Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Patient-derived head and neck cancer organoids allow treatment stratification and serve as a tool for biomarker validation and identification.

BACKGROUND: Organoids are in vitro three-dimensional structures that can be grown from patient tissue. Head and neck cancer (HNC) is a collective term used for multiple tumor types including squamous cell carcinomas and salivary gland adenocarcinomas. METHODS: Organoids were established from HNC patient tumor tissue and characterized using immunohistochemistry and DNA sequencing. Organoids were exposed to chemo- and radiotherapy and a panel of targeted agents. Organoid response was correlated with patient clinical response. CRISPR-Cas9-based gene editing of organoids was applied for biomarker validation. FINDINGS: A HNC biobank consisting of 110 models, including 65 tumor models, was generated. Organoids retained DNA alterations found in HNC. Comparison of organoid and patient response to radiotherapy (primary [n = 6] and adjuvant [n = 15]) indicated potential for guiding treatment options in the adjuvant setting. In organoids, the radio-sensitizing potential of cisplatin and carboplatin could be validated. However, cetuximab conveyed radioprotection in most models. HNC-targeted treatments were tested on 31 models, indicating possible novel treatment options with the potential for treatment stratification in the future. Activating PIK3CA mutations did not predict alpelisib response in organoids. Protein arginine methyltransferase 5 (PRMT5) inhibitors were identified as a potential treatment option for cyclin-dependent kinase inhibitor 2A (CDKN2A) null HNC. CONCLUSIONS: Organoids hold potential as a diagnostic tool in personalized medicine for HNC. In vitro organoid response to radiotherapy (RT) showed a trend that mimics clinical response, indicating the predictive potential of patient-derived organoids. Moreover, organoids could be used for biomarker discovery and validation. FUNDING: This work was funded by Oncode PoC 2018-P0003.

Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation.

Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach, small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels, satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment, which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens for genes that program cell fate.

Continuously Grooved Stent Struts for Enhanced Endothelial Cell Seeding.

PURPOSE: Implantation of pre-endothelialized stents could enhance cellular recovery of a damaged vessel wall provided attached cells remain viable, functional and are present in sufficient numbers after deployment. The purpose of this study was to evaluate the feasibility of grooved stainless steel (SS) stents as a primary endothelial cell (EC) carrier with potentially enhanced EC protection upon stent deployment. MATERIALS AND METHODS: Attachment and behavior of enzymatically harvested human adult venous ECs seeded onto gelatin-coated vascular stents were evaluated in an in vitro setting. Smooth and grooved SS stents and smooth nitinol stents were studied. RESULTS: All cells expressed EC markers vWF and CD31. Using rotational seeding for a period of 16-24 h, ECs attached firmly to the stents with sufficient coverage to form a confluent EC monolayer. The grooved SS wire design was found to enable attachment of three times the number of cells compared to smooth wires. This also resulted in an increased number of cells remaining on the stent after deployment and after pulsatile flow of 180 ml/min for 24 h, which did not result in additional EC detachment. CONCLUSIONS: The grooved stent provides a potential percutaneous means to deliver sufficient numbers of viable and functional cells to a vessel segment during vascular intervention. The grooves were found to offer a favorable surface for EC attachment and protection during stent deployment in an in vitro setting.