Derivation of human trophoblast stem cells from placentas at birth.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3ß) inhibitor CHIR99021, the transforming growth factor-beta (TGFß) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology.

Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions.

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFß) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFß signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFß inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFß inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro.

Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase.

The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting nonoverlapping epitopes on the target analyte were each fused to nonactive fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provides the foundation for the development of split luciferase assays for detection and quantification of various targets.

Yeast Display Guided Selection of pH-Dependent Binders.

pH-dependent antigen binding has proven useful in engineering next-generation therapeutics specifically via antibody recycling technology. This technology allows for half-life extension, thereby lowering the amount and frequency of dosing of therapeutics. Cell sorting, coupled with display techniques, has been used extensively for the selection of high-affinity binders. Herein, we describe a cell sorting methodology utilizing yeast surface display for selection of binding proteins with strong binding at physiological pH and weak to no binding at acidic pH. This methodology can be readily applied to engineer proteins and/or antibodies that do not have pH-dependent binding or for selection of de novo pH-dependent binders using library-based methods.

Discovery of Cyclic Peptide Binders from Chemically Constrained Yeast Display Libraries.

Cyclic peptides with engineered protein-binding activity have great potential as therapeutic and diagnostic reagents owing to their favorable properties, including high affinity and selectivity. Cyclic peptide binders have generally been isolated from phage display combinatorial libraries utilizing panning based selections. As an alternative, we have developed a yeast surface display platform to identify and characterize cyclic peptide binders from genetically encoded combinatorial libraries. Through a combination of magnetic selection and fluorescence-activated cell sorting (FACS), high-affinity cyclic peptide binders can be efficiently isolated from yeast display libraries. In this platform, linear peptide precursors are expressed as yeast surface fusions. To achieve cyclization of the linear precursors, the cells are incubated with disuccinimidyl glutarate, which crosslinks amine groups within the displayed linear peptide sequence. Here, we detail protocols for cyclizing linear peptides expressed as yeast surface fusions. We also discuss how to synthesize a yeast display library of linear peptide precursors. Subsequently, we provide suggestions on how to utilize magnetic selections and FACS to isolate cyclic peptide binders for target proteins of interest from a peptide combinatorial library. Lastly, we detail how yeast surface displayed cyclic peptides can be used to obtain efficient estimates of binding affinity, eliminating the need for chemically synthesized peptides when performing mutant characterization.

Isolation of Single-Domain Antibodies to Transmembrane Proteins Using Magnetized Yeast Cell Targets.

The isolation of binding ligands from yeast-displayed combinatorial libraries has typically relied on the use of a soluble, recombinantly expressed form of the target protein when performing magnetic selections or fluorescence-activated cell sorting. When identifying binding ligands, appropriate target protein expression and subsequent purification represents a significant bottleneck. As an alternative, we describe the use of target proteins expressed on the surface of magnetized yeast cells in the selection of yeast-displayed nanobody libraries. In this approach, yeast cells displaying the target protein also co-express an iron oxide-binding protein; incubation with iron oxide nanopowder results in magnetization of target-displaying cells. Alternatively, target-displaying cells are magnetized by nonspecific adsorption of iron oxide nanopowder. Subsequently, any library cells that interact with the magnetized target cells can be isolated using a magnet. Here, we detail protocols for the isolation of binders to membrane protein targets from a yeast display nanobody library using magnetized yeast cell targets. We provide guidance on how to generate magnetic yeast cell targets as well as library selection conditions to bias the isolation of high affinity binders. We also discuss how to assess the affinity and specificity of the isolated nanobodies using flow cytometry.

Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity.

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

Mapping the residue specificities of epigenome enzymes by yeast surface display.

Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here, we describe a versatile and scalable approach which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first use this system to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, a large panel of commercially available anti-acetylation antibodies are screened for their specificities, identifying many suitable and unsuitable reagents. Furthermore, this approach enables efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4 and uncovers residue interdependencies affecting p300 activity. These results show that using yeast surface display to study histone modifications is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.

Two distinct trophectoderm lineage stem cells from human pluripotent stem cells.

The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells-they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development.

Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands.

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 µM for YAP and 0.68 µM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.

Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions.

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

Use of Target-Displaying Magnetized Yeast in Screening mRNA-Display Peptide Libraries to Identify Ligands.

This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of select WW-YAP-binding peptides: KD values of 37 and 4 µM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 µM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.

Genetically encoded live-cell sensor for tyrosinated microtubules.

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display.

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated cross-linking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides using magnetic selection, followed by fluorescence activated cell sorting (FACS) to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ∼ 1.2-3.7 µM). The isolated peptides bind lysozyme selectively and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides can be used to efficiently obtain quantitative estimates of binding affinity, circumventing the need for chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that likely binds IL-17 in its receptor binding region with moderate apparent affinity (KD ∼ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.

Past, Present, and Future of Affinity-based Cell Separation Technologies.

Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. STATEMENT OF SIGNIFICANCE: Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs.

A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells.

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

Simultaneous Soluble Secretion and Surface Display of Proteins in Saccharomyces cerevisiae Using Inefficient Ribosomal Skipping.

Combinatorial library screening platforms, such as yeast surface display, typically identify several candidate proteins that need further characterization and validation using soluble recombinant protein. However, recombinant production of these candidate proteins involves tedious and time-consuming subcloning steps. This, in turn, limits the number of candidate proteins that can be characterized. To address this bottleneck, we have developed a platform that exploits inefficient ribosomal skipping by the F2A peptide for simultaneous soluble secretion and cell surface display of protein in the yeast Saccharomyces cerevisiae. Here we provide detailed protocols utilizing this F2A-based yeast display system. We discuss specific recommendations for the purification of the secreted protein. Additionally, we provide suggestions for testing the functionality and binding specificity of the soluble secreted proteins using flow cytometry analysis.

Screening Yeast Display Libraries against Magnetized Yeast Cell Targets Enables Efficient Isolation of Membrane Protein Binders.

When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity (KD ≈ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ≈ 272-1934 nM) and the extracellular domain of the c-Kit receptor (KD ≈ 93 to KD > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion-in contrast to recombinant soluble expression-makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.

Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation.

Live-cell fluorescence microscopy is broadly applied to study the dynamics of receptor-mediated cell signaling, but the availability of intracellular biosensors is limited. A biosensor based on the tandem SH2 domains from phospholipase C-γ1 (PLCγ1), tSH2-WT, has been used to measure phosphorylation of the epidermal growth factor receptor (EGFR). Here, we found that tSH2-WT lacked specificity for phosphorylated EGFR, consistent with the known promiscuity of SH2 domains. Further, EGF-stimulated membrane recruitment of tSH2-WT differed qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model suggested, and experiments confirmed, that the high avidity of tSH2-WT resulted in saturation of its target and interference with EGFR endocytosis. To overcome the apparent target specificity and saturation issues, we implemented two protein engineering strategies. In the first approach, we screened a combinatorial library generated by random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1 and isolated a mutant form (mSH2) with enhanced specificity for phosphorylated Tyr992 (pTyr992) of EGFR. A biosensor based on mSH2 closely reported the kinetics of EGFR phosphorylation but retained cross-reactivity similar to tSH2-WT. In the second approach, we isolated a pTyr992-binding protein (SPY992) from a combinatorial library generated by mutagenesis of the Sso7d protein scaffold. Compared to tSH2-WT and mSH2, SPY992 exhibited superior performance as a specific, moderate-affinity biosensor. We extended this approach to isolate a biosensor for EGFR pTyr1148 (SPY1148). This approach of integrating theoretical considerations with protein engineering strategies can be generalized to design and evaluate suitable biosensors for various phospho-specific targets.