Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.

Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.

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.

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.

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.

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae.

The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface. We show that binding proteins derived from the Sso7d scaffold and the homodimeric enzyme glucose oxidase can be simultaneously secreted solubly and expressed as yeast cell surface fusions using the F2A-based system. Furthermore, a combinatorial library of Sso7d mutants can be screened to isolate binders with higher affinity for a model target (lysozyme), and the pool of higher affinity binders can be characterized in soluble form. Significantly, we show that both N- and C-terminal fusions to Aga2 can be simultaneously secreted solubly and displayed on the cell surface; this is particularly advantageous because protein functionality can be affected by the specific position of Aga2 in the protein fusion. We expect that the F2A-based yeast surface display and secretion system will be a useful tool for protein engineering and enable efficient characterization of individual clones isolated from combinatorial libraries.

Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts.

Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development.