Journal of Biological Engineering

This study explores the potential of utilizing {beta}-cells, exemplified with R7T1 {beta}-cell pseudoislets, as a transplantable cell factory for on-demand recombinant protein therapeutic delivery. While mammalian cell lines are widely used for in vitro protein production, the commonly utilized constitutive secretion pathway poses challenges to in vivo cell therapy, especially for delivering proteins requiring precise exposure kinetics. The proposed approach capitalizes on unique aspects of {beta}-cells, including substantial vesicular protein storage capacity and electrochemically-regulated protein release, to facilitate timely and titratable in vivo therapeutic delivery. Examining a variety of strategies to acheive {beta}-cell glucagon or glucagon-like peptide 1 (GLP-1) storage and secretion, we devised a flexible {beta}-cell-based expression platform for efficient cellular peptide production and on-demand release. This platform utilizes the preproinsulin coding sequence as a template, wherein therapeutic peptides of interest (glucagon or GLP-1) are substituted for C-peptide while the A- and B-peptide insulin chains are mutated to prevent bio-active insulin production. This approach overcomes the challenge of efficient bio-active peptide expression by leveraging the endogenous {beta}-cell peptide expression, translation, processing, storage and secretion machinery. Furthermore, {beta}-cells provide a mechanism for scalable electyrochemnically-triggered peptide delivery. This transformative strategy, which may be extended to other proteins and peptide expression cassettes, holds significant promise for targeted and temporally controlled in vivo production and release of recombinant protein therapeutics. The study suggests potential applications in addressing challenges in metabolic disorders, blood disorders, and oncology. Future refinements may focus on optimizing vector design, peptide production, and in vivo adaptation.

Macrophages are abundant immune cells within the tumor microenvironment with intrinsic phagocytic capabilities, yet their antitumor functions are frequently suppressed by inhibitory signals. Synthetic biology enables the rational design of ligand-responsive genetic circuits to reprogram immune cell behavior. Here, we report the engineering of a synthetic Notch-based receptor that detects PD-L1, an immune-checkpoint broadly expressed by cancer cells. Upon PD-L1 engagement, the circuit triggers programmable outputs, including expression of a fluorescent reporter or CV1-Fc, that locally blocks the CD47 "dont eat me" signal. We show that circuit activation scales with PD-L1 levels, partially attenuates PD-1/PD-L1 signaling, and that conditional CV1-Fc expression enhances engulfment of SKOV-3 ovarian cancer cells by THP-1-derived macrophages in vitro. Collectively, this work reframes PD-L1 from an end-point therapeutic target to a primary input signal for synthetic circuit activation and establishes a modular framework for engineering macrophage behaviour through spatially confined, ligand-responsive control. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/700810v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1cbb391org.highwire.dtl.DTLVardef@7a0900org.highwire.dtl.DTLVardef@1e4f6d6org.highwire.dtl.DTLVardef@109838d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. A novel -PDL1 SNIPR receptor to program macrophage outputs. PD-L1 expressed on cancer cells is detected by -PD-L1 SNIPR-engineered macrophages, triggering release of the GAL4-VP64 transcription factor and activation of a programmable actuator. Circuit activation leads to customized outputs, including biosensing and enhanced macrophage phagocytosis via CD47 blockade, as well as immunomodulatory effects through interference with the PD-1/PD-L1 checkpoint axis. This sensor-actuator framework enables spatially confined and ligand-dependent reprogramming of macrophage function. C_FIG

Mesenchymal stromal cells (MSCs) are widely studied for their immunomodulatory and tissue reparative capabilities, but clinical translation has been hampered by inconsistent efficacy and limited standardization in manufacturing. While cytokine-based priming methods, such as interferon-gamma (IFN-{gamma}) stimulation, have shown promise in enhancing MSC potency, alternative approaches targeting distinct biological metabolism integral to secretome and membrane architecture have not been explored in MSCs. In this study, we investigate sphingomyelinase (SMase), an enzyme that generates ceramide from sphingomyelin, as a novel lipid-based priming strategy to modulate MSC function. Here, human MSCs were treated with SMase and high-content imaging and morphological profiling revealed that SMase-treated cells adopted a phenotype overlapping with IFN-{gamma}-licensed MSCs, including increased cell compactness and solidity. Lipidomic analysis showed broad alterations in sphingolipid species, and dynamic flux estimation (DFE) modeling predicted distinct metabolic shifts in SMase-treated cells compared to untreated controls. These changes were sustained up to 35 hours post-stimulation, indicating stable metabolic reprogramming. SMase priming also altered the MSC secretome, enriching for factors implicated in immune regulation. Functionally, SMase-primed MSCs retained the ability to suppress T-cell activation and promote anti-inflammatory macrophage phenotypes. Collectively, these findings demonstrate that SMase stimulation induces a durable, immunomodulatory-like state in MSCs through coordinated changes in lipid metabolism and secretory activity. This lipid-centric priming approach represents a promising alternative to cytokine-based licensing strategies and may support therapeutic MSC products.

mRNA-based chimeric antigen receptor (CAR)-T cells offer the promise of enhanced safety and simplified manufacturing. However, in vitro-transcribed (IVT) mRNA is known to trigger antiviral immune responses, inflammatory signaling, and apoptosis in transfected cells. To address these challenges and enable efficient IVT-mRNA expression, modified nucleosides, such as N1-methyl-pseudouridine (m1{Psi}), have become the gold standard for CAR-T cell production, albeit at increased cost. In this study, immune responses to IVT-mRNA were evaluated across five primary human cell types, including T-cells. Unexpectedly, T-cells, unlike other immune and non-immune cell types tested, exhibited no immune activation in response to unmodified mRNA. T-cell viability and cytokine secretion patterns remained unaffected, regardless of whether unmodified mRNA was delivered via lipid nanoparticles (LNPs) or electroporation. Furthermore, CAR expression levels in T-cells were not influenced by mRNA modification with m1{Psi} or 5-methoxy-uridine (5moU) nucleosides. The absence of nucleoside modifications did not compromise CAR-T cell cytotoxic potency, demonstrating that such modifications are not required for producing functional CAR-T cells. These findings eliminate the need for nucleoside modification in T-cell mRNA, simplifying and reducing the cost of CAR-T cell manufacturing while positioning IVT-mRNA as a highly efficient and minimally invasive tool for CAR-T cell engineering.

Modular methods for directing mammalian gene expression would enable advances in tissue regeneration, enhance cell-based therapeutics and improve modulation of immune responses. To address this challenge, engineered endosymbionts (EES) that escape endosomal destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to control host cell gene expression were developed. Microscopy confirmed that EES escape phagosomes, replicate within the cytoplasm, and can secrete reporter proteins into the cytoplasm that were then transported to the nucleus. Synthetic operons encoding the mammalian transcription factors, Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the EES genome. Using controlled induction, these EES were shown to direct gene expression in J774A.1 macrophage/monocyte cells and modulate the host cell fates. Expressing mammalian transcription factors from engineered intracellular bacteria as endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/463266v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@2b5935org.highwire.dtl.DTLVardef@1edd8edorg.highwire.dtl.DTLVardef@3cb5b9org.highwire.dtl.DTLVardef@1be9b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

The suspension culture of pluripotent stem (PS) cells in stirred bioreactors poses a delicate balance between maintaining homogeneous cell dispersion and avoiding excessive shear stress that can compromise cell viability and pluripotency. In this study, we used computational fluid dynamics (CFD) coupled with a discrete particle method (DPM) to simulate iPS cell behavior in a 5 mL delta-impeller stirred tank. Our analysis revealed that upward flow at the tank bottom and downward flow at the top are critical for maintaining a stable suspension. To optimize the stirring protocol, we applied Bayesian optimization to identify a time-dependent stirring schedule that begins with a high-speed phase for resuspension, followed by a low-speed phase for sustained suspension with minimal hydrodynamic stress. The optimized schedule demonstrated improved suspension ratio and reduced slip velocity, indicating lower mechanical stress on cells. These findings provide engineering insights into scalable bioreactor operation, contributing to the design of robust iPS cell manufacturing systems.

Adipose tissue is an active endocrine organ that can signal bidirectionally to many tissues and organ systems in the body. With obesity, adipose tissue is a source of low-level inflammation that contributes to various co-morbidities and damage to downstream effector tissues. The ability to synthesize genetically engineered adipose tissue could have critical applications in studying adipokine signaling and the use of adipose tissue for novel therapeutic strategies. This study aimed to develop a method for non-viral adipogenic differentiation of genome-edited murine induced pluripotent stem cells (iPSCs) and to test the ability of such cells to engraft in mice in vivo. Designer adipocytes were created from iPSCs, which can be readily genetically engineered using CRISPR-Cas9 to knock out or insert individual genes of interest. As a model system for adipocyte-based drug delivery, an existing iPSC cell line that transcribes interleukin 1 receptor antagonist under the endogenous macrophage chemoattractant protein-1 promoter was tested for adipogenic capabilities under these same differentiation conditions. To understand the role of various adipocyte subtypes and their impact on health and disease, an efficient method was devised for inducing browning and whitening of IPSC-derived adipocytes in culture. Finally, to study the downstream effects of designer adipocytes in vivo, we transplanted the designer adipocytes into fat-free lipodystrophic mice as a model system for studying adipose signaling in different models of disease or repair. This novel translational tissue engineering and regenerative medicine platform provides an innovative approach to studying the role of adipose interorgan communication in various conditions.

Standard differentiation assays sample cell states only at discrete time points, while the underlying progression unfolds continuously and heterogeneously across cells. As a result, different combinations of proliferation, commitment, and maturation dynamics can converge to similar endpoint measurements. This many-to-one mapping between latent trajectories and observable readouts constitutes a partially observed inverse problem that limits mechanistic interpretation. Although this ambiguity is inherent to many experimental systems, it is rarely examined using models that connect cell-state dynamics to assay-level quantities. We present OsteoMin, a coarse-grained cellular automaton that links stochastic transitions between pre-osteoblast and osteoblast states to experimentally measurable readouts of alkaline phosphatase activity, collagen deposition, and mineralization. Model parameters were constrained using literature-reported kinetics and evaluated against dexamethasone and menaquinone-4 perturbations. The frame-work reproduces qualitative assay trends and enables systematic analysis of how cell-state progression, matrix maturation, and external perturbations shape differentiation outcomes. Using this framework, we quantify the identifiability limits of endpoint assays and test whether standard measurements can distinguish underlying differentiation mechanisms. Distinct perturbation families often produce similar endpoint responses (macro-F1 {approx} 0.42), indicating limited discriminative power. Incorporating temporal trajectories improves separability (macro-F1 {approx} 0.78), demonstrating that most identifiable information resides in marker dynamics rather than terminal measurements. Sobol analysis shows early markers depend on proliferation timing, whereas late mineralization is governed by nonlinear matrix maturation and parameter interactions. Together, these results show that endpoint assays constrain overall progression but do not uniquely identify underlying mechanisms. OsteoMin provides a framework linking differentiation dynamics to assay observables and a basis for assessing identifiability in endpoint-driven systems.

Human pluripotent stem cells (hPSCs) hold significant promise for regenerative medicine, yet optimizing their expansion in three-dimensional bioreactor systems remains challenging due to complex interactions between mechanical forces, metabolic constraints, and aggregate formation dynamics. This study developed and validated a mechanistic mathematical model to predict hPSC proliferation dynamics in vertical-wheel bioreactor (VWBR) systems, incorporating the effects of shear stress and energy dissipation rate (EDR) on cell growth and aggregate dynamics. Seven model variants employing different kinetic formulations for shear stress and energy dissipation rate effects were systematically evaluated through model selection, identifiability analyses, and experimental validation. Experimental data from six bioreactor conditions varying in initial cell density (2 x 104-15 x 104 cells/mL), agitation rate (30-60 RPM), and working volume (100-500 mL) were used for model calibration and selection. Bayesian Information Criterion analysis identified a model combining Michaelis-Menten kinetics for shear stress inhibition with a EDR-mediated aggregate detachment formulation as the best-performing variant, achieving a Mean Relative Prediction Error of 13.97%, comparable to the experimental variability of 16.29%. Independent validation experiments using leave-out data gathered under different media exchange schedules confirmed model accuracy with prediction errors below 14%, consistent with observed experimental variability around 12%. The validated model was used to optimize the media exchange protocol, leading to a 37.5% reduction in media consumption with only a 13.5% reduction in final cell yield, demonstrating its utility for prospective, quantitative bioprocess design in VWBR systems.

Small extracellular vesicles (sEV) released by healthy beta cells are promising candidates for diabetes therapy thanks to their aptitude to modulate inflammation, to induce or maintain pancreatic function and to prevent pathogenic mechanisms. To advance the clinical development of therapeutics, there is a crucial need for scalable production methods. Stirred tank bioreactors (STR) are widely used in the industry due to their ability to provide homogeneous gas and nutrient supply, online monitoring, and efficient scale up. Anchorage- dependent cells can be cultured in STR on microcarriers or as spheroids, but may experience shear stress, which can affect sEV phenotype and function. Using pancreatic beta cells, this study identifies critical cell culturing parameters, including culture mode (monolayer vs. spheroids), medium formulation (with or without serum, glucose control), and process parameters (stirring, duration, cell density). The findings show that small spheroid culture promotes beta cell maturation without decreasing the yield of sEV per cell, despite a reduced cell surface exchange area. However, stirring increased expression of cellular stress markers and decreased cell viability. Set up of a three-step bioprocess allowed to maximize cell viability and sEV yields at high cell density over short production duration. sEV produced under these conditions maintained high purity, membrane integrity, and the aptitude to reduce T- lymphocyte proliferation and IFN-{gamma} cytokine secretion in a mixed lymphocyte reaction. Flow cytometry analysis revealed lower CD63/CD81 ratios in STR, indicating enhanced ectosome production. Switch from high glucose expansion to low glucose production medium further allowed to direct sorting of the antigen insulin into beta-sEV. This study demonstrates the feasibility of producing functional sEV from mature beta cells cultured as small spheroids, suitable for upscale. Production of sEV in STR may be particularly beneficial for ectosome- enriched compound loading for therapeutic applications. Graphical abstractRational development of a scalable bioprocess to control extracellular vesicles production & function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/611247v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@6eaefeorg.highwire.dtl.DTLVardef@a2da3borg.highwire.dtl.DTLVardef@1a58f35org.highwire.dtl.DTLVardef@5cfd1d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISmall 3D-spheroid culture promotes cell maturation C_LIO_LIStirring induces cellular stress responses & promotes ectosome release from the cytoplasmic membrane C_LIO_LIDesign of experiment efficiently enhances cell viability and EV yield with preserved immune-modulatory properties C_LIO_LIGlucose-starvation during the production phase directs insulin-sorting into extracellular vesicles C_LI

The ability to precisely control the activity of defined cell populations enables studies of their physiological roles and may provide therapeutic applications. While prior studies have shown that magnetic activation of ferritin-tagged ion channels allows cell-specific modulation of cellular activity, the large size of the constructs made the use of adeno-associated virus, AAV, the vector of choice for gene therapy, impractical. In addition, simple means for generating magnetic fields of sufficient strength have been lacking. Toward these ends, we first generated a novel anti-ferritin nanobody that when fused to transient receptor potential cation channel subfamily V member 1, TRPV1, enables direct binding of the channel to endogenous ferritin in mouse and human cells. This smaller construct can be delivered in a single AAV and we validated that it robustly enables magnetically induced cell activation in vitro. In parallel, we developed a simple benchtop electromagnet capable of gating the nanobody-tagged channel in vivo. Finally, we showed that delivering these new constructs by AAV to pancreatic beta cells in combination with the benchtop magnetic field delivery stimulates glucose-stimulated insulin release to improve glucose tolerance in mice in vivo. Together, the novel anti-ferritin nanobody, nanobody-TRPV1 construct and new hardware advance the utility of magnetogenetics in animals and potentially humans.

Despite accelerating interest in using human induced pluripotent stem cell (hiPSC)-derived products for disease modeling and therapeutic development, there is substantial evidence that conventional culture approaches do not fully recapitulate natural embryonic nor lineage-committed states. It remains poorly understood how in vitro environmental conditions cause divergence from natural developmental trajectories, and current strategies emphasize restricted characterization of phenotype without appreciating the complexity of biology in maintaining pluripotency and driving differentiation. To address this knowledge gap, we examined hiPSC cell state during short-term culture in stirred-suspension bioprocesses under varying oxygen and agitation conditions. We profiled intracellular metabolic, transcriptional, and proteomic changes to characterize cellular responses to engineered environments and implications for cell phenotype. Using a random forest framework, we modeled population dynamics over time across metabolic and transcriptional programs and mapped those predictions onto hallmark biological signatures. This integrative approach captures and identifies environmentally reinforced programs, offering a framework to guide optimization of pluripotent cell state maintenance and differentiation.

Pluripotent stem cells have a high potential for research and development applications in biomedicine. They are undifferentiated cells capable of unlimited self-renewal and maintenance of pluripotency, yet can be induced to differentiate into specific cell types through defined cues. Various protocols have been proposed to maintain mouse embryonic stem cells (mESCs) in the pluripotent state; state of the art cell culture protocols are based on serum-free media, and continuous provision of drugs inhibiting pathways associated to differentiation. We explore here the possibility to avoid continuous drug exposure. First, we employ an external feedback control microfluidics/microscopy platform to steer the expression of a pluripotency reporter. Control experiments show a significant delay in cell response to drugs; these dynamics are confirmed by flow-cytometry open-loop experiments. We then harness such delays to design novel culture protocols with limited cell exposure to small-molecule inhibitors; in so doing, we reduce cell exposure to drug, but we maintain pluripotency. Our results suggest new approaches to design cell culture protocols based on the quantitative analysis and control of cellular dynamics.

The production of recombinant proteins is a problem of major industrial and pharmaceutical importance. Secretion of the protein by the host cell considerably simplifies downstream purification processes. However, it is also the limiting production step for many hard-to-secrete proteins. Current solutions involve extensive chassis engineering to favor trafficking and limit protein degradation triggered by excessive secretion-associated stress. Here, we propose instead a regulation-based strategy in which induction is dynamically adjusted based on the current stress level of the cells. Using a small collection of hard-to-secrete proteins and a bioreactor-based platform with automated cytometry measurements, we demonstrate that the regulation sweet spot is indicated by the appearance of a bimodal distribution of internal protein and of secretory stress levels, when a fraction of the cell population accumulates high amounts of proteins, decreases growth, and faces significant stress, that is, experiences a secretion burn-out. In these cells, adaptations capabilities are overwhelmed by a too strong production. With these notions, we define an optimal stress level based on physiological readouts. Then, using real-time control, we demonstrate that a strategy that keeps the stress at optimal levels increases production of a single-chain antibody by 70%.

Large plasmids are often avoided in mammalian co-transfection due to the assumption that they transfect poorly, driving the use of multiple smaller plasmids. Here, we pair finite-state-projection modeling with flow cytometry experiments to compare one-, two-, and three-plasmid delivery of GFP/BFP/RFP. Estimated entry rates were size-independent from 4.9 to 16.4 kb, indicating that plasmid length is not the dominant barrier in this range. Our results suggest that using lipofectamine slightly increases co-transfection efficiency due to the ability of lipoplexes to contain multiple plasmids. However, this benefit is limited to only delivering two plasmids. Additionally, we show that contrary to current beliefs, putting all genes onto the same plasmid both increases the probability that a cell will express all genes of interest and results in a tighter correlation of gene expression levels compared to these multi-plasmid systems. Together, these results identify multi-cargo delivery and not plasmid size as the key constraint on co-transfection and show that single-plasmid designs are generally preferable for applications such as viral-vector production. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/675629v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@251928org.highwire.dtl.DTLVardef@196d1cborg.highwire.dtl.DTLVardef@a781e9org.highwire.dtl.DTLVardef@1420928_HPS_FORMAT_FIGEXP M_FIG C_FIG

Gene therapies using adeno-associated viruses (AAVs) for central nervous system (CNS) disorders face challenges due to host immune responses not represented in classical preclinical models. Here, we present a human-induced pluripotent stem cell (hiPSC)-derived innate immunocompetent 3D CNS model that recapitulates neuroinflammatory hallmarks, serving as a platform for preclinical gene therapy development. Utilizing various scales of stirred-tank bioreactor systems, we generated (neurospheroids) iNSpheroids composed of neurons, astrocytes, and oligodendrocytes, alongside microglial cells (iMGL) to mimic the neuro-immune axis. These systems enabled large-scale production of iNSpheroids and subsequent miniaturization for co-culture experiments and screening of inflammatory stimuli, while maintaining a highly controlled environment. The iMGL-iNSpheroids demonstrated active neuron-microglia crosstalk and exhibited distinct inflammatory responses to a series of neuroinflammatory factors. iMGL-iNSpheroids mounted a mild and transient response to rAAV9, mediated by the activation of inflammatory pathways (e.g., TNF-via NF-{kappa}B activation) in glial cell populations. This model offers a valuable tool to dissect neuroinflammatory mechanisms, accelerating gene therapy development. TeaserImmune-competent 3D human CNS model recapitulates glial responses to rAAVs, enabling reliable preclinical gene therapy screening.

Non-viral approaches to transfection have emerged a viable option for gene transfer. Electro-mechanical transfection involving use of electric fields coupled with high fluid flow rates is a scalable strategy for cell therapy development and manufacturing. Unlike purely electric field-based or mechanical-based delivery methods, the combined effects result in delivery of genetic material at high efficiencies and low toxicity. This study focuses on delivery of reporter mRNA to show electro-mechanical transfection can be used successfully in human T cells. Rapid optimization of delivery to T cells was observed with efficiency over 90% and viability over 80%. Confirmation of optimized electro-mechanical transfection parameters was assessed in multiple use cases including a 50-fold scale up demonstration. Transcriptome and ontology analysis show that delivery, via electro-mechanical transfection, does not result in gene dysregulation. This study demonstrates that non-viral electro-mechanical transfection is an efficient and scalable method for cell and gene therapy engineering and development. One Sentence SummaryThis study demonstrates that non-viral electro-mechanical transfection is an efficient and scalable method for development of engineered cellular therapies.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/666879v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@9139c3org.highwire.dtl.DTLVardef@673bc1org.highwire.dtl.DTLVardef@1842210org.highwire.dtl.DTLVardef@1d86903_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Therapeutic proteins are produced frequently by mammalian cells in large-scale bioreactors. As a result, producer cells are exposed to a chemically (nutrients, gas exchange, target protein overexpression) and physically (shear due to mixing) stressful environment, which can lead to loss of proteostasis and endoplasmic reticulum (ER) stress. In response, cells activate the unfolded protein response (UPR). The UPR includes activation of autophagy and proteasomes, both of which target unfolded/misfolded proteins for degradation. To investigate the impacts of autophagy and proteasome activity on secreted protein production in ER-stressed cells, we used HeLa and MDA-MB-231 cells transfected to express Gaussia luciferase (as a model for therapeutic protein production) and exposed to tunicamycin (TM) (to activate ER stress). As expected, TM exposure decreased protein production and secretion. Inhibiting autophagy improved secretion in stressed cells as expected. However, counterintuitively, increasing proteasomal degradation improved secretion while inhibiting proteasomal activity decreased secretion, that is proteasomal activity was directly correlated to secretion. Taken together, our results demonstrate that protein secretion can be improved through control of autophagy and proteasomal activity, providing insight into strategies for improving yield from protein production bioprocesses. Key PointsO_LITunicamycin induced ER stress reduced protein production. C_LIO_LIAutophagy inhibition improved secretion in ER stressed cells. C_LIO_LIActivation of proteasomal degradation improved secretion in ER stressed cells. C_LI

Precise regulation of gene expression in batch bacterial cultures is challenging because the underlying dynamics vary with cellular physiological state over time. Although cell-silicon systems enable rapid, real-time optogenetic control, disturbance rejection remains difficult in batch culture because the plant dynamics shift across growth phases, limiting the effectiveness of fixed-gain controllers designed under constant-growth assumptions. Here, we present a multiscale model-guided feedback control framework for disturbance rejection in batch E. coli cultures. Frequency-response analysis shows that the input-output dynamics of gene expression depend strongly on growth phase, revealing operating-point-dependent limits on the disturbance rejection performance of a fixed-gain PID controller. To address this limitation, we develop two growth-aware control strategies: a gain-scheduled PID (PID-GS) controller that adapts to cellular physiological state, and a gain-scheduled feedback-feedforward controller (PID-GS-FF) that further compensates for growth perturbations. We also introduce a controller evaluation framework that identifies three distinct operating regimes for targeted experimental validation. Together, these results show that accounting for growth-state-dependent dynamics is necessary for robust disturbance rejection in batch culture and provide a control-oriented framework for regulating living systems with shifting operating conditions.

During development, state transitions are coordinated through changes in the identity of molecular regulators in a cell state- and dose specific manner. The ability to rationally engineer such functions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein we report the generation of synthetic gene circuits that can detect a discrete cell state, and upon state detection, produce fine-tuned effector proteins in a programmable manner. Effectively, these gene circuits convert a discrete (digital-like) cell state into an analog signal by merging AND-like logic integration of endogenous miRNAs (classifiers) with a miRNA-mediated output fine-tuning technology (miSFITs). Using an automated miRNA identification and model-guided circuit optimization approach, we were able to produce robust cell state specific and graded output production in undifferentiated hPSC. We further finely controlled the levels of endogenous BMP4 secretion, which allowed us to document the effect of endogenous factor secretion in comparison to exogenous factor addition on early tissue development using the hPSC-derived gastruloid system. Our work provides the first demonstration of a discrete-to-analog signal conversion circuit operating in living hPSC, and a platform for customized cell state-specific control of desired physiological factors, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/467377v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@150da71org.highwire.dtl.DTLVardef@135554borg.highwire.dtl.DTLVardef@72b5b3org.highwire.dtl.DTLVardef@f70f84_HPS_FORMAT_FIGEXP M_FIG C_FIG