Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2.

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.

Tissue-Engineering the Intestine: The Trials before the Trials.

Building complex tissues requires the development of innovative interdisciplinary engineering solutions. In this Forum, the INTENS Consortium discuss experimental considerations and challenges for generating a tissue-engineered intestine for the treatment of short bowel syndrome, taking into account cell source, scaffold choice, and design strategy for achieving proper assembly and function.

A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.

In the germarium of the Drosophila ovary, germline cysts are encapsulated one at a time by a follicular epithelium derived from two follicle stem cells (FSCs). Ovaries in flies mutant for the serine/threonine kinase Pak exhibit a novel phenotype, in which two side-by-side cysts are encapsulated at a time, generating paired egg chambers. This striking phenotype originates in the pupal ovary, where the developing germarium is shaped by the basal stalk, a stack of cells formed by cell intercalation. The process of basal stalk formation is not well understood, and we provide evidence that the cell intercalation is driven by actomyosin contractility of DE-Cadherin-adhered cells, leading to a column of disk-shaped cells exhibiting a novel radial cell polarity. Cell intercalation fails in Pak mutant ovaries, leading to abnormally wide basal stalks and consequently wide germaria with side-by-side cysts. We present evidence that Pak mutant germaria have extra FSCs, and we propose that contact of a germline cyst with the basal stalk in the pupal ovary contributes to FSC niche formation. The wide basal stalk in Pak mutants enables the formation of extra FSC niches which are mispositioned and yet functional, indicating that the FSC niche can be established in diverse locations.

FACS purification of Drosophila larval neuroblasts for next-generation sequencing.

Elegant tools are available for the genetic analysis of neural stem cell lineages in Drosophila, but a methodology for purifying stem cells and their differentiated progeny for transcriptome analysis is currently missing. Previous attempts to overcome this problem either involved using RNA isolated from whole larval brain tissue or co-transcriptional in vivo mRNA tagging. As both methods have limited cell type specificity, we developed a protocol for the isolation of Drosophila neural stem cells (neuroblasts, NBs) and their differentiated sibling cells by FACS. We dissected larval brains from fly strains expressing GFP under the control of a NB lineage-specific GAL4 line. Upon dissociation, we made use of differences in GFP intensity and cell size to separate NBs and neurons. The resulting cell populations are over 98% pure and can readily be used for live imaging or gene expression analysis. Our method is optimized for neural stem cells, but it can also be applied to other Drosophila cell types. Primary cell suspensions and sorted cell populations can be obtained within 1 d; material for deep-sequencing library preparation can be obtained within 4 d.

The Par complex and integrins direct asymmetric cell division in adult intestinal stem cells.

The adult Drosophila midgut is maintained by intestinal stem cells (ISCs) that generate both self-renewing and differentiating daughter cells. How this asymmetry is generated is currently unclear. Here, we demonstrate that asymmetric ISC division is established by a unique combination of extracellular and intracellular polarity mechanisms. We show that Integrin-dependent adhesion to the basement membrane induces cell-intrinsic polarity and results in the asymmetric segregation of the Par proteins Par-3, Par-6, and aPKC into the apical daughter cell. Cell-specific knockdown and overexpression experiments suggest that increased activity of aPKC enhances Delta/Notch signaling in one of the two daughter cells to induce terminal differentiation. Perturbing this mechanism or altering the orientation of ISC division results in the formation of intestinal tumors. Our data indicate that mechanisms for intrinsically asymmetric cell division can be adapted to allow for the flexibility in lineage decisions that is required in adult stem cells.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation.

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

Fly stem cell research gets infectious.

To maintain tissue homeostasis, stem cells need to increase their proliferation rate to repair tissue damage caused by stress or infection. In this issue, Jiang et al. (2009) describe a regulatory feedback mechanism involving the Jak/Stat signaling pathway that enables stem cells of the fly midgut to accomplish this task.

The serine/threonine kinase dPak is required for polarized assembly of F-actin bundles and apical-basal polarity in the Drosophila follicular epithelium.

During epithelial development cells become polarized along their apical-basal axis and some epithelia also exhibit polarity in the plane of the tissue. Mutations in the gene encoding a Drosophila Pak family serine/threonine kinase, dPak, disrupt the follicular epithelium that covers developing egg chambers during oogenesis. The follicular epithelium normally exhibits planar polarized organization of basal F-actin bundles such that they lie perpendicular to the anterior-posterior axis of the egg chamber, and requires contact with the basement membrane for apical-basal polarization. During oogenesis, dPak becomes localized to the basal end of follicle cells and is required for polarized organization of the basal actin cytoskeleton and for epithelial integrity and apical-basal polarity. The receptor protein tyrosine phosphatase Dlar and integrins, all receptors for extracellular matrix proteins, are required for polarization of the basal F-actin bundles, and for correct dPak localization in follicle cells. dpak mutant follicle cells show increased beta(Heavy)-spectrin levels, and we speculate that dPak regulation of beta(Heavy)-spectrin, a known participant in the maintenance of membrane domains, is required for correct apical-basal polarization of the membrane. We propose that dPak mediates communication between the basement membrane and intracellular proteins required for polarization of the basal F-actin and for apical-basal polarity.

dPak is required for integrity of the leading edge cytoskeleton during Drosophila dorsal closure but does not signal through the JNK cascade.

The Pak kinases are effectors for the small GTPases Rac and Cdc42 and are divided into two subfamilies. Group I Paks possess an autoinhibitory domain that can suppress their kinase activity in trans. In Drosophila, two Group I kinases have been identified, dPak and Pak3. Rac and Cdc42 participate in dorsal closure of the embryo, a process in which a hole in the dorsal epidermis is sealed through migration of the epidermal flanks over a tissue called the amnioserosa. Dorsal closure is driven in part by an actomyosin contractile apparatus at the leading edge of the epidermis, and is regulated by a Jun amino terminal kinase (JNK) cascade. Impairment of dPak function using either loss-of-function mutations or expression of a transgene encoding the autoinhibitory domain of dPak led to disruption of the leading edge cytoskeleton and defects in dorsal closure but did not affect the JNK cascade. Group I Pak kinase activity in the amnioserosa is required for correct morphogenesis of the epidermis, and may be a component of the signaling known to occur between these two tissues. We conclude that dorsal closure requires Group I Pak function in both the amnioserosa and the epidermis.