Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

Gamma secretase modulators and BACE inhibitors reduce Aß production without altering gene expression in Alzheimer's disease iPSC-derived neurons and mice.

In drug discovery, as well as in the study of disease biology, it is fundamental to develop models that recapitulate aspects of a disorder, in order to understand the pathology and test therapeutic approaches. Patient-derived induced pluripotent stem cells (iPSCs) offer the potential of obtaining tissue-specific cells with a given human genotype. Here we derived neural cultures from Alzheimer's disease patient iPSCs and characterized their response to three classes of compounds that reduce the production of Aß42, a major driving force of this pathology. We characterized their effect on the cells, looking at Tau proteostasis and gene expression changes by RNAseq. ß-secretase inhibitor and γ-secretase modulators left the transcriptional balance of the cells virtually unaffected, while γ-secretase inhibitors caused drastic gene expression changes due to Notch inhibition. We observed similar effects in vivo, treating mice with the same compound classes. Our results show that ß-secretase inhibitors and γ-secretase modulators are attractive candidates for modulating Aß production in Alzheimer's disease. Moreover, we demonstrate that the response to compounds obtained with iPSC-derived neurons is similar to the one observable in vivo.

Large-Scale Production of Human iPSC-Derived Macrophages for Drug Screening.

Tissue-resident macrophages are key players in inflammatory processes, and their activation and functionality are crucial in health and disease. Numerous diseases are associated with alterations in homeostasis or dysregulation of the innate immune system, including allergic reactions, autoimmune diseases, and cancer. Macrophages are a prime target for drug discovery due to their major regulatory role in health and disease. Currently, the main sources of macrophages used for therapeutic compound screening are primary cells isolated from blood or tissue or immortalized or neoplastic cell lines (e.g., THP-1). Here, we describe an improved method to employ induced pluripotent stem cells (iPSCs) for the high-yield, large-scale production of cells resembling tissue-resident macrophages. For this, iPSC-derived macrophage-like cells are thoroughly characterized to confirm their cell identity and thus their suitability for drug screening purposes. These iPSC-derived macrophages show strong cellular identity with primary macrophages and recapitulate key functional characteristics, including cytokine release, phagocytosis, and chemotaxis. Furthermore, we demonstrate that genetic modifications can be readily introduced at the macrophage-like progenitor stage in order to interrogate drug target-relevant pathways. In summary, this novel method overcomes previous shortcomings with primary and leukemic cells and facilitates large-scale production of genetically modified iPSC-derived macrophages for drug screening applications.

Modeling the Effects of Severe Metabolic Disease by Genome Editing of hPSC-Derived Endothelial Cells Reveals an Inflammatory Phenotype.

The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we used edited human pluripotent stem cells (hPSCs) that carried either a homozygous deletion of AKT2 (AKT2 KO) or a dominant active mutation (AKT2 E17K), which, along with the parental wild type (WT), were differentiated into ECs. Profiling of EC lines indicated an increase in proinflammatory and a reduction in anti-inflammatory fatty acids, an increase in inflammatory chemokines in cell supernatants, increased expression of proinflammatory genes, and increased binding to the EC monolayer in a functional leukocyte adhesion assay for both AKT2 KO and AKT2 E17K. Collectively, these findings suggest that vascular endothelial inflammation that results from dysregulated insulin signaling (homeostasis) may contribute to coronary artery disease, and that either downregulation or upregulation of the insulin pathway may lead to inflammation of endothelial cells. This suggests that the standard of care for patients must be expanded from control of metabolic parameters to include control of inflammation, such that endothelial dysfunction and cardiovascular disorders can ultimately be prevented.

Genetic engineering of mammalian cells by direct delivery of FLP recombinase protein.

Protein transduction is based on the ability of certain peptides, designated as cell penetrating peptides (CPPs), to intracellularly deliver cargo molecules, such as peptides and proteins. In combination with site specific recombination, CPP-mediated delivery of recombinases enables a precise and highly efficient control of gene expression in cultured cells and mice. Herein, we provide detailed protocols for engineering and purification of a cell-permeant FLP recombinase protein. Two examples describe the use of cell permeant FLP for excising prespecified fragments from transgenes expressed in fibroblasts and mouse embryonic stem cells. A third example describes the combined use of cell-permeant Cre and FLP recombinases to reversibly induce transgenes in embryonic stem cells. We anticipate that the protocols described herein will be widely used for various genetic interventions addressing complex biological questions.

Engineering cell-permeant FLP recombinase for tightly controlled inducible and reversible overexpression in embryonic stem cells.

Combined application of DNA recombinases Cre and FLP enables tightly controlled independent and/or sequential gene regulations. However, in practice, such dual recombinase strategies are hampered by the comparably low efficiency of the FLP recombinase. Here, we present the engineering of a recombinant cell-permeant FLP protein (TAT-FLP) that induces recombination in >75% of fibroblasts and mouse as well as human embryonic stem (ES) cells. We show that TAT-FLP ideally complements the strength of cell-permeant Cre recombinase for genetic engineering as exemplified by FLP-ON-Cre-OFF, an inducible transgene expression cassette that enables tightly controlled expression in a reversible manner. We exemplify this concept by conditional overexpression of LacZ and the caudal-related homeobox transcription factor CDX2. We expect our FLP transduction system to become widely useful for numerous genetic interventions addressing complex biological questions and the generation of transgene-free therapeutically applicable ES cell-derived cells.

Ligand-dependent kinase activity of MERTK drives efferocytosis in human iPSC-derived macrophages.

Removal of apoptotic cells by phagocytes (also called efferocytosis) is a crucial process for tissue homeostasis. Professional phagocytes express a plethora of surface receptors enabling them to sense and engulf apoptotic cells, thus avoiding persistence of dead cells and cellular debris and their consequent effects. Dysregulation of efferocytosis is thought to lead to secondary necrosis and associated inflammation and immune activation. Efferocytosis in primarily murine macrophages and dendritic cells has been shown to require TAM RTKs, with MERTK and AXL being critical for clearance of apoptotic cells. The functional role of human orthologs, especially the exact contribution of each individual receptor is less well studied. Here we show that human macrophages differentiated in vitro from iPSC-derived precursor cells express both AXL and MERTK and engulf apoptotic cells. TAM RTK agonism by the natural ligand growth-arrest specific 6 (GAS6) significantly enhanced such efferocytosis. Using a newly-developed mouse model of kinase-dead MERTK, we demonstrate that MERTK kinase activity is essential for efferocytosis in peritoneal macrophages in vivo. Moreover, human iPSC-derived macrophages treated in vitro with blocking antibodies or small molecule inhibitors recapitulated this observation. Hence, our results highlight a conserved MERTK function between mice and humans, and the critical role of its kinase activity in homeostatic efferocytosis.

Secreted retrovirus-like GAG-domain-containing protein PEG10 is regulated by UBE3A and is involved in Angelman syndrome pathophysiology.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology.

Incorporation of stem cell-derived astrocytes into neuronal organoids to allow neuro-glial interactions in toxicological studies.

Human cell-based neural organoids are increasingly being used for investigations of neurotoxicity, and to study the pathophysiology of neurodegenerative diseases. Here, we present a fast and robust method to generate 3D cultured human dopaminergic neurons (LUHMES) for toxicity testing and long-term culture. Moreover, a plating step was introduced to allow generation of neurite networks with defined 2D orientation and several mm length, while all cell bodies (somata) remained in a 3D, dome-like structure. These cultures, named here 2.5D (for 2.5 dimensional), offer new approaches to quantify toxicant effects on organoids by standard technology and high throughput. For instance, the system reacted to the parkinsonian model toxicants MPP+, rotenone, MG-132 and the ferroptosis-inducer erastin. Moreover, stable incorporation of human stem cell-derived astrocytes or microglia was possible. Added astrocytes stabilized the post mitotic state of the LUHMES neurons and thereby allowed the formation of a stable micro-physiological system. We observed neuroprotection against the proteasome inhibitor MG-132 and the ferroptosis-inducer erastin by such glia. This exemplifies the crucial protective role of astrocytes in neurodegeneration. The modularity of the system was further employed to incorporate microglia together with astrocytes into the organoids. Such ratio-defined, three cell type-based organoids will allow new approaches to study human pathophysiology and toxicology of the nervous system.

Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia.

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.

Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance.

Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB. We used our previously established method to  generate ECs from human pluripotent stem cells (hPSCs), and then we overexpressed the candidate TFs in hPSC-ECs and measured barrier resistance and integrity using electric cell-substrate impedance sensing, trans-endothelial electrical resistance and FITC-dextran permeability assays. SOX18 and TAL1 were the strongest EC barrier-inducing TFs, upregulating Wnt-related signaling and EC junctional gene expression, respectively, and downregulating EC proliferation-related genes. These TFs were combined with SOX7 and ETS1 that together effectively induced EC barrier resistance, decreased paracellular transport and increased protein expression of tight junctions and induce mRNA expression of several genes involved in the formation of EC barrier and transport. Our data shows identification of a transcriptional network that controls barrier resistance in ECs. Collectively this data may lead to novel approaches for generation of in vitro models of the BBB.

Monolayer Generation of Vascular Endothelial Cells from Human Pluripotent Stem Cells.

The use of human pluripotent stem cells (hPSCs) for modeling human diseases and therapeutic applications requires differentiation methods that generate physiologically relevant cell types in a robust and standardized way. Herein, we describe an efficient and scalable monolayer protocol to convert pluripotent stem cells into vascular endothelial cells using defined culture conditions.The combinatorial use of small molecule compounds, growth factors as well as morphogens directs human pluripotent stem cells toward endothelial cells within 6 days. The protocol has the capacity to generate endothelial cells with high efficiencies of up to 80%. An additional immunomagnetic cell purification step that is based on the surface marker VE-cadherin results in a virtually pure population of endothelial cells. In a subsequent expansion step human PSC-derived endothelial cells can be further propagated, while maintaining their endothelial identity. Thus, our differentiation protocol enables the generation of hPSC-derived endothelial cells at a scale that is relevant for drug discovery campaigns or clinical applications.

Requirements for Using iPSC-Based Cell Models for Assay Development in Drug Discovery.

A prevalent challenge in drug discovery is the translation of findings from preclinical research into clinical success. Currently, more physiological in vitro systems are being developed to overcome some of these challenges. In particular, induced pluripotent stem cells (iPSCs) have provided the opportunity to generate human cell types that can be utilized for developing more disease-relevant cellular assay models. As the use of these complex models is lengthy and fairly complicated, we lay out our experiences of the cultivation, differentiation, and quality control requirements to successfully utilize pluripotent stem cells in drug discovery.

Reproducibility of Molecular Phenotypes after Long-Term Differentiation to Human iPSC-Derived Neurons: A Multi-Site Omics Study.

Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells.

Cell-penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy-independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well-studied CPP TAT fused to a protein (>50 amino acids) or peptide (<50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21(WAF/Cip) and DNA Ligase I measured in living cells.

Generation of a homozygous GBA deletion human embryonic stem cell line.

We describe the generation of a biallelic GBA deletion human embryonic stem cell line using zinc finger nuclease-mediated gene targeting. The homozygous targeting of exon 4 of the GBA locus leads to a complete loss of glucocerebrosidase (GCase) protein expression.

Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aß Levels Mediated by Brain Shuttle Transport.

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidß (Aß) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aß levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aß. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Generation of functional podocytes from human induced pluripotent stem cells.

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro.

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis.

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.