ABSTRACT
Uncovering the stimulus-response histories that give rise to cell fates and behaviors is an area of great interest in developmental biology, tissue engineering, and regenerative medicine. A comprehensive accounting of cell experiences that lead to the development of organs and tissues can help us to understand developmental anomalies that may underly disease. Perhaps more provocatively, such a record can also reveal clues as to how to drive cell collective decision-making processes, which may yield predictable cell-based therapies or facilitate production of tissue substitutes for transplantation or in vitro screening of prospective therapies to mitigate disease. Toward this end, various methods have been applied to molecularly trace developmental trajectories and record interaction histories of cells. Typical methods involve artificial gene circuits based on recombinases that activate a suite of fluorescent reporters or CRISPR-Cas9 genome writing technologies whose nucleic acid-based record keeping serves to chronicle cell-cell interactions or past exposure to stimuli of interests. Exciting expansions of the synthetic biology toolkit with artificial receptors that permit establishment of defined input-to-output linkages of cell decision-making processes opens the door to not only record cell-cell interactions, but to also potentiate directed manipulation of the outcomes of such interactions via regulation of carefully selected transgenes. Here, we combine CRISPR-based strategies to genetically and epigenetically manipulate cells to express components of the synthetic Notch receptor platform, a widely used artificial cell signaling module. Our approach gives rise to the ability to conditionally record interactions between human cells, where the record of engagement depends on expression of a state-specific marker of a subset of cells in a population. Further, such signal-competent interactions can be used to direct differentiation of human embryonic stem cells toward pre-selected fates based on assigned synNotch outputs. We also implemented CRISPR-based manipulation of native gene expression profiles to bias outcomes of cell engagement histories in a targeted manner. Thus, we present a useful strategy that gives rise to both state-specific recording of cell-cell interactions as well as methods to intentionally influence products of such cell-cell exchanges.
ABSTRACT
Objective: Investigational cell therapies have been developed as disease-modifying agents for the treatment of osteoarthritis (OA), including those that inducibly respond to inflammatory factors driving OA progression. However, dysregulated inflammatory cascades do not specifically signify the presence of OA. Here, we deploy a synthetic receptor platform that regulates cell behaviors in an arthritis-specific fashion to confine transgene expression to sites characterized by cartilage degeneration. Methods: An scFv specific for type II collagen (CII) was used to produce a synthetic Notch (synNotch) receptor that enables "CII-synNotch" mesenchymal stromal cells (MSCs) to recognize CII fibers exposed in damaged cartilage. Engineered cell activation by both CII-treated culture surfaces and on primary tissue samples was measured via inducible reporter transgene expression. TGFß3-expressing cells were assessed for cartilage anabolic gene expression via qRT-PCR. In a co-culture with CII-synNotch MSCs engineered to express IL-1Ra, ATDC5 chondrocytes were stimulated with IL-1α, and inflammatory responses of ATDC5s were profiled via qRT-PCR and an NF-κB reporter assay. Results: CII-synNotch MSCs are highly responsive to CII, displaying activation ranges over 40-fold in response to physiologic CII inputs. CII-synNotch cells exhibit the capacity to distinguish between healthy and damaged cartilage tissue and constrain transgene expression to regions of exposed CII fibers. Receptor-regulated TGFß3 expression resulted in upregulation of Acan and Col2a1 in MSCs, and inducible IL-1Ra expression by engineered CII-synNotch MSCs reduced pro-inflammatory gene expression in chondrocytes. Conclusion: This work demonstrates proof-of-concept that the synNotch platform guides MSCs for spatially regulated, disease-dependent delivery of OA-relevant biologic drugs.
ABSTRACT
In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems. Key features ⢠Generation of an inducible CRISPRoff Sleeping Beauty transposon system. ⢠Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation. ⢠Strategy to downregulate an essential gene at different stages of oligodendrocyte development.
ABSTRACT
BACKGROUND: The hormone leptin exerts its function in the brain to reduce food intake and increase energy expenditure to prevent obesity. However, most obese subjects reflect the resistance to leptin even with elevated serum leptin. Considering that leptin must cross the blood-brain barrier (BBB) in several regions to enter the brain parenchyma, altered leptin transport through the BBB might play an important role in leptin resistance and other biological conditions. Here, we report the use of a human induced pluripotent stem cell (iPSC)-derived BBB model to explore mechanisms that influence leptin transport. METHODS: iPSCs were differentiated into brain microvascular endothelial cell (BMEC)-like cells using standard methods. BMEC-like cells were cultured in Transwell filters, treated with ligands from a nuclear receptor agonist library, and assayed for leptin transport using an enzyme-linked immune sorbent assay. RNA sequencing was further used to identify differentially regulated genes and pathways. The role of a select hit in leptin transport was tested with the competitive substrate assay and after gene knockdown using CRISPR techniques. RESULTS: Following a screen of 73 compounds, 17ß-estradiol was identified as a compound that could significantly increase leptin transport. RNA sequencing revealed many differentially expressed transmembrane transporters after 17ß-estradiol treatment. Of these, cationic amino acid transporter-1 (CAT-1, encoded by SLC7A1) was selected for follow-up analyses due to its high and selective expression in BMECs in vivo. Treatment of BMEC-like cells with CAT-1 substrates, as well as knockdown of CAT-1 expression via CRISPR-mediated epigenome editing, yielded significant increases in leptin transport. CONCLUSIONS: A major female sex hormone, as well as an amino acid transporter, were revealed as regulators of leptin BBB transport in the iPSC-derived BBB model. Outcomes from this work provide insights into regulation of hormone transport across the BBB.
