Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

Design of protein-binding proteins from the target structure alone.

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1-5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

Design of biologically active binary protein 2D materials.

Ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers6,7, but with the exception of a single lattice formed with flexible linkers8, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality9-12. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.

F-domain valency determines outcome of signaling through the angiopoietin pathway.

Angiopoietins 1 and 2 (Ang1 and Ang2) regulate angiogenesis through their similar F-domains by activating Tie2 receptors on endothelial cells. Despite the similarity in the underlying receptor-binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of AKT, strengthens cell-cell junctions, and enhances endothelial cell survival while Ang2 can antagonize these effects, depending on cellular context. To investigate the molecular basis for the opposing effects, we examined the phenotypes of a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: Scaffolds presenting 3 or 4 F-domains have Ang2-like activity, upregulating pFAK and pERK but not pAKT, while scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity, upregulating pAKT and inducing migration and vascular stability. The scaffolds with 6 or more F-domains display super-agonist activity, producing stronger phenotypes at lower concentrations than Ang1. Tie2 super-agonist nanoparticles reduced blood extravasation and improved blood-brain barrier integrity four days after a controlled cortical impact injury.

Small RNAs: keeping stem cells in line.

Stem cells and RNA silencing have emerged as areas of intense interest for both basic and clinical research. Recently these fields have converged with reports implicating small regulatory RNAs in the maintenance and pluripotency of stem cells.

Metabolic remodeling during the loss and acquisition of pluripotency.

Pluripotent cells from the early stages of embryonic development have the unlimited capacity to self-renew and undergo differentiation into all of the cell types of the adult organism. These properties are regulated by tightly controlled networks of gene expression, which in turn are governed by the availability of transcription factors and their interaction with the underlying epigenetic landscape. Recent data suggest that, perhaps unexpectedly, some key epigenetic marks, and thereby gene expression, are regulated by the levels of specific metabolites. Hence, cellular metabolism plays a vital role beyond simply the production of energy, and may be involved in the regulation of cell fate. In this Review, we discuss the metabolic changes that occur during the transitions between different pluripotent states both in vitro and in vivo, including during reprogramming to pluripotency and the onset of differentiation, and we discuss the extent to which distinct metabolites might regulate these transitions.

First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

Wnt/ß-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. In human embryonic stem cells (hESCs), Wnt/ß-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/ß-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of ß-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/ß-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/ß-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.

microRNAs Regulating Human and Mouse Naïve Pluripotency.

microRNAs are ~22bp nucleotide non-coding RNAs that play important roles in the post-transcriptional regulation of gene expression. Many studies have established that microRNAs are important for cell fate choices, including the naïve to primed pluripotency state transitions, and their intermediate state, the developmentally suspended diapause state in early development. However, the full extent of microRNAs associated with these stage transitions in human and mouse remain under-explored. By meta-analysis of microRNA-seq, RNA-seq, and metabolomics datasets from human and mouse, we found a set of microRNAs, and importantly, their experimentally validated target genes that show consistent changes in naïve to primed transitions (microRNA up, target genes down, or vice versa). The targets of these microRNAs regulate developmental pathways (e.g., the Hedgehog-pathway), primary cilium, and remodeling of metabolic processes (oxidative phosphorylation, fatty acid metabolism, and amino acid transport) during the transition. Importantly, we identified 115 microRNAs that significantly change in the same direction in naïve to primed transitions in both human and mouse, many of which are novel candidate regulators of pluripotency. Furthermore, we identified 38 microRNAs and 274 target genes that may be involved in diapause, where embryonic development is temporarily suspended prior to implantation to uterus. The upregulated target genes suggest that microRNAs activate stress response in the diapause stage. In conclusion, we provide a comprehensive resource of microRNAs and their target genes involved in naïve to primed transition and in the paused intermediate, the embryonic diapause stage.

Metabolic remodeling in early development and cardiomyocyte maturation.

Aberrations in metabolism contribute to a large number of diseases, such as diabetes, obesity, cancer, and cardiovascular diseases, that have a substantial impact on the mortality rates and quality of life worldwide. However, the mechanisms leading to these changes in metabolic state--and whether they are conserved between diseases--is not well understood. Changes in metabolism similar to those seen in pathological conditions are observed during normal development in a number of different cell types. This provides hope that understanding the mechanism of these metabolic switches in normal development may provide useful insight in correcting them in pathological cases. Here, we focus on the metabolic remodeling observed both in early stage embryonic stem cells and during the maturation of cardiomyocytes.

Let-7 family of microRNA is required for maturation and adult-like metabolism in stem cell-derived cardiomyocytes.

In metazoans, transition from fetal to adult heart is accompanied by a switch in energy metabolism-glycolysis to fatty acid oxidation. The molecular factors regulating this metabolic switch remain largely unexplored. We first demonstrate that the molecular signatures in 1-year (y) matured human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are similar to those seen in in vivo-derived mature cardiac tissues, thus making them an excellent model to study human cardiac maturation. We further show that let-7 is the most highly up-regulated microRNA (miRNA) family during in vitro human cardiac maturation. Gain- and loss-of-function analyses of let-7g in hESC-CMs demonstrate it is both required and sufficient for maturation, but not for early differentiation of CMs. Overexpression of let-7 family members in hESC-CMs enhances cell size, sarcomere length, force of contraction, and respiratory capacity. Interestingly, large-scale expression data, target analysis, and metabolic flux assays suggest this let-7-driven CM maturation could be a result of down-regulation of the phosphoinositide 3 kinase (PI3K)/AKT protein kinase/insulin pathway and an up-regulation of fatty acid metabolism. These results indicate let-7 is an important mediator in augmenting metabolic energetics in maturing CMs. Promoting maturation of hESC-CMs with let-7 overexpression will be highly significant for basic and applied research.

Derivation of naive human embryonic stem cells.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition.

The function of metabolic state in stemness is poorly understood. Mouse embryonic stem cells (ESC) and epiblast stem cells (EpiSC) are at distinct pluripotent states representing the inner cell mass (ICM) and epiblast embryos. Human embryonic stem cells (hESC) are similar to EpiSC stage. We now show a dramatic metabolic difference between these two stages. EpiSC/hESC are highly glycolytic, while ESC are bivalent in their energy production, dynamically switching from glycolysis to mitochondrial respiration on demand. Despite having a more developed and expanding mitochondrial content, EpiSC/hESC have low mitochondrial respiratory capacity due to low cytochrome c oxidase (COX) expression. Similarly, in vivo epiblasts suppress COX levels. These data reveal EpiSC/hESC functional similarity to the glycolytic phenotype in cancer (Warburg effect). We further show that hypoxia-inducible factor 1α (HIF1α) is sufficient to drive ESC to a glycolytic Activin/Nodal-dependent EpiSC-like stage. This metabolic switch during early stem-cell development may be deterministic.

Genetic elevation of sphingosine 1-phosphate suppresses dystrophic muscle phenotypes in Drosophila.

Duchenne muscular dystrophy is a lethal genetic disease characterized by the loss of muscle integrity and function over time. Using Drosophila, we show that dystrophic muscle phenotypes can be significantly suppressed by a reduction of wunen, a homolog of lipid phosphate phosphatase 3, which in higher animals can dephosphorylate a range of phospholipids. Our suppression analyses include assessing the localization of Projectin protein, a titin homolog, in sarcomeres as well as muscle morphology and functional movement assays. We hypothesize that wunen-based suppression is through the elevation of the bioactive lipid Sphingosine 1-phosphate (S1P), which promotes cell proliferation and differentiation in many tissues, including muscle. We confirm the role of S1P in suppression by genetically altering S1P levels via reduction of S1P lyase (Sply) and by upregulating the serine palmitoyl-CoA transferase catalytic subunit gene lace, the first gene in the de novo sphingolipid biosynthetic pathway and find that these manipulations also reduce muscle degeneration. Furthermore, we show that reduction of spinster (which encodes a major facilitator family transporter, homologs of which in higher animals have been shown to transport S1P) can also suppress dystrophic muscle degeneration. Finally, administration to adult flies of pharmacological agents reported to elevate S1P signaling significantly suppresses dystrophic muscle phenotypes. Our data suggest that localized intracellular S1P elevation promotes the suppression of muscle wasting in flies.

miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure.

microRNAs (miRNAs) are crucial for cellular development and homeostasis. In order to better understand regulation of miRNA biosynthesis, we studied cleavage of primary miRNAs by Drosha. While Drosha knockdown triggers an expected decrease of many mature miRNAs in human embryonic stem cells (hESC), a subset of miRNAs are not reduced. Statistical analysis of miRNA secondary structure and fold change of expression in response to Drosha knockdown showed that absence of mismatches in the central region of the hairpin, 5 and 9-12 nt from the Drosha cutting site conferred decreased sensitivity to Drosha knockdown. This suggests that, when limiting, Drosha processes miRNAs without mismatches more efficiently than mismatched miRNAs. This is important because Drosha expression changes over cellular development and the fold change of expression for miRNAs with mismatches in the central region correlates with Drosha levels. To examine the biochemical relationship directly, we overexpressed structural variants of miRNA-145, miRNA-137, miRNA-9, and miRNA-200b in HeLa cells with and without Drosha knockdown; for these miRNAs, elimination of mismatches in the central region increased, and addition of mismatches decreased their expression in an in vitro assay and in cells with low Drosha expression. Change in Drosha expression can be a biologically relevant mechanism by which eukaryotic cells control miRNA profiles. This phenomenon may explain the impact of point mutations outside the seed region of certain miRNAs.

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.

BACKGROUND: Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. METHODS AND RESULTS: A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. CONCLUSIONS: Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens.

Hypoxia induces re-entry of committed cells into pluripotency.

Adult stem cells reside in hypoxic niches, and embryonic stem cells (ESCs) are derived from a low oxygen environment. However, it is not clear whether hypoxia is critical for stem cell fate since for example human ESCs (hESCs) are able to self-renew in atmospheric oxygen concentrations as well. We now show that hypoxia can govern cell fate decisions since hypoxia alone can revert hESC- or iPSC-derived differentiated cells back to a stem cell-like state, as evidenced by re-activation of an Oct4-promoter reporter. Hypoxia-induced "de-differentiated" cells also mimic hESCs in their morphology, long-term self-renewal capacity, genome-wide mRNA and miRNA profiles, Oct4 promoter methylation state, cell surface markers TRA1-60 and SSEA4 expression, and capacity to form teratomas. These data demonstrate that hypoxia can influence cell fate decisions and could elucidate hypoxic niche function.

Protocol for generating three-dimensional induced early ameloblasts using serum-free media and growth factors.

Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta. For complete details on the use and execution of this protocol, please refer to Alghadeer et al.1.

Next-generation direct reprogramming.

Tissue repair is significantly compromised in the aging human body resulting in critical disease conditions (such as myocardial infarction or Alzheimer's disease) and imposing a tremendous burden on global health. Reprogramming approaches (partial or direct reprogramming) are considered fruitful in addressing this unmet medical need. However, the efficacy, cellular maturity and specific targeting are still major challenges of direct reprogramming. Here we describe novel approaches in direct reprogramming that address these challenges. Extracellular signaling pathways (Receptor tyrosine kinases, RTK and Receptor Serine/Theronine Kinase, RSTK) and epigenetic marks remain central in rewiring the cellular program to determine the cell fate. We propose that modern protein design technologies (AI-designed minibinders regulating RTKs/RSTK, epigenetic enzymes, or pioneer factors) have potential to solve the aforementioned challenges. An efficient transdifferentiation/direct reprogramming may in the future provide molecular strategies to collectively reduce aging, fibrosis, and degenerative diseases.