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A fully automated high throughput-workflow for human neural organoids

ABSTRACT: Recent advances in stem cell technology have led to the development of three-dimensional (3D) culture systems called organoids, which have fueled hopes to bring about the next generation of more physiologically relevant high throughput screens (HTS). However, the adaptation of established organoid protocols for HTS applications has so far been elusive. Here, we present a fully scalable, HTS-compatible workflow for the automated generation, maintenance, whole mount staining, clearing, and optical analysis of human neural organoids generated from neural precursor cells in a standard 96-well format. By combining organoid generation and analysis steps in an automated fashion, we can perform quantitative whole-organoid high content imaging with single cell resolution. The resulting organoids are highly homogeneous with regard to their morphology, size, global gene expression, cellular composition, and structure. Calcium imaging suggests organoid-wide synchronized functional coupling. The scalability of our approach has the potential to form the basis for 3D tissue-based screening in a variety of applications including drug development, toxicology studies, and disease modeling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE119060 | GEO | 2020/09/28

REPOSITORIES: GEO

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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. Sample Nomenclature - Description ------------------------------------------------------------------------- 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). 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. Fetal progenitor organoid 3D organoid structures that arose from culturing isolated fetal epithelial bud tips. 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. Patterned lung organoid (PLO) lung organoids that were generated by differentiating hPSCs, as described throughout the manuscript. Bud tip organoid organoids derived from PLOs, enriched for SOX2/SOX9 co-expressing cells, and grown/passaged in 3F medium.

Project description:We previously established long-term 3D organoid culture systems for several murine tissues (intestine, stomach, pancreas and liver) as well as human intestine and pancreas. Here, we describe culture conditions to generate long-term 3D culture from human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. Human gastric cultures can expand indefinitely in 3D Matrigel. Cultures can be generated from normal tissue, from single sorted stem cells, or from tumor tissue. Organoids maintain many characteristics of the respective tissue in terms of histology, marker expression and euploidy. Organoids from normal tissue express markers of four lineages of the stomach and self-organize in gland and pit-domains. They can be directed to specifically express either lineages of the gastric gland, or the gastric pit by addition of Nicotinamide and withdrawal of Wnt. While gastric pit lineages react marginally to bacterial infection, gastric gland lineages mount a strong inflammatory response. The gastric culture system provides a unique tool to study gastric pathologies. We generated 2 sets of experiments. The first set contains organoids in 4 conditions: (1) organoids in expansion condition ENRWFGNiTi ("gland-type organoids") from 3 donors, (2) organoids as in 1 but differentiated for 4 days in differentiation condition ENR_FGNiTi ("pit'type organoids"), (3) organoids as in 1 but infected with Helicobacter pylori strain P12 MOI 50 for 2 h, (4) organoids as in 2 but infected as in 3. All 4 conditions were tested on the same organoid line in parallel. This experiment was conducted independently with cultures from 3 different donors. The second set of experiments compares freshly isolated glands with organoids. Samples from 2 patients were analyzed. Each patient received a total gastrectomy. From each patient, glands from corpus region or pyloric antrum were isolated. From each isolation, one aliquod was stored for microarray analysis and one aliquod used to generate organoids. Organoids and glands were subsequently lysed and analyzed in parallel.

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A fully automated high throughput-workflow for human neural organoids

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