Analog Intracellular-Phase Tuning of Cyborg Stem Cells

Introduction: : Chronic wounds in aging patients are particularly difficult to treat due to the accumulation of senescent cells, which produce pro-inflammatory signals that impair healing and increase the risk of tumorigenesis. Placental-derived mesenchymal stem cell therapies offer regenerative potential through secretion of anti-inflammatory and pro-repair factors like IL-10 and TGF-β, but their clinical use is limited by poor survival, phenotypic instability, and risk of aberrant growth in vivo. Current encapsulation and growth-control methods compromise cell function, underscoring the urgent need for a stable, modular system to decouple replication from therapeutic secretion. To address these limitations, we present a novel intracellular PEGDA-based hydrogel that forms directly inside live cells, generating non-dividing but metabolically active “Cyborg Cells” (Figure 1A). These cells enter a unique semi-synthetic state, maintaining metabolic functions and protein secretion for extended periods while exhibiting increased resistance to apoptotic signals (Figure 1B). This platform functions as a “one-time” implantable therapy, offering stable and programmable cytokine delivery without the risks associated with uncontrolled proliferation. Our results demonstrate tunable control of molecular diffusion and secretion through intracellular hydrogel networks. Additionally, engineered Cyborg PMSCs maintained secretion of native and synthetic immune-modulatory proteins in co-culture with human-derived macrophages, corroborating their potential for targeted anti-inflammation and wound-repair. Our Cyborg Cell platform enables tunable control over intracellular environments, supporting consistent cytokine production independent of cell state. This strategy establishes a foundation for engineering semi-synthetic stem cells with programmable secretion, with broad implications for wound healing and long-term inflammation control.

Materials and

Methods: : Cyborg Cells were generated by suspending HEK293FT cells in a hydrogel buffer, then washed and UV crosslinked to generate the intracellular hydrogel (Figure 1A). To study the effects on intracellular transport, we expressed genetically encoded multimeric crowding sensors (GEMs), derived from Pyrococcus furiosus encapsulin fused to the Staygold fluorescent protein. These formed large fluorescent complexes, for visualization of molecular crowding in live Cyborg Cells. Diffusion dynamics were quantified using 20 ms time-lapse fluorescence microscopy and analyzed through kymography under varying osmotic and crowding conditions. In addition, we tracked nuclear localizing RFP-labeled mRNA (RFP) from 1 kb to 4 kb size to monitor nuclear membrane trafficking. Fluorescence microscopy was then used to track the movement of particles and measure cytoplasmic:nuclear fluorescence intensity. To assess secretory function, Cyborg PMSCs were evaluated using a 45-plex Luminex cytokine assay. Signal peptide motifs associated with enhanced secretion were identified using MEME Suite, SignalP 5.0, and Clustal Omega. A synthetic library of signal sequences was engineered by extracting conserved motifs from proteomic comparisons between Cyborg and non-gelated controls. These motifs were cloned into SEAP reporters and validated by expression assays. Lastly, bulk RNA-seq was conducted seven days post-gelation to identify sustained transcriptional responses, adaptation signatures, and molecular resistance pathways. As a next step, high-secretion synthetic signals were used to enhance production of immune-modulatory products IL-10, TGF-β, and Peptide14 in Cyborg PMSCs. As a proof-of-concept, the engineered cells were co-cultured with THP-1 macrophages and tested for their ability to reduce LPS-induced inflammatory signaling and restrict macrophage activation.

Results, Conclusions, and Discussions:: We engineered Cyborg PMSCs as a novel non-dividing but metabolically active system capable of controlled and enhanced therapeutic secretion, which can survive and regulate pro-inflammatory conditions. We first demonstrated quantitative and analog control over the intracellular hydrogel, and its impact on molecular diffusion. We altered intracellular PEGDA hydrogel density by changing concentration, incubation time, and extracellular osmolarity, then quantified immobilized proteins by number and area. Increased PEGDA concentration and osmolarity increased GEM immobilization, demonstrating tunable control over hydrogelation and intracellular transport (Figure 1C). To examine how intracellular hydrogelation affects mRNA transport, we tracked 1 kb - 4 kb labeled mRNA. Nuclear export of 2kb transcripts displayed similar RFP intensity ratios between WT and Cyborg conditions, corroborating the maintenance of synthesis and secretion functions with intracellular hydrogelation (Figure 1D). To demonstrate tunable protein secretion in Cyborg Cells, we identified conserved secretory motifs of >40 secreted proteins across gelation conditions and designed synthetic signal peptides, which enhanced SEAP secretion in WT and Cyborg PMSCs, enabling tunable, intracellular-gelation-responsive therapeutic expression (Figure 1E). To understand how intracellular gelation “differentiates” cells, we conducted bulk RNA-seq. Cyborg Cells displayed a unique transcriptional state with downregulation of apoptotic and proliferative genes, including Caspase-3, DNA polymerases, and Dna2, alongside suppression of ER-Golgi transport and upregulation of Clathrin. The Cyborg profile shows enrichment in stress adaptation and vesicle transport, supporting a restructured, secretion-competent cell state (Figure 1F). For biomedical application in anti-inflammatory function, we engineered Cyborg PMSCs with synthetic signal peptides to express IL-10, PDGFB, and Peptide14, which are key immunomodulatory and regenerative proteins. We then tested Cyborg PMSCs’ ability to modulate immune activation under chronic inflammation. This study establishes intracellular hydrogelation as a tool for engineering stem cells with tunable transport, enhanced secretion, and stress resilience. Cyborg Cells remain active while non-replicating, enabling predictive and safe therapeutics. We defined a modular framework using synthetic signal peptides for sustained secretion of immune-modulating proteins. Our work enables the development of regenerative therapies, including chronic wound repair and treatment of ischemia-related tissue failure.

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