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:There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac).
Project description:Organoids are in vitro 3D models that are generated using stem cells to study organ development and regeneration. Despite the extensive research on lung organoids, there is limited information on pig lung cell generation or development. Here, we identified five epithelial cell types along with their characteristic markers using scRNA-seq. Additionally, we found that NKX2.1 and FOXA2 acted as the crucial core transcription factors in porcine lung development. The presence of SOX9/SOX2 double positive cells was identified as a key marker for lung progenitor cells. The Monocle algorithm was used to create a pseudo-temporal differentiation trajectory of epithelial cells, leading to the identification of signaling pathways related to porcine lung development. Moreover, we established the differentiation method from porcine pluripotent stem cells (pPSCs) to SOX17+FOXA2+ definitive endoderm and NKX2.1+FOXA2+CDX2- anterior foregut endoderm (AFE). The AFE are further differentiated into lung organoids while closely monitoring the differentiation process. We showed that NKX2.1 overexpression facilitated the induction of lung organoids and supported subsequent lung differentiation and maturation. This model offers valuable insights into studying the interaction patterns between cells and the signaling pathways during the development of the porcine lung.
Project description:Purpose: The goal of this study is to establish and molecularly characterize non-small cell lung cancer (NSCLC) organoids. Generation of NSCLC organoids would provide additional preclinical models for drug screening and biomarker discovery. Methods: Patient lung tumors and previously established patient-derived xenografts (PDX) were processed to generate organoids. Total RNA was extracted and subjected to RNA-seq to examine the gene expression similarity between patient/PDX/organoid of the same model using t-SNE clustering. Results: Through RNA-sequencing, we generated TPM values of patient, PDX and organoid samples of 5 models. t-SNE analysis showed that the patient/PDX/organoid of the same models clustered together. The lung adenocarcinoma samples formed a separate cluster from the squamous cell carcinoma samples based on gene expression. Conclusions: Our study introduces the establishment of NSCLC organoids and demonstrates the gene expession similarity of the organoid model to its corresponding PDX and patient sample.