mRNA vaccines encoding membrane-anchored RBDs of SARS-CoV-2 mutants induce strong humoral responses and can overcome immune imprinting.

We investigated mRNA vaccines encoding a membrane-anchored receptor-binding domain (RBD), each a fusion of a variant RBD, the transmembrane (TM) and cytoplasmic tail fragments of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. In naive mice, RBD-TM mRNA vaccines against SARS-CoV-2 variants induced strong humoral responses against the target RBD. Multiplex surrogate viral neutralization (sVNT) assays revealed broad neutralizing activity against a range of variant RBDs.

Single nuclei transcriptomics of the in situ human limbal stem cell niche.

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery.

Necroptosis does not drive disease pathogenesis in a mouse infective model of SARS-CoV-2 in vivo.

Necroptosis, a type of lytic cell death executed by the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) has been implicated in the detrimental inflammation caused by SARS-CoV-2 infection. We minimally and extensively passaged a single clinical SARS-CoV-2 isolate to create models of mild and severe disease in mice allowing us to dissect the role of necroptosis in SARS-CoV-2 disease pathogenesis. We infected wild-type and MLKL-deficient mice and found no significant differences in viral loads or lung pathology.

SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates.

The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-β signaling.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

Background: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines.

Methods: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain.

Biparatopic nanobodies targeting the receptor binding domain efficiently neutralize SARS-CoV-2.

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus.

Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation.

TINC- A Method to Dissect Regulatory Complexes at Single-Locus Resolution- Reveals an Extensive Protein Complex at the Nanog Promoter.

Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques.

Differentiation of Retinal Glial Cells From Human Embryonic Stem Cells by Promoting the Notch Signaling Pathway.

Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway.

Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells.

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures.

Fine Tuning of Canonical Wnt Stimulation Enhances Differentiation of Pluripotent Stem Cells Independent of β-Catenin-Mediated T-Cell Factor Signaling.

The canonical Wnt/β-catenin pathway is crucial for early embryonic patterning, tissue homeostasis, and regeneration. While canonical Wnt/β-catenin stimulation has been used extensively to modulate pluripotency and differentiation of pluripotent stem cells (PSCs), the mechanism of these two seemingly opposing roles has not been fully characterized and is currently largely attributed to activation of nuclear Wnt target genes. Here, we show that low levels of Wnt stimulation via ectopic expression of Wnt1 or administration of glycogen synthase kinase-3 inhibitor CHIR99021 significantly increases PSC differentiation into neurons, cardiomyocytes and early endodermal intermediates.

Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells.

In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state.

Stem Cell Surface Marker Expression Defines Late Stages of Reprogramming to Pluripotency in Human Fibroblasts.

Unlabelled: Our current understanding of the induction of pluripotency by defined factors indicates that this process occurs in discrete stages characterized by specific alterations in the cellular transcriptome and epigenome. However, the final phase of the reprogramming process is incompletely understood. We sought to generate tools to characterize the transition to a fully reprogramed state.

Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells.

The pluripotent state in mouse and human.

In the mouse, naïve pluripotent stem cells (PSCs) are thought to represent the cell culture equivalent of the late epiblast in the pre-implantation embryo, with which they share a unique defining set of features. Recent studies have focused on the identification and propagation of a similar cell state in human. Although the capture of an exact human equivalent of the mouse naïve PSC remains an elusive goal, comparative studies spurred on by this quest are lighting the path to a deeper understanding of pluripotent state regulation in early mammalian development.

Characterization of the retinal pigment epithelium in Friedreich ataxia.

We assessed structural elements of the retina in individuals with Friedreich ataxia (FRDA) and in mouse models of FRDA, as well as functions of the retinal pigment epithelium (RPE) in FRDA using induced pluripotent stem cells (iPSCs). We analyzed the retina of the FRDA mouse models YG22R and YG8R containing a human FRATAXIN (FXN) transgene by histology. We complemented this work with post-mortem evaluation of eyes from FRDA patients.

Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells.

Glaucoma, the worldwide leading cause of irreversible blindness, is characterized by progressive degeneration of the optic nerve and loss of retinal ganglion cells. Research into glaucoma pathogenesis has been hampered by difficulties in isolating and culturing retinal ganglion cells in vitro. However, recent improvements in laboratory techniques have enabled the generation of a variety of mature cell types from pluripotent stem cells, including retinal ganglion cells.

Human pluripotent stem cell strategies for age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the Western world and is increasing exponentially as the population ages. Despite enormous worldwide efforts, the earliest pathogenic pathways involved in AMD are still not fully understood. It is essential to develop research tools for effective modeling of AMD pathogenesis and for subsequent drug discovery and cell or molecular therapies.

Wnt/β-catenin signaling suppresses DUX4 expression and prevents apoptosis of FSHD muscle cells.

Facioscapulohumeral muscular dystrophy is a dominantly inherited myopathy associated with chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4. DUX4 is encoded within each unit of the D4Z4 array where it is normally transcriptionally silenced and packaged as constitutive heterochromatin. Truncation of the array to less than 11 D4Z4 units (FSHD1) or mutations in SMCHD1 (FSHD2) results in chromatin relaxation and a small percentage of cultured myoblasts from these individuals exhibit infrequent bursts of DUX4 expression.

LRP-6 is a coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts that are inhibited by DKK-1.

Fibrosis of vital organs is a major public health problem with limited therapeutic options. Mesenchymal cells including microvascular mural cells (pericytes) are major progenitors of scar-forming myofibroblasts in kidney and other organs. Here we show pericytes in healthy kidneys have active WNT/β-catenin signaling responses that are markedly up-regulated following kidney injury.

WIKI4, a novel inhibitor of tankyrase and Wnt/ß-catenin signaling.

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells.

Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4.

Signal transduction pathways play diverse, context-dependent roles in vertebrate development. In studies of human embryonic stem cells (hESCs), conflicting reports claim Wnt/β-catenin signaling promotes either self-renewal or differentiation. We use a sensitive reporter to establish that Wnt/β-catenin signaling is not active during hESC self-renewal.

Acute effect of endothelins on intercellular communication of human embryonic stem cells.

Endothelin (ET) family comprises three isoforms, ET-1, ET-2 and ET-3 that bind to two receptors ET-A and ET-B. Upon hESC differentiation, ET-1 and ET-B are respectively up- and down-regulated, suggesting a potential role of ETs in hESC biology. Here we show expression of ET receptors in hESC and demonstrate that ET-1 and ET-2 inhibit gap junctional intercellular communication (GJIC), while ET-3 does not.

Wnt3a regulates survival, expansion, and maintenance of neural progenitors derived from human embryonic stem cells.

Many reports describe the efficient derivation and expansion of neural progenitors (NP) from human embryonic stem cells (hESC). However, little is known about the signaling factors found within the neurosphere microenvironment that regulate NP maintenance and differentiation. We show that Wnt ligand and receptor transcripts are endogenously upregulated within neurospheres derived from noggin-primed hESC.