for the clinical assessment of endometrial function. Paired organoid cultures derived from scratch biopsies and ensuing menstrual flow share the same transcriptome signature, responses to early pregnancy hormones, and pathological changes in cases of early-pregnancy loss. The technique opens new avenues for investigating endometrial function in cases of subfertility and gynaecological disorders.
Project description:The aim of this study was to determine the effects of TGFβ at the premalignant stage of CRC development. Organoid cultures were isolated from normal colon and from tubular adenomas. One normal colon culture was genetically engineered using the CRISPR-Cas9 system to carry the BRAFV600E mutation. Organoid cultures of three different tubular adenomas and two normal colon samples, and one BRAFV600E-mutant organoid culture received TGFβ or control treatment after which gene expression was analyzed by microarray (Affymetrix U133+ PM Genechip Array).
Project description:Murine small intestinal organoids were cultured in the presence or absence of 5µM inhibitor MS023 (PubChem CID 92136227), which targets type I protein arginine methyltransferases Prmt1, Prmt3, Prmt4, Prmt6, Prmt8. Organoids were grown in culture media containing EGF, Noggin and R-spondin (ENR), media was changed after 48h, and organoids were harvested after 120h.