Biology subjects

Stone, M. L.

Publications and source records attributed to Stone, M. L..

4 recordsLinked to original sources

bioRxiv2026

Electrical stimulation combined with p27Kip1 inactivation drives proliferative neurogenic reprogramming of Mueller glia in the adult mouse retina

Mueller glial reprogramming studies demonstrate that mammalian Mueller glia can be induced to proliferate and/or engage in neural differentiation, as occurs naturally in teleost fish. A major objective is the identification of combined strategies that promote both robust proliferation and neurogenesis. These studies would benefit from a translatable screening platform that enables controlled perturbation, maintained tissue context and longitudinal analysis, such as 3D culture for first tier analysis of reprogramming strategies. Here, we validate a 3D retinal culture for Mueller glial reprogramming studies by recapitulating key signatures of an in vivo reprogramming paradigm. Next, we find that electrical stimulation (E-stim) as a tunable, extrinsic cue is sufficient to activate endogenous Ascl1 expression, indicating a state transition favorable for neurogenesis, while Mueller glia-specific p27Kip1 inactivation promotes robust, prolonged proliferation. Utilization of the lineage-tracing proliferation-history reporter H3.1-iCOUNT enabled longitudinal proliferation analysis and assessment of reprogramming outcomes within the proliferative, Mueller-derived population. With this model, we find that E-stim and p27Kip1 inactivation in combination (ESPI) increases proliferation, endogenous Ascl1 expression, and neurogenesis of Mueller-derived cells across modalities. Together, this work establishes a 3D culture framework for discovery of combinatorial reprogramming strategies within a proliferative context and identifies ESPI as an efficient approach to proliferative, neurogenic Mueller glial reprogramming.

developmental biology↗

bioRxiv2025

Systemic administration of a reported extracellular vesicle inhibitor, dimethyl amiloride, induces preterm birth and fetal growth restriction in pregnant mice.

Successful pregnancy is dependent on extensive coordination between maternal, placental, and fetal units. Alterations in maternal-placental signaling or abnormal placental development can lead to a wide variety of pregnancy complications. This commonly includes preterm birth and fetal growth restriction, which are associated with a high rate of maternal and fetal morbidity and mortality. Maternal-placental signaling is in part mediated by the release of extracellular vesicles. These lipid-bilayer nanoparticles are secreted by various cell types and act as key regulators of both normal and pathogenic cell-cell communication. In a healthy pregnancy, maternal plasma extracellular vesicle concentrations increase as gestation progresses. However, in the pregnancy-associated disorder, preeclampsia, excessive extracellular vesicle secretion occurs and may be involved in the pathogenesis of the condition. Thus, there is a critical need - as a first step - to understand if modifying extracellular vesicle concentration, specifically during pregnancy can attenuate or exacerbate gestational pathogenesis. In this study, we evaluated the effects of administering a reported extracellular vesicle inhibitor, dimethyl amiloride, on maternal extracellular vesicle concentrations in healthy pregnant mice and assessed maternal and fetal outcomes. Maternal administration of dimethyl amiloride resulted in a significant decrease in maternal weight as well as fetal growth and substantially increased the rate of preterm birth. In contrast to previous reports in non-pregnant animals, we found that dimethyl amiloride did not significantly reduce maternal extracellular vesicle concentrations in pregnant mice. Our data demonstrate that systemic administration of dimethyl amiloride drastically impacts the mother and fetus during gestation and caution is suggested against its use during pregnancy.

developmental biology↗

bioRxiv2025

Diet gel-based oral drug delivery system for controlled dosing of small molecules for microglia depletion and inducible Cre recombination in mice

Small molecules like PLX5622 for microglia depletion and Tamoxifen for inducible Cre recombination are commonly used in mouse research. Traditional application methods, such as chow or oral gavage and injections, have limitations, including uncontrolled dosage and risk of injury. To address this issue, we have developed an alternative oral drug delivery system using a gel-based rodent maintenance diet that allows for controlled consumption and adjustment of dosage and is suitable for water-insoluble small molecules. We tested DietGel(R) 93M (93M) infused with PLX5622 (0.8 mg/g and 2.0 mg/g) in the Cx3cr1gfp/+ retinal microglia reporter mouse and Tamoxifen-infused 93M (0.3125 mg/g) in the Rlbp1-CreERT2;Rosaai14 mouse with an inducible tdTomato reporter in retinal Muller glia. Mice were single-caged and received daily batches of PLX5622-infused 93M over 14 days or Tamoxifen-infused 93M for one or three days followed by a 14-day observation period. Longitudinal scanning laser ophthalmoscopy in vivo and fixed tissue imaging were used to track GFP and tdTomato expression. Following evaluation of a suitable 93M consumption rate (g/d) to sustain body weight, the PLX5622-93M diet at both concentrations showed a 94% microglia depletion rate at 3 days and >99% after one and two weeks. The Tamoxifen-93M diet confirmed suitability for inducible Cre recombination, with significant treatment-time dependent efficacy and a positive correlation between total Tamoxifen dose and tdTomato expression. This study demonstrates that a diet gel-based drug delivery system offers a controllable and less invasive alternative to current drug application methods for PLX5622 and Tamoxifen.

cell biology↗

bioRxiv2023

An agarose disk electroporation method for ex vivo retinal tissue cultured at the air-liquid interface reveals electrical stimulus-induced cell cycle reentry in retinal cells

It is advantageous to culture the ex vivo murine retina along with many other tissue types at the air-liquid interface. However, gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery, but typically requires submergence in a liquid buffer to allow electric current flow. We have developed a submergence-free electroporation technique using that allows for efficient gene delivery to the ex vivo murine retina. This method advances our ability to use ex vivo retinal tissue for genetic studies and can easily be adapted for any tissue cultured at an air-liquid interface. Use of this method has revealed valuable insights on the state of ex vivo retinal tissues and the effects of electrical stimulation on retinal cells. MotivationTissues cultured at the air-liquid interface, such as retinal tissue, are adhered to a filter membrane with media underneath but not fully submerged. If tissues are fully submerged in liquid, they detach from the membrane and become damaged. Electroporation typically requires tissue submergence for electric current flow. We have developed a submergence-free electroporation method using an agarose disk for electric current delivery that can efficiently and consistently electroporate ex vivo retinal tissue cultured at an air-liquid interface without the need to submerge the tissue and disrupt the culture system.

cell biology↗