Cell Stem Cell

Genetically engineered human induced pluripotent stem cells (hiPSCs) represent a promising platform for regenerative medicine and next-generation immunotherapies. While recent advances enable stroma-free differentiation of hiPSCs into mature CD3TCR{beta} cytotoxic T lymphocytes (CTLs), overall efficiency remains limited. Here, we identify small-molecule modulators that enhance T cell output, particularly at the ProT cell stage. Targeted and stage-specific inhibition of AHR, DOT1L, or GSK3 drives robust maturation from ProT to CD4 immature single-positive (ISP) cells, markedly increasing CD4CD8 populations and augmenting CTL production of up to 2000 fold. hiPSC-derived T (iT) cells matured under these conditions display superior activity in cytotoxicity assays using AMG-701 (BCMAxCD3) or Blinatumomab (CD19xCD3). These effects were reproducible across independent hiPSC lines, diverse hematopoietic progenitor generation methods, and multiple stroma-free differentiation platforms, and were further validated in cord blood CD34 cells. Notably, AHR inhibition enhanced T cell development and promoted B lymphopoiesis, revealing shared regulatory pathways in lymphoid lineage specification. We also demonstrate that the Oct4-activating compound OAC1 functions as a weak AHR inhibitor, partially recapitulating the effects of canonical AHR blockers in both cellular and zebrafish AHR reporter systems. Collectively, our findings define key molecular circuits governing human lymphoid differentiation and establish practical strategies to optimize the yield and function of hiPSC-derived cytotoxic T cells. This work advances the development of both universal and autologous hiPSC-derived T cell therapies, offering a path forward even for patient-specific hiPSC lines with suboptimal T cell differentiation potential.

The lungs are highly susceptible to chronic disease in advanced age, likely due to the uniquely compromised repair function of alveolar type II (AT2) cells, facultative progenitor cells that maintain the gas exchange surface. Using aging mouse models, single-cell sequencing, and ex vivo organoid assays, we found that homeostatic aged AT2 cells exhibited an Interferon {gamma} (IFN{gamma}) response associated with IFN{gamma}+ CD8+ T cells in tertiary lymphoid structures (TLS). Aged AT2 cells exhibit impaired regeneration in organoid assays and lost markers of an IFN{gamma} response outside the lung microenvironment, demonstrating that elevated local IFN{gamma} influences the state of AT2 cells. Neutralization of IFN{gamma} signaling and immunoproteasome knockout mice with attenuated IFN{gamma} levels partially rescued aged AT2 cell regeneration. Our findings demonstrate that local IFN{gamma} signaling in aging lungs actively represses alveolar regeneration, establishing chronic inflammatory signaling as a cause of age-related decline in the lung. Halting chronic inflammatory processes restored alveolar regeneration and may provide a means to improve lung health in old age.

Colonic stem cells reside in a microenvironment enriched in epidermal growth factor, which is essential for their survival and can activate both PI3K-AKT and MAPK-ERK pathways. This predicts co-activation of both pathways within the growth factor-high stem cell compartment at the base of crypts. However, in patient-derived human colonic organoids and normal human tissue, stem cells maintain robust AKT activity while suppressing ERK signaling despite active EGFR engagement. As stem cells differentiate, they activate pulsatile Erk signaling, which is essential for migration, survival, and maintenance of barrier function. We show that AKT-dependent phosphorylation of Raf-1 at serine 259 establishes a post-receptor checkpoint that maintains ERK temporal dynamics in stem cells. Acute activation of ERK in stem cells triggers rapid global differentiation. Disruption of the ERK checkpoint via mutation of serine 259 leads to sustained AKT and ERK co-activation in stem cells. Unlike ERK/AKT coactivation driven by apoptosis, co-activation in the stem cell compartment results in the emergence of a neoplastic, architecturally disorganized cell population dominating the cell fate profile. Incredibly, introducing brief ERK pulses through Akt inhibition or ERK activation triggers re-differentiation of neoplastic cells. Consistent with duration-dependent MAPK encoding principles, these data demonstrate that regardless of baseline signaling amplitude, ERK signaling dynamics are epistatic to total kinase signaling load in human colonic stem cells.

Emerging evidence indicates that a subset of cancer cells enriched for stemness-related gene signatures possess distinct immunomodulatory capacities, enabling these tumor-initiating stem cells (tSCs) to more effectively evade or resist anti-tumor immunity. Despite these advances, the tSC-specific molecular circuits orchestrating their specialized immune privilege program are not well defined. Here, in squamous cell carcinomas of the skin and oral cavity, we comprehensively delineate the unique immune-evasive properties of tSCs and dissect the transcriptional regulation shaping their immunomodulatory programs. By integrating transcriptome profiling, chromatin landscape mapping, genetic perturbation, and single-cell RNA sequencing, we found that the tSC-specific immune program is broadly governed by SOX2, a stemness-associated transcription factor. We demonstrate that SOX2 enables tSCs to sustain immature tumor-associated neutrophils (TANs) and subsequently trigger these myeloid cells to foster the development of tumor-associated macrophages (TAMs). This SOX2-directed tSC-TAN-TAM axis establishes a localized immunosuppressive niche for protecting tSC. SIGNIFICANCEHere, we uncover SOX2 as a master regulator that orchestrates conserved immune modulatory circuits in tSCs to sustain pro-tumor myeloid cell states. These findings place tSCs at the apex of immune landscape remodeling, asserting a central role of stemness-associated program in organizing the immunosuppressive tumor microenvironment.

Synovial fibroblasts (SF) drive joint pathology in rheumatoid arthritis (RA). Difficult-to-treat RA frequently exhibits a fibroblast-rich synovial pathotype, enriched in DKK3{square} and CD34{square} SF, highlighting a critical therapeutic gap. Through multicohort transcriptomic analysis of synovial tissues and mechanistic in vitro studies, we identified SF as principal targets of Janus kinase (JAK) inhibition in RA. We demonstrated that JAK inhibitors (JAKi) can target multiple core aspects of fibroblast pathobiology - therapeutic refractoriness, cartilage destruction, and inflammation - offering a mechanistic rationale for JAKi superiority in difficult-to-treat RA. JAK1 was the dominantly expressed JAK across synovial pathotypes and SF subsets, including DKK3{square} and CD34{square} populations. A STAT1-interferon type I gene program was enriched in matrix-destructive PRG4{square} SF, consistent with JAKi efficacy in erosive RA. In contrast, canonical IL-6 signaling predominated in IL6-expressing inflammatory CXCL12high and HLA-DR+ SF, and was reproduced in cytokine-stimulated cultured SF, underscoring the autocrine nature of synovial IL-6 signaling. These data inferred a heightened JAKi sensitivity of PRG4{square}, CXCL12high, and HLA-DR+ SF subsets, informing precision therapeutic strategies. We uncovered a strong synergy between TNF and IL-6 trans-signaling, profoundly amplifying fibroblast inflammation. In high and synergistic cytokine milieu, STAT1/3 phosphorylation and IL-6 secretion persisted in SF despite tofacitinib treatment, revealing tofacitinibs functional ceiling. This could explain reduced tofacitinib efficacy and adherence in patients with high baseline arthritis activity. Finally, inflamed SF partially uncoupled STAT3 activation from sustained JAK1 phosphorylation, limiting inflammatory output. Similar uncoupling in tofacitinib-treated SF, likely drove rapid STAT1/3 reactivation following tofacitinib washout. These data aligned with JAKi withdrawal complications and clinical recommendations for gradual JAKi tapering. Collectively, our study identifies SF as key cellular targets of JAK inhibition and delineates cytokine- and drug-driven mechanisms that may constrain the efficacy and safety profiles of JAKi in RA.

Individuals homozygous for the SERPINA1 "Z" mutation with alpha-1 antitrypsin deficiency (AATD) are highly susceptible to emphysema. This predisposition has classically been attributed to a relative deficiency of circulating alpha-1 antitrypsin (AAT) reaching the lungs and associated protease-antiprotease imbalance. Accumulating evidence suggests that the presence of misfolded Z-AAT protein either in the circulation, the lung interstitium, or within resident lung cells could contribute to emphysema pathogenesis. We have shown that type 2 alveolar epithelial cells (AT2s), progenitor cells of the lung alveolus, heterogeneously retain Z-AAT and exhibit a transcriptional disease signature in AATD patient samples. However, a lack of model systems that faithfully recapitulate AT2 biology and associated Z-AAT expression has limited our ability to study this phenomenon. Here, we apply syngeneic induced pluripotent stem cell-derived AT2s (iAT2s) and a novel mouse model featuring AT2-specific inducible human SERPINA1 expression to interrogate the cell-instrinsic consequences of Z-AAT expression, validating findings in an independent dataset of human COPD lung tissue comparing ZZ to MM SERPINA1 genotypes. We find further evidence of Z-AAT retention within AT2s and identify shared AT2 transcriptomic disease signatures conserved across model systems, characterized by innate immune and inflammatory signaling, NF-{kappa}B activation, and endoplasmic reticulum stress. Mice with AT2-specific Z-AAT expression additionally demonstrate increased susceptibility to elastase-induced emphysema, providing functional evidence for AT2-intrinsic contributions to AATD-associated lung disease. Within iAT2s, a subpopulation of Z-AAT expressing cells exhibits activation of the PERK-eIF2 signaling axis and markers of an alveolar basal intermediate (ABI) state, emerging cell-autonomously in the absence of mesenchymal co-culture.Together, these data provide evidence that Z-AAT expression in AT2s induces heterogenous cell-intrinsic stress responses including proteotoxic stress, inflammatory signaling, and aberrant cell fate adoption, and is sufficient to result in predisposition to injury, supporting a potential contribution of AT2-intrinsic Z-AAT toxicity to human AATD-associated emphysema pathogenesis.

Oral squamous cell carcinomas (OSCC) account for [~]90% of all oral malignancies and have devastating effects on overall health and quality of life. However, little is known about the early initiating events that drive the development of oral leukoplakia-like premalignant lesions (OPLs) and disease progression. Here, we create a mouse model of tobacco-related premalignant OSCC that takes into consideration its primary risk factors, including advancing age and male sex. This model notably recapitulates the age variant patterns of OSCC risk observed in humans, with a higher prevalence of oral premalignant lesions in older mice. In addition, by building a transcriptomic atlas with this system, we reveal genetic signatures associated with oncogenic progression in the tongue and buccal epithelium, and their resident somatic tissue stem cells. We also identify several novel transcriptomic signatures of premalignancy in OSCC, including enhanced immune response and expansion and dysregulation of head and neck tissue stem cells. These findings offer a new framework for investigating physiologically-relevant risk factors and drivers of OSCC and illuminate novel biological pathways underlying its pathology.

Stem cell niches are dynamic microenvironments that regulate tissue homeostasis. Epidermal stem cells (EpiSC) preferentially localize to concave regions of epidermal rete ridges, which serve as primary niches for stem cell maintenance. EpiSC number and functional integrity decline during chronological aging. A defining feature of aged skin is epidermal atrophy, in which the prominent rete ridges present in young skin become flattened. Whether such topographical alterations influence EpiSC homeostasis and differentiation remains unclear. To address this, we generated anatomically accurate rete ridge structures using 3D bioprinting of collagen matrices as an ex vivo model and compared EpiSC cultured within concave topography to those maintained on a flat matrix resembling aged skin. Transcriptomic analysis revealed that concave niches promoted keratinocyte differentiation, marked by increased type I and II keratin gene expression and downregulation of cell cycle-associated genes. ATAC-seq identified topography-dependent chromatin accessibility changes enriched for transcription factors regulating epidermal differentiation, including upregulation of KLF4 and GRHL3 and downregulation of SOX9, HOXA1, and ETS1. Consistently, aged human skin showed reduced KLF4 and GRHL3 and increased SOX9 compared with young skin. Our findings demonstrate that concave niche topography imposes a spatially defined EpiSC microenvironment that promotes differentiation, alters cell cycle, and when perturbed, potentially contributes to the aging process. We conclude that spatial localization within rete ridge regions significantly affects epidermal progenitor stemness properties as fundamental differences in the physical microenvironment appear to influence cell fate decisions, thus, form shapes function of EpiSC.

We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (NCT06186401). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro, integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. One Sentence SummaryOur spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.

Adult zebrafish exhibit scarless repair and functional recovery following spinal cord injury. Their regenerative capacity is attributed to potent stem-like progenitors that mediate neuronal and glial repair. Zebrafish are thought to lack anti-regenerative extracellular matrix (ECM) components abundant in mammalian SCI, but the positive contributions of ECM to spontaneous spinal cord repair are less understood. By employing cross-species single-cell transcriptomics, we found the hyaluran modifying enzyme Hapln1 is upregulated in zebrafish progenitors but not in mouse progenitors following injury. Loss-of-function of hapln1a/b and ablation of hapln1+ cells reduce progenitor cell activation and hinder spontaneous recovery from injury. Using a series of in vivo and in vitro assays, we show that Hapln1 is required for hyaluran-cd44b mediated progenitor cell proliferation. This study reveals that, in addition to lacking anti-regenerative ECM components around SC lesions, zebrafish can also leverage pro-regenerative ECM molecules to enhance progenitor cell potency and promote repair.

Myelofibrosis in patients with myeloproliferative neoplasms (MPNs) is traditionally characterized by bone marrow fibrosis and osteosclerosis, with de novo bone formation commonly attributed to impaired osteoclast-mediated resorption. Here, we challenge this paradigm by demonstrating that a solitary clonal driver mutation simultaneously induces pathological bone formation and resorption, with osteosclerosis acting to conceal localized and active bone destruction rather than inhibiting it. Through population analysis; clinical imaging; patient-derived multi-tissue sequencing; murine models and organ-on-a-chip systems, we demonstrate that spatial and ontogeny-dependent remodeling in mesoderm- and neural crest-derived bones is mechanistically interconnected via a previously unidentified osteochondral stromal injury program. Neural crest-derived stromal cells suppress osteogenic programs and undergo injury-induced lineage plasticity with ectopic chondrogenesis, mirroring pathological remodeling in mesoderm-derived growth plate regions. This shared injury response promotes osteoclastogenesis and is mediated by a conserved Thrombospondin 1+ (THBS1+) stromal population that links fibrotic remodeling to bone loss. Combined pharmacological inhibition of THBS1 and JAK signaling reduces myeloproliferation, halts fibrosis progression, and restores two developmentally distinct bones, establishing THBS1 as a unifying therapeutic target in myelofibrosis.

Adoptive cell therapy (ACT) in solid tumors is limited by tumor microenvironment (TME)-imposed resistance mechanisms that are inadequately addressed by conventional systems. We developed tissue-preserving patient-derived explants (PDEs) from lung and ovarian cancer to interrogate redirected T cell immunity in intact human tissue. Using mesothelin-targeting bispecific T cell engager (BiTE(R), Amgen trademark)-secreting T cells, we observed antigen-dependent but heterogeneous responses across lesions. An integrated ex vivo response score stratified responder and non-responder TMEs, revealing that resistance associates with reduced antigen density, stromal dominance, and limited myeloid licensing rather than baseline lymphocyte abundance. Elevated prostaglandin E2 (PGE2) inversely correlated with BiTE-induced T cell activation, identifying the COX/PGE2 axis as a tissue-imposed constraint. COX inhibition amplified interferon-driven immune programs enhanced intratumoral CD8 infiltration, and increased tumor-restricted apoptosis. Spatial transcriptomics localized these effects to tumor-proximal immune hubs in responders, whereas non-responders remained stromally insulated. These findings position PDEs as human-based new approach methodologies enabling combinatorial ACT pharmacodynamics and stratification. Statement of significancePatient-derived explants provide a human-based new approach methodology to interrogate adoptive immunotherapy pharmacodynamics within intact tumor microenvironments in NSCLC and HGSOC. We uncover a COX/PGE2-mediated tissue ceiling that limits BiTE-driven T cell function and demonstrate that COX inhibition reactivates tumor-proximal immune hubs to enhance intratumoral CD8 infiltration and tumor-restricted apoptosis, informing patient stratification and rational combinations.

Infections are the most common cause of non-relapse mortality in multiple myeloma (MM), but the basis of persistent immune dysfunction is obscured by patient heterogeneity and complex treatment regimens, including autologous stem cell transplant (ASCT). We performed longitudinal multi-omic profiling of matched bone marrow and peripheral blood from MM patients across diagnosis, induction, ASCT, and recovery. We found the tumor imposes a compartment-specific immune program where the marrow exhibits metabolic and inflammatory changes that bias hematopoiesis and alter cytotoxic effector programs not mirrored in blood. Adaptive immune reconstitution is impaired up to two years post-ASCT. Half of patients fail to mount IgG responses to high-dose non-adjuvanted influenza vaccine, a defect overcome by the lipid nanoparticle (LNP) adjuvanted COVID mRNA vaccine, which elicited responses in all patients, supporting adjuvanted influenza vaccine strategies in MM. Together these findings define how myeloma and its treatment durably reshape immunity from the marrow outward. HighlightsO_LIMultiple Myeloma marrow and blood show opposing metabolic and inflammatory states C_LIO_LIInduction therapy selects durable myeloma plasma-cell transcriptional states C_LIO_LIB cell and follicular helper T deficits blunt antigen responses after transplant C_LIO_LICOVID-19 vaccination builds immune memory with variable responses to flu vaccination C_LI eTOCMultiple myeloma and its treatment leave a lasting imprint on the bone marrow niche. By profiling bone marrow and blood longitudinally at diagnosis, through induction, autologous transplant, and recovery, we show that marrow-local metabolic and inflammatory constraints persist and help explain why influenza vaccination often fails while mRNA vaccination succeeds.

Although small cell lung cancer (SCLC) comprises transcription factor (TF)-defined molecular subtypes (ASCL1, NEUROD1, POU2F3), the extent to which these subtypes predict response to clinically effective therapy in patients--and whether therapy can select for subtype switching--remains unknown. The recent approval of the DLL3xCD3 bispecific T-cell engager tarlatamab represents one of the first meaningful advances in relapsed small cell lung cancer (SCLC) in decades, yet responses remain heterogeneous and resistance is inevitable. Here, we inferred SCLC gene expression from circulating chromatin in prospectively collected patient plasma (46 patients; 167 samples), enabling interrogation of response and acquired resistance to tarlatamab. Parallel development of the first immunocompetent syngeneic mouse model to study tarlatamab response and resistance enabled functional validation. Across species, findings converged on a central principle: TF subtype governs both initial response and acquired resistance. Therapeutic response was significantly associated with ASCL1-subtype tumors, whereas NEUROD1-subtype tumors exhibited inferior responses and POU2F3-subtype tumors were uniformly resistant, consistent with DLL3 being a direct ASCL1 transcriptional target and most highly expressed in ASCL1-positive tumors. Strikingly, one mode of acquired resistance revealed therapeutic selection for a NEUROD1-high state with concomitant DLL3 downregulation. Other resistant tumors exhibited enrichment of regulatory and exhausted T-cell programs, highlighting tarlatamabs dual-targeting mechanism of action. Together, these results reveal that tarlatamab exerts selective pressure against ASCL1-driven lineages, facilitating resistance through loss of an antigen intrinsically linked to that state. These findings underscore the clinical relevance of TF-defined molecular subtypes in human SCLC. More broadly, they highlight the power of integrating longitudinal in vivo plasma transcriptional profiling from patient plasma with functional mouse modeling to uncover clinical and biological mechanisms of response and resistance to cell-surface-targeted therapies.

The COVID-19 epidemic and success of mRNA-LNP vaccines demonstrated the transformative potential of mRNA therapeutics. Beyond vaccination, mRNA delivery offers a platform for transient on-demand expression of therapeutic proteins for both rare and common diseases. While delivery of therapeutics to the liver is relatively straightforward, targeted delivery of mRNA-LNPs to the central nervous system (CNS) remains a significant challenge. Here we show that intranasal mRNA-LNP delivery results in localized mRNA cargo uptake and functional expression in the respiratory and olfactory epithelium, where the encoded cargo protein is secreted and can enter the CNS. Guided by genomic data of pain-associated gene expression, we identified secreted proteins as candidate mRNA-encoded analgesics. Intranasal mRNA-LNP encoding a synthetic oxytocin transcript (OXT) resulted in bioactive oxytocin peptide delivery to the CNS. Functionally, intranasal OXT mRNA-LNP enhanced social behaviour and attenuated pain responses across multiple behavioural paradigms, without impairing motor coordination. Importantly, repeated dosing was well tolerated and intranasal mRNA-LNP did not elicit an inflammatory response or alter overall health. Together, these findings establish intranasal mRNA-LNP delivery of secreted ligands as a safe, non-invasive route to target the CNS, unlocking a new class of mRNA therapeutics for pain or other disorders of the brain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/711938v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@131252aorg.highwire.dtl.DTLVardef@17efb79org.highwire.dtl.DTLVardef@1afe479org.highwire.dtl.DTLVardef@c0f2f5_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryIntranasal administration of mRNA-LNP enables local transfection in nasal epithelium and subsequent secretion of therapeutic oxytocin peptides into the brain, promoting social interactions and reducing pain.

Declines in nicotinamide adenine dinucleotide (NAD+) are linked to mitochondrial dysfunction, genomic instability, and metabolic stress that accompany aging and associated processes. While precursor-based approaches elevate systemic NAD, their clinical translation can be constrained by biosynthetic bottlenecks and first-pass metabolism. RENEWAL-NAD+ (ClinicalTrials.gov NCT07336836; retrospectively registered 01/04/2026) was a double-blind, randomized, placebo-controlled Phase 0/1b trial in healthy adults aged 45-75 years (60 randomized; primary analysis n=50) evaluating 5 days of oral LathMized NAD+ (LNAD+), a proprietary physiochemically modulated formulation designed to alter the supramolecular organization and solution behavior of NAD+ while preserving its native molecular structure. The primary endpoints were change in intracellular NAD (icNAD), measured in whole blood, and circulating NAD (cirNAD), measured in separated plasma, relative to baseline. At Day 6, icNAD increased by 53% versus placebo (p=5.48e-14; Hedges g=3.66), while cirNAD was unchanged (p=0.60), demonstrating compartment-selective intracellular NAD+ delivery. Plasma NAD catabolites increased substantially (1-methyl-nicotinamide, MeNAM p=5.39e-13; N1-methyl-2-pyridone-5-carboxamide, 2PY p=2.95e-16), consistent with engagement of downstream NAD metabolic flux. Exploratory analyses identified non-overlapping correlates for the two compartments (cirNAD tracking inflammatory and metabolic markers, icNAD tracking red blood cell indices and NAM). Treatment was very well tolerated: symptom incidence was comparable between groups (p=0.68), only one mild adverse event (nausea, Grade 1) occurred in the LNAD+ arm, and no secondary clinical laboratory, vital sign, wellbeing, or wearable-derived endpoint survived multiplicity correction. These data demonstrate rapid intracellular NAD augmentation after oral LNAD+ dosing with pharmacodynamic evidence of downstream metabolism, compartment-specific physiological signatures, and a favorable short-term safety profile.

Allogeneic cell therapies require the coordinated expression of multiple immunomodulatory genes, yet multigene circuits that function in permissive cell lines often fail in differentiated human tissues for unclear reasons. Here, we systematically dissect how transcriptional architecture governs functional immunoregulation in engineered human keratinocyte and fibroblast lines. Using site-specific large-cargo integration (eePASSIGE) as an enabling tool, we determined that genomic insertion efficiency was not the limiting factor for phenotype; rather, promoter arrangement and gene order dictated expression hierarchy. A single-promoter EF1-IDO1-T2A-GFP design that expressed robustly in HEK293T cells was nearly silent in skin-derived cells, preventing reporter-based enrichment. In dual- and tri-modular cassettes, we observed severe transcriptional interference: a downstream CMV promoter driving GFP or PD-L1/iCasp9 (via EMCV-IRES) markedly suppressed the upstream EF1-IDO1 unit, despite intact integration (resulting in [~]175-625-fold attenuation), demonstrating strong promoter interference within the circuit. Functionally, co-culture assays revealed a hierarchical immunomodulatory logic: high IDO1 expression proved to be a requisite threshold for T-cell suppression, whereas PD-L1 provided measurable benefit only against highly activated, PD-1+ T cells in vitro. Collectively, these data establish a site-specific framework for generating immune-tuned skin cells and define essential design rules for avoiding promoter interference in next-generation translational skin substitutes.

To elucidate how cancer cells generate resistance and defined forms of tissue structure in response to therapeutic stress, we tracked the temporal dynamics of life cycle of polyploid giant cancer cells (PGCCs) induced by the mitotic destabilizer vincristine (VCR). Live-cell fluorescence imaging revealed that VCR activated a distinct endoreplication-based life cycle, which replaced canonical mitosis. PGCCs exhibited reduced proliferative activity, enhanced epithelial-mesenchymal transition (EMT), progressive acquisition of blastomere-like features, and broad multilineage differentiation potential. Both PGCC populations and single PGCC-derived progeny displayed time- and dose-dependent acquisition of malignant traits in vitro and tumorigenic capacity in vivo. PGCC-derived spheroids exhibited ability to differentiate into the cells of origin from three germ layers. Importantly, pre-budding PGCCs induced by higher VCR concentrations exhibited enhanced ability for glandular structure formation and tissue differentiation. Morphologically, the nuclei of PGCC-derived spheroids underwent growth in size, formation of luminal structure, and followed by maturation of lumen. Mechanistically, PGCCs entered a senescent state characterized by elevated senescence-associated secretory phenotype (SASP)-manifested by rich proinflammatory cytokines. Notably, silencing IL1{beta}, IL6, or IL8, or pharmacological inhibition of their receptors, suppressed PGCC formation, budding, EMT, and blastomere-like reprogramming into structured tissue. Our studies provide novel mechanistic insights into early embryogenesis and tumorigenesis at the tissue structural developmental level.

Pulmonary fibrosis (PF) involves excessive collagen accumulation, yet mechanisms shifting the balance of synthesis and degradation toward net deposition remain unclear. Myeloperoxidase (MPO) inversely correlates with survival in PF. Using the bleomycin model, we found MPO knockout (MPOko) mice were protected from fibrosis, and pharmacological MPO inhibition after peak inflammation (day 7) recapitulated this protection. MPO persisted in lung tissue 21 days post-injury despite neutrophil efflux, linking acute inflammation to sustained remodeling. Mechanistically, we identified that MPO inhibits Cathepsin K (CatK), a potent collagenolytic enzyme involved in fibrosis resolution. Notably, CatK gene expression (CTSK) is elevated in PF, suggesting post-translational inhibition of CatK. MPOko and inhibitor-treated mice exhibited elevated CatK activity after bleomycin; exogenous addition of pathophysiologic concentrations of MPO reduced CatK activity in mouse precision-cut lung slices and human fibroblasts. Biochemically, MPO reduced CatK activity to 33% of control. In two distinct cohorts of PF patients, we observed significantly increased MPO protein levels in platelet poor plasma and in lung tissue. In PF patients, 62% had MPO levels in platelet poor plasma exceeding healthy controls, while lung tissue from other PF patients showed significantly elevated MPO staining. Plasma levels were inversely correlated with decreased survival, FVC, and DLCO. These findings establish MPO as a post-translational inhibitor of CatK-mediated collagenolysis, revealing a mechanism linking acute inflammation to sustained fibrosis and suggest a patient subpopulation that may benefit from MPO-targeted therapy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/713467v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@d8fc5eorg.highwire.dtl.DTLVardef@1a088fcorg.highwire.dtl.DTLVardef@818b7dorg.highwire.dtl.DTLVardef@ecdca0_HPS_FORMAT_FIGEXP M_FIG C_FIG Myeloperoxidase persists in lung tissue after injury and inhibits cathepsin K activity, impairing collagen degradation and promoting extracellular matrix accumulation during pulmonary fibrosis.

Cellular therapies, toxicity screening, and regenerative medicine depend on selecting mammalian cell types with optimal lifespan, persistence post-transplant, immunogenicity, and chemical resilience. This review synthesizes data from over 50 immune, parenchymal, stem, and emerging engineered cell populations--including gamma-delta T cells, iNKT cells, CAR-macrophages, and hypoimmune iPSC derivatives--drawing from in vivo lifespan studies (including 1{blacksquare}C birth-dating and deuterium labeling), engraftment dynamics, immune rejection risk, and stress sensitivity profiles. We introduce a Programmability & Persistence Score (PPS; 0-20) that integrates these features into a unified metric, complemented by Pareto frontier analysis to visualize multi-objective trade-offs. High-PPS cell types (e.g., HLA-matched HSCs, hypoimmune iPSCs, chondrocytes) are suited for long-term regenerative applications, while low-PPS sentinels (e.g., neutrophils, enterocytes) serve acute assays. We discuss mathematical extensions including multi-criteria decision analysis, fuzzy membership functions, and Bayesian frameworks that address limitations of linear additive scoring. Together, these integrated profiles support cell selection for gene editing, organ-on-chip systems, in vivo cell programming, and immunotherapy, bridging cell biology with translational engineering.