Biology subjects

Mack, D. L.

Publications and source records attributed to Mack, D. L..

13 recordsLinked to original sources

bioRxiv2026

Genetic control couples TGFβ-independent pluripotent stem cell renewal to deterministic forward programming across germ layers

Reproducible and scalable manufacture of human induced pluripotent stem cell (hiPSC) derivatives requires control over expansion and differentiation. Transcription factor-mediated forward programming can place hiPSC differentiation under genetic control, but expansion still relies on extracellular growth-factor supplementation, increasing process complexity and operator dependence. Here we introduce GERALT (Genetically Enhanced Renewal and Autonomous Lineage Transdifferentiation), a push-pull genetic circuit assembled through targeted safe-harbour integration that couples NANOG-supported, TGF{beta}-independent renewal to doxycycline-inducible lineage programming and simultaneous NANOG silencing. GERALT maintained pluripotency during extended TGF{beta} withdrawal in both adherent and suspension culture, yet remained programmable towards myogenic, neuronal, and hepatic fates. Compared with the established OPTi-OX platform in myogenesis, GERALT enabled differentiation in simplified medium while canalising and accelerating commitment. GERALT also supported direct myogenic programming in suspension and enabled direct casting of hiPSCs into hydrogel followed by in-tissue programming, yielding electrically responsive engineered muscle tissues. These results extend genetic control upstream into stem cell renewal, facilitating deployment of new approach methodologies and other biomanufacturing applications.

synthetic biology↗

bioRxiv2026

Designed Minibinders Rewire Receptor Signaling to Enable Functional Human Myogenic Reprogramming

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.

biochemistry↗

bioRxiv2026

Engineered muscle tissues with enhanced maturation enable the identification of clinically relevant rAAV capsids

Developing in vitro models that recapitulate both the structure and function of native human tissues is crucial for a better understanding of pathophysiology and for improving the reliability of preclinical studies. Here, we demonstrate that engineered muscle tissues derived from human pluripotent stem cells can serve as an in vitro platform for gene therapy. Recombinant vectors derived from the adeno-associated virus transduce engineered muscle tissues with high efficiency and in a dose-dependent manner, allowing long term assessment of transgene expression in a human cellular context. We next used this model to conduct a comparative analysis of 8 natural AAV capsids and showed that their relative efficiency depends on engineered muscle tissue maturation level. In more mature tissues subjected to uniaxial mechanical stretch, AAV9 performed better, which is reminiscent of its high clinical potential in patients with neuromuscular disorders. Finally, our model also confirmed the higher efficiency of artificial MyoAAV variants specifically developed to have an improved muscle transduction. Altogether, this work demonstrates the potential of human engineered muscle tissues in the preclinical testing of AAV vectors, paving the way for the development of personalized gene therapy platforms.

bioengineering↗

bioRxiv2025

Inference and validation of a dynamic gene regulatory network influencing cell fate during Duchenne muscular dystrophy initiation

The onset of gene regulatory disturbances leading to developmental disorders remains poorly understood, particularly when symptoms begin after birth. Using induced pluripotent stem cells, single-cell transcriptomics and computational modeling, we determined the gene expression waves guiding early myogenesis and how they are affected by a disease-causing mutation. We uncovered a dynamic gene regulatory network organized in two kernels that function as a toggle-switch controlling stochastic cell fate decisions. In cells derived from patients with Duchenne muscular dystrophy, these waves of regulation are less pronounced and the switch remains in the OFF state with a higher probability, resulting in convergence into a restricted cell fate. Experimental reactivation of the switch through RNA interference led to a rescued transcriptomic state and to a refined network topology. This work uncovers a dynamic regulatory mechanism underlying early cell fate divergence and provides a general framework to infer and validate dynamic gene regulatory networks involved in disease initiation.

bioinformatics↗

bioRxiv2025

Voluntary Running Sustains the Correction of Inflammation-Related Gene Expression Conferred by AAV Gene Therapy in Mdx Mice

Duchenne Muscular Dystrophy is a fatal disease characterized by persistent skeletal muscle degeneration, inflammation, and fibrosis. Gene therapy using an adeno-associated virus derived vector and a microdystrophin transgene is currently under investigation in patients but the impact of physical activity on long-term therapeutic outcome remains poorly understood. Recently, we reported 21-weeks of voluntary wheel running complemented the positive endurance and muscle function outcomes of gene therapy in mdx mice. In the present study, we performed a transcriptomic analysis of the gene expression changes associated with functional recovery in the diaphragm. RNA-Sequencing and bioinformatic analysis revealed 2881 dysregulated genes in untreated and unexercised mdx mice including inflammatory and fibrotic signaling pathways frequently affected in Duchenne Muscular Dystrophy patients. Among the dysregulated genes, 774 were rescued towards WT after adeno-associated virus microdystrophin injection. Importantly, 93% of the rescued genes were maintained by voluntary running, which indicates that physical exercise has no significant impact on the outcome of gene therapy in the mdx diaphragm. Our study provides vital information that could help guide DMD patient follow-up protocols after treatment with gene therapy.

pathology↗

bioRxiv2024

YAP dysregulation triggers hypertrophy by CCN2 secretion and TGFβ uptake in human pluripotent stem cell-derived cardiomyocytes

Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in b-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet, how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway, specifically its effector molecule YAP, has been demonstrated to be reactivated in pathological hypertrophic growth. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, YAP, is altered 1) by changes in the biomechanics of HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates to confirm the hypercontractile phenotype in MYH7 mutants. We next modulate contractility in healthy and disease hiPSC-CMs by treatment with positive and negative inotropic drugs and demonstrate a correlative relationship between contractility and YAP activity. We further demonstrate the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation. We conclude that the overactivation of YAP, possibly initiated and driven by hypercontractility, correlates with excessive CCN2 secretion (connective tissue growth factor), enhancing cardiac fibroblast/myofibroblast transition and production of known hypertrophic signaling molecule TGF{beta}. Our study suggests YAP being an indirect player in the initiation of hypertrophic growth and fibrosis in HCM. Our results provide new insights into HCM progression and bring forth a testbed for therapeutic options in treating HCM. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/597045v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@3357aforg.highwire.dtl.DTLVardef@c1c210org.highwire.dtl.DTLVardef@abb72forg.highwire.dtl.DTLVardef@187aa80_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Far-red and sensitive sensor for monitoring real-time H2O2 dynamics with subcellular resolution and inmulti-parametric imaging applications.

H2O2 is a key oxidant in mammalian biology and a pleiotropic signaling molecule at the physiological level, and its excessive accumulation in conjunction with decreased cellular reduction capacity is often found to be a common pathological marker. Here, we present a red fluorescent Genetically Encoded H2O2 Indicator (GEHI) allowing versatile optogenetic dissection of redox biology. Our new GEHI, oROS-HT, is a chemigenetic sensor utilizing a HaloTag and Janelia Fluor (JF) rhodamine dye as fluorescent reporters. We developed oROS-HT through a structure-guided approach aided by classic protein structures and recent protein structure prediction tools. Optimized with JF635, oROS-HT is a sensor with 635 nm excitation and 650 nm emission peaks, allowing it to retain its brightness while monitoring intracellular H2O2 dynamics. Furthermore, it enables multi-color imaging in combination with blue-green fluorescent sensors for orthogonal analytes and low auto-fluorescence interference in biological tissues. Other advantages of oROS-HT over alternative GEHIs are its fast kinetics, oxygen-independent maturation, low pH sensitivity, lack of photo-artifact, and lack of intracellular aggregation. Here, we demonstrated efficient subcellular targeting and how oROS-HT can map inter and intracellular H2O2 diffusion at subcellular resolution. Lastly, we used oROS-HT with the green fluorescent calcium indicator Fluo-4 to investigate the transient effect of the anti-inflammatory agent auranofin on cellular redox physiology and calcium levels via multi-parametric, dual-color imaging.

bioengineering↗

bioRxiv2024

Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROSs capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via A{beta}-putriscine-MAOB axis, highlighting the sensors relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.

bioengineering↗

bioRxiv2023

Dystrophin deficiency impairs cell junction formation during embryonic myogenesis

Mutations in the DMD gene lead to Duchenne muscular dystrophy, a severe X-linked neuromuscular disorder that manifests itself as young boys acquire motor functions. DMD is typically diagnosed at 2 to 4 years of age, but the absence of dystrophin negatively impacts muscle structure and function before overt symptoms appear in patients, which poses a serious challenge in the optimization of standards of care. In this report, we investigated the early consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Here, dystrophin deficiency was linked to marked dysregulations of cell junction protein families involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.

pathology↗

bioRxiv2022

Interferon-gamma Elicits Pathological Hallmarks of ALS in Human Motor Neurons

Neuroinflammation is an established factor contributing to amyotrophic lateral sclerosis (ALS) pathology, implicating the possible detrimental effects of inflammatory cytokines on motor neurons. The RNA/DNA-binding protein TDP-43 has emerged as a pivotal actor in ALS, because TDP-43 mutations cause familial ALS and loss of nuclear TDP-43, associated with its redistribution into cytoplasmic aggregates (TDP-43 proteinopathy) in motor neurons occurs in 97% of ALS cases. However, mechanisms linking neuroinflammation to TDP-43 mislocalization have not been described. Programmed death-ligand 1 (PD-L1) is an immune-modulatory protein, highly expressed on cell surfaces following acute inflammatory stress. To determine which inflammatory cytokines might impact motor neuron function, seven cytokines known to be elevated in ALS patients cerebrospinal fluid were tested for their effects on PD-L1 expression in human iPSC-derived motor neurons. Among the tested cytokines, only interferon-{gamma} (IFN-{gamma}) was found to strongly promote PD-L1 expression. Thus, we hypothesized that excessive exposure to IFN-{gamma} may contribute to motor neuron degeneration in ALS. We observed that neuronal populations exposed to IFN-{gamma} exhibited severe TDP-43 cytoplasmic aggregation and excitotoxic behavior correlated with impaired neural firing activity, hallmarks of ALS pathology, in both normal and ALS mutant (TARDB1K+/-) neurons. Single-cell RNA sequencing revealed possible mechanisms for these effects. Motor neurons exposed to IFN-{gamma} exhibited an extensive shift of their gene expression profile toward a neurodegenerative phenotype. Notably, IFN-{gamma} treatment induced aberrant expression levels for 70 genes that are listed in the recent literature as being dysregulated in various ALS subtypes. Additionally, we found that genes related to neuronal electrophysiology, protein aggregation, and TDP-43 misregulation were abnormally expressed in IFN-{gamma} treated cells. Moreover, IFN-{gamma} induced a significant reduction in the expression of genes that encode indispensable proteins for neuromuscular synapse development and maintenance, implying that the continuous cytokine exposure could directly impair signal transmission between motor axons and muscle membranes. Our findings suggest that IFN-{gamma} could be a potent upstream pathogenic driver of ALS and provide potential candidates for future therapeutic targets to treat sporadic forms of ALS, which account for roughly 90% of reported cases.

neuroscience↗

bioRxiv2022

Perpendicular Shear Stresses Drive Transmural Helical Remodeling in Engineered Human Ventricular Models

Tissue engineering with human induced pluripotent stem cell-derived cardiomyocytes enables unique opportunities for creating physiological models of the heart in vitro. However, there are few approaches available that can recapitulate the complex structure-function relationships that govern cardiac function at the macroscopic organ level. Here, we report a down-scaled, conical human 3D ventricular model with controllable cellular organization using multilayered, patterned cardiac sheets. Tissue engineered ventricles whose cardiomyocytes were pre-aligned parallel or perpendicular to the long axis outperformed those whose cardiomyocytes were angled or randomly oriented. Notably, the inner layers of perpendicular cardiac sheets realigned over 4 days into a parallel orientation, creating a helical transmural architecture, whereas minimal remodeling occurred in the parallel or angled sheets. Finite element analysis of engineered ventricles demonstrated that circumferential alignment leads to maximal perpendicular shear stress at the inner layer, whereas longitudinal orientation leads to maximal parallel stress. We hypothesize that cellular remodeling occurs to reduce perpendicular shear stresses in myocardium. This advanced platform provides evidence that physical forces such as shear stress drive self-organization of cardiac architecture.

bioengineering↗

bioRxiv2022

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

bioengineering↗

bioRxiv2018

AAV-mediated gene transfer restores a normal muscle transcriptome in a canine model of X-linked myotubular myopathy

Multiple clinical trials employing recombinant adeno-associated viral (rAAV) vectors have been initiated for neuromuscular disorders, including Duchenne and limb-girdle muscular dystrophies, spinal muscular atrophy, and recently X-linked myotubular myopathy (XLMTM). Previous work from our laboratory on a canine model of XLMTM showed that a single rAAV8-cMTM1 systemic infusion corrects structural abnormalities within the muscle and restores contractile function, with affected dogs surviving more than four years post injection. This exceptional therapeutic efficacy presents a unique opportunity to identify the downstream molecular drivers of XLMTM pathology, and to what extent the whole muscle transcriptome is restored to normal after gene transfer. Herein, RNA-sequencing was used to examine the transcriptomes of the Biceps femoris and Vastus lateralis in a previously-described canine cohort showing dose-dependent clinical improvements after rAAV8-cMTM1 gene transfer. Our analysis confirmed several dysregulated genes previously observed in XLMTM mice, but also identified new transcripts linked to XLMTM pathology. We demonstrated XLMTM transcriptome remodeling and dose-dependent normalization of gene expression after gene transfer and created new metrics to pinpoint potential biomarkers of disease progression and correction.

pathology↗