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A major aim in the field of synthetic biology is developing tools capable of responding to user-defined inputs by activating therapeutically relevant cellular functions. Gene transcription and regulation in response to external stimuli are some of the most powerful and versatile of these cellular functions being explored. Motivated by the success of chimeric antigen receptor (CAR) T-cell therapies, transmembrane receptor-based platforms have been embraced for their ability to sense extracellular ligands and to subsequently activate intracellular signal transduction. The integration of transmembrane receptors with transcriptional activation platforms has not yet achieved its full potential. Transient expression of plasmid DNA is often used to explore gene regulation platforms in vitro. However, applications capable of targeting therapeutically relevant endogenous or stably integrated genes are more clinically relevant. Gene regulation may allow for engineered cells to traffic into tissues of interest and secrete functional proteins into the extracellular space or to differentiate into functional cells. Transmembrane receptors that regulate transcription have the potential to revolutionize cell therapies in a myriad of applications, including cancer treatment and regenerative medicine. In this review, we will examine current engineering approaches to control transcription in mammalian cells with an emphasis on systems that can be selectively activated in response to extracellular signals. We will also speculate on the potential therapeutic applications of these technologies and examine promising approaches to expand their capabilities and tighten the control of gene regulation in cellular therapies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Sebastian Vega reports financial support was provided by National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Effective screening methodologies for cells are challenged by the divergent and heterogeneous nature of phenotypes inherent to stem cell cultures, particularly on engineered biomaterial surfaces. In this study, we showcase a high-content, confocal imaging-based methodology to parse single-cell phenotypes by quantifying organizational signatures of specific subcellular reporter proteins and applied this profiling approach to three human stem cell types (embryonic-human embryonic stem cell [hESC], induced pluripotent-induced pluripotent stem cell [iPSC], and mesenchymal-human mesenchymal stem cell [hMSC]). We demonstrate that this method could distinguish self-renewing subpopulations of hESCs and iPSCs from heterogeneous populations. This technique can also provide insights into how incremental changes in biomaterial properties, both physiochemical and mechanical, influence stem cell fates by parsing the organization of stem cell proteins. For example, hMSCs cultured on polymeric films with varying degrees of poly(ethylene glycol) to modulate osteogenic differentiation were parsed using high-content organization of the cytoskeletal protein F-actin. In addition, hMSCs cultured on a self-assembled monolayer platform featuring compositional gradients were screened and descriptors obtained to correlate substrate variations with adipogenic lineage commitment. Taken together, high-content imaging of structurally sensitive proteins can be used as a tool to identify stem cell phenotypes at the single-cell level across a diverse range of culture conditions and microenvironments.