Sarcopenia, loss of muscle mass is a considerable health burden that demands immediate societal attention. Direct myogenic somatic cell reprogramming, a potential muscle regeneration method is constrained by an inability to control the signaling logic that governs cell fate. Here, we show that this barrier can be overcome using AI-designed receptor modulators. Screening de novo minibinders, we identify a synthetic protein cocktail, C6-DPC, that drives efficient human fibroblast-to-muscle transdifferentiation with robust structural and metabolic maturation. C6-DPC reprograms extracellular signaling by activating pro-myogenic FGFR1/2c pathways while suppressing anti-myogenic inputs through ALK1 and TGFBR2; targeted depletion of ALK1 is sufficient to lower the reprogramming barrier. Inflammatory signaling via gp130 emerges as a dominant checkpoint, and its inhibition further enhances conversion. Engineered tissues generate high twitch and tetanic forces in both wild-type and dystrophin-deficient human cells. These findings demonstrate that programmable synthetic ligands can rewrite receptor-level signaling to direct cell fate and enable functional tissue regeneration.

The Angiopoietin–Tie2 pathway is a key regulator of postnatal vascular maintenance and remodeling, regulating vascular barrier function and integrity. While the opposing roles of the ligands Angiopoietin-1 (Ang 1) and Angiopoietin-2 (Ang 2) have been recognized for decades, the structural mechanism governing their distinct signaling outputs has only recently been elucidated. As artificial intelligence and protein design continue to develop, emerging evidence suggests that ligand valency and receptor clustering are key determinants of Tie2 pathway activation and endothelial cell function; that is, “form follows function”. This review summarizes the latest discovery in the structural biology and signaling mechanism of the Tie2 pathway using protein design to decode the ligand–receptor interactions. Probing the underlying molecular basis of Tie2 offers new therapeutic opportunities for targeting diseases, featuring vascular dysfunctions such as sepsis, traumatic brain injury, acute respiratory diseases, chronic inflammation, and cancer. This also highlights the next generation of AI-designed protein therapeutics.

Background and aims:Increased levels of α-tubulin and its post-translational modifications (PTMs) are found in human heart failure and could initiate diastolic dysfunction by modulating cardiomyocyte stiffness. How these modifications occur and how they may underlie cardiac dysfunction remains unknown. Upstream kinases may play a critical role, but this has not been explored. Methods and results:Here we address this question by, for the first time ever, determining levels of the enzymes involved in microtubule (MT) detyrosination and acetylation (αTAT1, HDAC6) in a well-characterized cohort of patients with hypertrophic cardiomyopathy (HCM). In HCM patients (N=10-11), protein levels of detyrosination enzymes remain unaltered, whilst levels of αTAT1 and HDAC6 were decreased and increased, respectively. Phosphoproteomics in HCM (N=24) and control (N=8) myocardium identified significant differences in over 1900 serine/threonine and 160 tyrosine phosphosites, in addition to increased EGFR/IGF1R-MAPK signaling in HCM. We subsequently showed that MT repolymerization was increased in HCM MYBPC3 Arg943X hiPSC-CMs. Isoprenaline-mediated PKA activation decreased MT repolymerization in hiPSC-CMs and revealed CLASP1 , MAST4 and MAP1A as potential MT modifiers in HCM. Conclusions:We show that the altered HCM MT code cannot be attributed to levels of key MT-modifying enzymes. By combining kinome analyses in human HCM hearts with hiPSC-CM studies on MT dynamics, PTMs and contractility we unveiled a regulatory role for MTs in the cardiomyocyte response to beta-adrenergic receptor stimulation. Disease-mediated changes in the MT code thereby exert both a direct, and indirect effect on cardiac function via mediating the response to adrenergic activation. Graphical Abstract created with BioRender.com.

Directed differentiation of pluripotent stem cells (PSCs) into pancreatic islets is a cornerstone strategy for diabetes cell therapy. This process relies on growth factor-driven activation of core transcriptional regulators, notably PDX1 and NGN3, to restrict the multi-lineage potential of definitive endoderm to pancreatic progenitors and endocrine cell types. Yet differentiation efficiency and lineage fidelity vary markedly across PSC lines. Here, we demonstrate that a dominant constraint is persistent Polycomb Repressive Complex 2 (PRC2)-mediated epigenetic repression at the PDX1 and NGN3 loci, limiting endocrine specification despite inductive signaling. To directly test whether chromatin states at the PDX1 and NGN3 loci gate developmental competence, we deployed a computationally engineered epigenetic effector (EBdCas9) to transiently and sequentially remove H3K27me3 at those loci during defined developmental windows. Targeted epigenetic resolution robustly enhanced endocrine lineage commitment and accelerated β-cell differentiation across genetically diverse PSC lines. In contrast, direct transcriptional activation with VP64dCas9 increased PDX1 and NGN3 expression but did not improve differentiation outcomes. Integrated cell population studies and genome-wide chromatin and transcriptomic analyses reveal that PRC2-targeted remodeling preferentially activates endocrine gene networks while limiting progenitor expansion and lineage-inappropriate programs. These findings establish that gene-targeted manipulation of PRC2-mediated repression at PDX1 and NGN3 can be used to control cell lineage competence. Collectively, our study reframes variability in PSC differentiation as a failure of epigenetic resolution rather than transcriptional insufficiency and introduces locus-specific chromatin remodeling as a generalizable strategy to enforce developmental fidelity.

Schizophrenia has been linked to severely damaging de novo mutations in synaptic junction proteins, neurotransmitter receptors, transcription factors, and chromatin remodeling proteins. In a patient with schizophrenia in the absence of a family history of severe mental illness, we identified de novo nonsense mutation, INO80D p.Q568X, associated with both a truncated protein and partial nonsense-mediated decay. Three experiments were undertaken to evaluate the consequences of the mutation. 1) In neural stem cells (iNSCs) differentiated from WTC11 iPSCs, CRISPRi knockdown of INO80D led to downregulation of three subunits of the AMPA-glutamate receptor, of multiple genes mutant in schizophrenia, and of genes of synaptic function. 2) INO80D p.Q568X iNSCs and neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) had significantly lower expression of neurogenesis genes compared to patient-derived cells with the mutation corrected by CRISPR-Cas9 gene editing. Patient-derived INO80D p.Q568X neurons had significantly higher expression of cell division genes compared to lines with the mutation corrected, consistent with the possibility that some of these cells may be undergoing mitosis, which is not normal for neurons. 3) Finally, on microelectrode array (MEA) plates, WTC11-derived glutamatergic neurons with reduced expression of INO80D had more rapid firing rate and increased average network burst duration, both features of neurons derived from patients with neurodevelopmental disorders. Overall, these findings suggest that partial loss of INO80D function due to de novo mutation may have disrupted normal neurodevelopment and contributed to the schizophrenia of this patient.

Enamel, the hardest mineralized material in the human body, protects the underlying living tissues, the dentin and pulp of the tooth. However, over 90% of adults have lost or damaged enamel and cannot regenerate the protective structure due to lack of enamel-producing cells, ameloblasts. iPSC-derived secretory Ameloblasts (isAM) have promise in future regenerative dentistry. Today, it is not known why iAM maturation requires intimate contact with the dentin-producing cell type, odontoblast. Here, we reveal that one of the critical signaling ligands emanating from odontoblasts for ameloblast maturation is Delta, the ligand for Notch receptor. We showed that our designed, soluble Notch agonist can induce iAM organoid maturation in an unprecedented manner, without interactions with odontoblast layer. Notably, soluble Notch agonist induces the iAM maturation to a novel, WDR72-positive mature secretory AM stage (ismAM) in our ameloblast organoid model. When transplanted under the kidney capsule of NOD-SCID mice, these ismAM organoids generated enamel-like calcified material, as confirmed by microCT analysis, marking the first demonstration that Notch-activated iAM organoids can form such tissue in vivo. This novel maturation procedure enabled us to analyze the specific requirements of DLX3 function in ameloblasts, independent of its known function in odontoblasts. We now show that DLX3, a gene associated with Amelogenesis Imperfecta, is required on a cell-autonomous manner in human ameloblasts for the expression of Enamelin, MMP20, and WDR72, a role not previously demonstrated in mouse models.

Abstract The Angiopoietin–Tie2 pathway is a key regulator of vascular stability, but therapeutic exploitation has been limited by the poor developability of Angiopoietin-1 (Ang1), and there are key unresolved mechanistic questions. Ang1 binds both Tie2 and α5β1 integrin, and the role of the α5β1 interaction in signaling has been unclear. We used RFdiffusion to design a stable, high-affinity Tie2 minibinder that has no affinity for α5β1. The binder is a selective antagonist in monomeric form, and a potent agonist when assembled into an octavalent architecture (H8mb) which drives Tie2 clustering. H8mb signals as potently as Ang1, indicating that integrin engagement is not required for Tie2 activation. However, the duration of signaling is reduced, and internalization of H8mb-Tie2 complexes is more rapid, suggesting that integrin may function as a co-receptor for Ang1 that prolongs signaling by extending the lifetime of the receptor ligand complex on the cell surface. In a mouse model of acute respiratory distress syndrome (ARDS), H8mb markedly improved survival. These results demonstrate that de novo designed receptor binders can enable dissection of co-receptor control of signaling dynamics, and the more stable and manufacturable H8mb provides routes to more developable therapeutic candidates.

Late Onset Alzheimer’s Disease (LOAD) is the most common neurodegenerative disorder. Carriers of an ɛ4 allele of the apolipoprotein E gene (APOE) have significantly increased risk of developing LOAD. LOAD is also strongly sex biased. Besides the well-established role of sex hormones, the impact of genetic sex combined with APOE genotypes has not been studied. Sex chromosome dosage (SCD) is the main genetic difference between females (XX) and males (XY). Despite dosage compensation by X chromosome inactivation (XCI), some X-linked genes remain expressed from the inactive X (Xi) and are female biased in expression. Many of these genes are related to neurological and immune functions and could contribute to neuroinflammation in AD. To understand the genetic interactions between autosomal risk (APOE) and sex-bias in AD, we are generating isogenic sets of hiPSCs with various sex chromosome complements and APOE genotypes. We have derived XXY/XY, XY/X, and XX/X isogenic pairs by reprogramming cells from naturally mosaic individuals or by selective removal of the Xi in vitro. These lines uniquely position us to differentiate the effects of X chromosome dosage and/or presence of a Y chromosome, while minimizing genetic variability and environmental or hormonal confounders. Currently, we are also constructing APOE ɛ3 and ɛ4 alleles in our paired hiPSC lines with differing sex chromosome content using gene editing. Starting from an XXY/XY isogenic pair, we have generated an hiPSC series with six different genotypes of APOE ɛ3/ɛ3, ɛ3/ɛ4 or ɛ4/ɛ4 alleles with either the XXY or XY genotype. Preliminary results in differentiated neural precursor cells show differential expression of sex-linked genes in relation to SCD. We also observed genome-wide changes in gene expression and DNA methylation. Subsets of autosomal sex-biased genes were differentially affected either by X chromosome dosage, presence of a Y, or both. The new hiPSC lines are being differentiated to AD-relevant cell types, including neurons, microglia, and cortical organoids, for transcriptomic and functional analyses to elucidate how APOE alleles and genetic sex interact to modulate risk in LOAD pathology.

Growth factor induced receptor dimerization and activation of downstream pathways can modulate cell fate decisions. Here, we investigate the potential of de novo designed synthetic ligands, termed Novokines, to reprogram cell identity by inducing proximity of novel pairs of receptor subunits. We find that a design, H2F, that brings together HER2 (which has no known natural ligand) and the FGF receptor has potent signaling activity. H2F induces robust signaling and reprograms fibroblasts into myogenic cells. Unlike native FGF ligands, H2F selectively activates the MAPK pathway without engaging PLCγ-mediated Ca²⁺ signaling. FRET assays confirm H2F-mediated HER2-FGFR proximity, and phosphoproteomic analysis reveals activation of MAPK effectors. H2F-induced ERK phosphorylation is abolished in cells expressing a kinase-dead FGFR1 (K514M) mutant, confirming the requirement for FGFR catalytic activity. H2F treatment significantly increases myofiber formation from adult patient-derived primary myoblasts, demonstrating its capacity to promote myogenic regeneration. Our findings demonstrate that synthetic receptor pairings can rewire signaling outputs to drive regeneration, providing a programmable platform for cell fate engineering.

Congenital neutropenia is characterized by a reduced neutrophil count, decreased innate immunity and increased susceptibility to recurrent infections. While congenital neutropenia has various genetic causes, recent studies have linked TCIRG1 mutations to this condition. TCIRG1, a key component of the vacuolar ATPase (V-ATPase) complex, is essential for osteoclast function, but its role in hematopoiesis remains unclear. We previously identified heterozygous TCIRG1 mutations, including R736S, R736C, R736P, and E722D, in individuals with congenital neutropenia. However, the mechanism by which these mutations lead to impaired granulopoiesis remains unknown. To investigate the functional consequences of TCIRG1 mutations, we generated induced pluripotent stem cells (iPSCs) from affected individuals and healthy controls. Using in vitro differentiation protocols, we assessed hematopoietic progenitor formation, proliferation, survival, and neutrophil differentiation. We observed significant defects in myeloid differentiation and increased cell death in patient-derived iPSC lines. CRISPR/Cas9-mediated correction of the R736C mutation restored normal neutrophil differentiation, confirming its pathogenic role. Immunofluorescence analysis revealed reduced expression and altered intracellular localization of the TCIRG1 protein, characterized by a more diffuse cytosolic distribution in the mutant cell lines. Our findings suggest that TCIRG1 mutations impair neutrophil development, likely through structural and functional disruption of the V-ATPase complex. This study provides new insights into the molecular basis of TCIRG1-associated neutropenia and highlights potential avenues for therapeutic intervention.

The R403Q mutation in the sarcomere protein beta-myosin heavy chain (β-MHC) is a known genetic cause of hypertrophic cardiomyopathy (HCM), associated with ventricular hypercontractility, impaired relaxation, and cardiac arrhythmias. Despite extensive research, the mutations impact on myosin contractile properties remains unclear partly due to discrepancies across different model systems. In this study, we used a multidisciplinary approach to explore mutational effects using two distinct heterozygous R403Q systems: a Yucatan minipig model and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). X-ray diffraction of R403Q minipig ventricular muscle demonstrated reduced order of the thick filament, suggesting destabilization of the inhibited OFF (vs. ON) state of myosin in relaxed muscle, which correlated with elevated force at submaximal calcium. Super-resolution, single-molecule fluorescence microscopy indicated elevated ATPase activity in thick filament zones lacking cardiac myosin binding protein-C (cMyBP-C). Furthermore, R403Q myofibrils exhibited slower activation and relaxation kinetics, with reduced sensitivity to ADP. Molecular dynamics simulations suggested that altered interactions at the actomyosin interface contribute to these effects, rather than changes at the nucleotide binding pocket, typically associated with ADP release. Human engineered heterozygous R403Q hiPSC-CMs exhibited reduced maximal myofibril force, slowed relaxation kinetics, and hypercontraction in engineered heart tissue constructs-consistent with HCM phenotypes observed in the heterozygous porcine model. Our results demonstrate that the R403Q mutation induces contractile dysfunction within the early stages of stem cell derived cardiomyocyte development and in juvenile minipigs, and that hypercontractility and slower contractile kinetics may result from a combination of an increased population of activated (ON) myosin heads and delayed detachment during cross-bridge cycling, respectively.

Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular performance when delivered acutely post-myocardial infarction but are ineffective in chronic myocardial infarction/heart failure. 2’-deoxy-ATP (dATP) activates cardiac myosin and potently increases contractility. Here we engineered hPSC-CMs to overexpress ribonucleotide reductase, the enzyme controlling dATP production. In vivo, dATP-producing CMs formed new myocardium that transferred dATP to host cardiomyocytes via gap junctions, increasing their dATP levels. Strikingly, when transplanted into chronically infarcted hearts, dATP-producing grafts increased left ventricular function, whereas heart failure worsened with wild-type grafts or vehicle injections. dATP-donor cells recipients had greater voluntary exercise, improved cardiac metabolism, reduced pulmonary congestion and pathological cardiac hypertrophy, and improved survival. This combination of remuscularization plus enhanced host contractility offers a novel approach to treating the chronically failing heart. One Sentence Summary Transplanting gene-edited dATP-donor cardiomyocytes in chronically infarcted heart restores their cardiac function, improving both exercise tolerance and survival.

Enamel, the hardest material in the human body, is required to protect our living organ, tooth. However, over 90% of adults have lost or damaged enamel and cannot regenerate the protective structure due to lack of enamel producing cells, ameloblasts. iPSC derived mature Ameloblasts (iAM) have promise in future regenerative dentistry. Today it is not known why iAM maturation requires intimate contact with the dentin producing cell type, odontoblast. Here we reveal that one of the critical signaling ligands emanating from odontoblasts for ameloblast maturation is Delta, the ligand for Notch receptor. We showed that our designed, soluble Notch agonist can induce iAM organoid maturation in an unprecedented manner, without interactions with odontoblast layer. This novel maturation procedure enables us to analyze the specific requirements of DLX3 function in ameloblasts, independent of its known function in odontoblasts. We now show that DLX3, the gene associated with Amelogenesis Imperfecta, is required on a cell-autonomous manner in ameloblasts for the expression of Enamelin and MMP20.

Salivary glands are highly susceptible to injury and degeneration. To facilitate studies in human salivary gland disease, we developed a rapid protocol for 3D hiPSC-derived salivary gland organoids that recapitulate human fetal salivary gland gene expression and function, have both ductal and acinar cell types, secretory capacity, and the ability to respond to cholinergic agonism. Oral health issues resulting from diabetes mellitus have been attributed to salivary gland dysfunction, leading to chronic xerostomia and increased dental caries. To study diabetic salivary gland hypofunction, we further developed a diabetic model, demonstrating diabetic hallmarks including FOXO1 nuclear localization, AGE-RAGE signaling, and defective oxidative phosphorylation, which were by treatment with the diabetic drug Metformin. Our model has implications for the development of effective therapeutics against salivary gland dysfunction in diabetes and other metabolic diseases in exocrine tissues.

The clinical use of Nerve Growth Factor (NGF) for neuronal regeneration has been hampered by pain sensitization side effects. NGF signals through the receptor tyrosine kinase TrkA and the co-receptor p75NTR; pain sensitization is thought to involve p75NTR. We sought to overcome this limitation by de novo design of a TrkA agonist that does not bind p75NTR. We designed homodimeric TrkA engaging constructs that dimerize TrkA subunits in a variety of geometries, and identified those eliciting the strongest signaling. The resulting designed agonists are able to stimulate transdifferentiated neurons and neuroblastoma cell lines, leading to neurite outgrowth and neuronal differentiation, with considerably reduced transcription of inflammation and pain related genes. These agonists are promising candidates for promoting neuronal regeneration without adverse side effects. Highlights ![Figure][1] ### Competing Interest Statement The authors plan to file a patent application. D.K.S. is a consultant and/or collaborator with ThermoFisher Scientific, AI Proteins, Genentech, and Matchpoint Therapeutics. [1]: pending:yes

Animal models of the neuromuscular junction (NMJ) have been widely studied but exhibit critical differences from human biology limiting utility in drug and disease modelling. Challenges with scarcity, scalability, throughput, and ethical considerations further limit the suitability of animal models for preclinical screening. Engineered models have emerged as alternatives for studying NMJ functionality in response to genetic and/or pharmacological challenge. However, these models have faced challenges associated with their poorly scalable creation, sourcing suitable cells, and the extraction of reliable, quantifiable metrics. We present a turnkey iPSCbased model of the NMJ employing channelrhodopsin-2 expression within the motor neuron (MN) population driving muscle contraction in response to blue light. MNs co-cultured with engineered skeletal muscle tissues produced twitch forces of 34.7 +/- 22.7 mu N in response to blue light, with a response fidelity > 92 %. Histological analysis revealed characteristic punctate acetylcholine receptor staining co-localized with the presynaptic marker synaptic vesicle protein-2. Dose-response studies using botulinum neurotoxin showed loss of function in a dose- and time-dependent manner (EC50 - 0.11 +/- 0.015 mu g). Variability of the EC50 values between 2 different iPSC differentiations of both cell types and 2 users was less than 2 %. Further testing with the acute neurotoxins acetylcholine mustard and d-tubocurarine validated the biological relevance of the postsynaptic machinery of the model. This model marks a meaningful progression of 3D engineered models of the NMJ, providing engineered tissues at a throughput relevant to potency and screening applications with an abundant iPSC cell source and standardized hardware-software ecosystem allowing technology transfer across laboratories.

Oral facial cleft (OFC) comprises cleft lip with or without cleft palate (CL/P) or cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) in many genomic loci where the presumed effector gene (for example, IRF6 in the 1q32 locus) is expressed in embryonic oral epithelium. To identify candidates for functional SNPs at eight such loci we conduct a massively parallel reporter assay in a fetal oral epithelial cell line, revealing SNPs with allele-specific effects on enhancer activity. We filter these SNPs against chromatin-mark evidence of enhancers and test a subset in traditional reporter assays, which support the candidacy of SNPs at loci containing FOXE1, IRF6,  MAFB, TFAP2A, and TP63. For two SNPs near IRF6 and one near FOXE1, we engineer the genome of induced pluripotent stem cells, differentiate the cells into embryonic oral epithelium, and discover allele-specific effects on the levels of effector gene expression, and, in two cases, the binding affinity of transcription factors FOXE1 or ETS2. Conditional analyses of GWAS data suggest the two functional SNPs near IRF6 account for the majority of risk for CL/P at this locus. This study connects genetic variation associated with OFC to mechanisms of pathogenesis.

Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta.For complete details on the use and execution of this protocol, please refer to Alghadeer et al.1

Genome wide association studies of isolated orofacial cleft (OFC) have identified common single nucleotide polymorphisms (SNPs) at the 1q32/ IRF6 locus and seven other loci where, like IRF6 , the presumed OFC-relevant gene is expressed in oral epithelium. To identify the functional subset of SNPs at these loci we conducted a massively parallel reporter assay (MPRA) in a cell line derived from fetal oral mucosa. This assay identified SNPs with allele-specific effects on reporter activity levels at all eight loci; at four of the loci, these SNPs were within chromatin marked as an active enhancer in human embryonic faces. Results from traditional luciferase reporter assays concorded with those from the MPRA at nine of eleven SNPs tested. We separately engineered two promising SNPs from the 1q32/ IRF6 locus to homozygosity in the genome of induced pluripotent stem cells and then differentiated the cells into embryonic oral epithelium. We found that cells homozygous for the risk-associated alleles had lower levels of IRF6 expression, and that these alleles favored the binding of transcriptional repressors, FOXE1 and ETS2, respectively. Mutations in FOXE1 also cause a syndromic form of OFC, and ETS2 suppresses differentiation of epithelia. Conditional analyses of a meta-analysis of multi-ethnic genome wide association studies suggest that the two SNPs account for the majority of risk for OFC associated with variation at 1q32/ IRF6 . This study connects genetic variation associated with orofacial cleft to mechanisms of pathogenesis. ### Competing Interest Statement KB is co-founder of Matchstick Technologies, Inc. KB is a co-inventor of PIXUL (US Patents 10809166, 11592366).