Microarrayed human bone marrow organoids for modeling blood stem cell dynamics.

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such an endothelial compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays.

Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays.

BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

Diagnostic tools and CFTR functional assays in cystic fibrosis: utility and availability in Switzerland.

Cystic fibrosis (CF) is a genetic disease caused by a bi-allelic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. When the diagnosis cannot be confirmed by a positive sweat test or/and the identification of two CF-causing variants, international guidelines recommend the use of CFTR functional assays. These tests assess whether CFTR activity is normal or diminished/absent through measurement of CFTR-mediated chloride secretion/absorption. CFTR functional assays are not only useful for diagnostic purposes but can also serve as a surrogate outcome for clinical trials of CFTR modulators, which are emerging therapeutic agents designed to correct the malfunctioning protein. In the near future they could also be used as precision-medicine techniques, to help guidance and optimisation of treatment. Until now, sweat testing has been the only CFTR functional assay available in Switzerland. Since 2020, the Centre Hospitalier Universitaire Vaudois (CHUV) at Lausanne and the Lucerne Children’s Hospital perform nasal potential difference measurement. Moreover, The Ecole Polytechnique Fédérale de Lausanne (EPFL) established a reliable procedure to generate adult intestinal organoids, i.e., stem cell-derived in-vitro grown mini tissues, extracted from rectal biopsies, which can be used to assess CFTR function in vitro. This narrative review describes the most popular CFTR functional assays, as well as their indications, limitations and availability in Switzerland.

High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays.

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

Smart Hydrogels for the Augmentation of Bone Regeneration by Endogenous Mesenchymal Progenitor Cell Recruitment.

The treatment of bone defects with recombinant bone morphogenetic protein-2 (BMP-2) requires high doses precluding broad clinical application. Here, a bioengineering approach is presented that strongly improves low-dose BMP-2-based bone regeneration by mobilizing healing-associated mesenchymal progenitor cells (MPCs). Smart synthetic hydrogels are used to trap and study endogenous MPCs trafficking to bone defects. Hydrogel-trapped and prospectively isolated MPCs differentiate into multiple lineages in vitro and form bone in vivo. In vitro screenings reveal that platelet-derived growth factor BB (PDGF-BB) strongly recruits prospective MPCs making it a promising candidate for the engineering of hydrogels that enrich endogenous MPCs in vivo. However, PDGF-BB inhibits BMP-2-mediated osteogenesis both in vitro and in vivo. In contrast, smart two-way dynamic release hydrogels with fast-release of PDGF-BB and sustained delivery of BMP-2 beneficially promote the healing of bone defects. Collectively, it is shown that modulating the dynamics of endogenous progenitor cells in vivo by smart synthetic hydrogels significantly improves bone healing and holds great potential for other advanced applications in regenerative medicine.

Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis.

Modeling the interaction between the supportive stroma and the hematopoietic stem and progenitor cells (HSPC) is of high interest in the regeneration of the bone marrow niche in blood disorders. In this work, we present an injectable co-culture system to study this interaction in a coherent in vitro culture and in vivo transplantation model. We assemble a 3D hematopoietic niche in vitro by co-culture of supportive OP9 mesenchymal cells and HSPCs in porous, chemically defined collagen-coated carboxymethylcellulose microscaffolds (CCMs). Flow cytometry and hematopoietic colony forming assays demonstrate the stromal supportive capacity for in vitro hematopoiesis in the absence of exogenous cytokines. After in vitro culture, we recover a paste-like living injectable niche biomaterial from CCM co-cultures by controlled, partial dehydration. Cell viability and the association between stroma and HSPCs are maintained in this process. After subcutaneous injection of this living artificial niche in vivo, we find maintenance of stromal and hematopoietic populations over 12 weeks in immunodeficient mice. Indeed, vascularization is enhanced in the presence of HSPCs. Our approach provides a minimalistic, scalable, biomimetic in vitro model of hematopoiesis in a microcarrier format that preserves the HSPC progenitor function, while being injectable in vivo without disrupting the cell-cell interactions established in vitro.

Artificial niche microarrays for probing single stem cell fate in high throughput.

To understand the regulatory role of niches in maintaining stem-cell fate, multifactorial in vitro models are required. These systems should enable analysis of biochemical and biophysical niche effectors in a combinatorial fashion and in the context of a physiologically relevant cell-culture substrate. We report a microengineered platform comprised of soft hydrogel microwell arrays with modular stiffness (shear moduli of 1-50 kPa) in which individual microwells can be functionalized with combinations of proteins spotted by robotic technology. To validate the platform, we tested the effect of cell-cell interactions on adipogenic differentiation of adherent human mesenchymal stem cells (MSCs) and the effect of substrate stiffness on osteogenic MSC differentiation. We also identified artificial niches supporting extensive self-renewal of nonadherent mouse neural stem cells (NSCs). Using this method, it is possible to probe the effect of key microenvironmental perturbations on the fate of any stem cell type in single cells and in high throughput.