Biology subjects

Isom, L. L.

Publications and source records attributed to Isom, L. L..

5 recordsLinked to original sources

bioRxiv2025

Cryopreservation of Human Cortical Organoids Using Vitrification.

Cryopreservation at ultra-low temperatures is a valuable tool for preserving cells and tissues used in research. However, few protocols exist for the preservation of brain organoid models. Current methods for preserving human cortical organoids (hCOs) rely on conventional slow cooling approaches with organoids suspended in a medium containing a cocktail of cryoprotectants. In contrast, we have optimized a vitrification technique previously used to cryopreserve human embryos and oocytes for application to hCOs. We have successfully cryopreserved hCOs that were generated by two different protocols. The vitrified organoids demonstrate a growth rate, cytoarchitecture, cell type composition and electrical activity comparable to non-vitrified controls. Our hCO cryopreservation method provides a useful alternative approach for bio-banking and cross-institutional collaboration using cortical organoids as their model system. Highlights- Human pluripotent stem cell (hPSC) derived hCOs can be cryopreserved using vitrification. - Vitrified hCOs develop similarly to non-vitrified controls. - Vitrified hCOs can be stored long-term and shipped to other institutions. MotivationMany methods of cryopreservation have been developed for the maintenance of cell lines, organoid models and tissues. Most organoid models have been successfully preserved using conventional slow-cooling methods; however, human cortical organoids have been difficult to preserve. Our findings demonstrate that the vitrification of cortical organoids preserves their structure, cell type diversity and function upon rewarming and continued culture.

neuroscience↗

bioRxiv2021

Subcellular dynamics and functional activity of the cleaved Na+ channel β1 subunit intracellular domain

The voltage-gated Na+ channel {beta}1 subunit, encoded by SCN1B, regulates cell surface expression and gating of subunits, and participates in cell adhesion. {beta}1 is cleaved by /{beta} and {gamma}-secretases, releasing an extracellular domain and intracellular domain (ICD) respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on {beta}1 function in breast cancer cells. Using a series of GFP-tagged {beta}1 constructs, we show that {beta}1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal {beta}1-GFP levels occurred following {gamma}-secretase inhibition, implicating endosomes, and/or the preceding plasma membrane, as important sites for secretase processing. Using live-cell imaging, we report {beta}1ICD-GFP accumulation in the nucleus. Furthermore, {beta}1-GFP and {beta}1ICD-GFP both increased Na+ current, whereas {beta}1STOP-GFP, which lacks the ICD, did not, thus highlighting that the {beta}1-ICD was necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is TTX-resistant (carried by Nav1.5), the Na+ current increased by {beta}1-GFP or {beta}1ICD-GFP was TTX-sensitive. In addition, {beta}1-GFP increased mRNA levels of the TTX-sensitive subunits SCN1A/Nav1.1 and SCN9A/Nav1.7. Taken together, this work suggests that the {beta}1-ICD is a critical regulator of subunit function in cancer cells. Our data further highlight that {gamma}-secretase may play a key role in regulating {beta}1 function in breast cancer.

cell biology↗

bioRxiv2021

Biomechanics and Myofibrillar Alignment Enhance Contractile Development and Reproducibility in Stem Cell Derived Cardiac Muscle

Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) allow novel investigations of human cardiac disease, but disorganized mechanics and immaturity of hPSC-CMs on two-dimensional (2D) surfaces have been hurdles for efficient and reproducible study of these cells. Here, we developed a platform of micron-scale 2D cardiac tissues (M2DCTs) to precisely control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips in 2D. By defining geometry and workload in M2DCTs in this reductionist platform that does not incorporate other cell types, we show that myofibrillar alignment and auxotonic contractions at physiologic workload critically drive maturation of cardiac contractile function, calcium handling, and electrophysiology. Additionally, the organized biomechanics in this system facilitates rapid and automated extraction of contractile kinetic parameters from brightfield microscopy images, increasing the reproducibility and throughput of pharmacologic testing. Finally, we show that M2DCTs enable precise and efficient dissection of contractile kinetics in cardiomyopathy disease models.

cell biology↗

bioRxiv2020

Dscam gene triplication causes neocortical overinhibition in Down syndrome

A growing number of molecules have been identified as regulators of inhibitory synapse development, but whether dysregulated expression of these molecules contribute to brain disorders is poorly understood. Here we show that Down syndrome cell adhesion molecule (Dscam) regulates the inhibition of neocortical pyramidal neurons (PyNs) in a level-dependent fashion. Loss of Dscam impairs inhibitory neuron development and function. In the Ts65Dn mouse model for Down syndrome, where Dscam is overexpressed, GABAergic innervation of cortical PyNs by chandelier and basket cells is increased. Genetic normalization of Dscam expression rescues the excessive GABAergic innervation and the increased inhibition of PyNs. These findings demonstrate excessive GABAergic innervation and inhibition in the neocortex of Down syndrome mouse model and identify Dscam overexpression as the cause. They also implicate dysregulated Dscam levels as a potential pathogenic driver in related neurological disorders.

neuroscience↗

bioRxiv2018

Ankyrin-G Regulates Forebrain Connectivity and Network Synchronization via Interaction with GABARAP

GABAergic circuits are critical for the synchronization and higher order function of brain networks, and defects in this circuitry are linked to neuropsychiatric diseases, including bipolar disorder, schizophrenia, and autism. Work in cultured neurons has shown that ankyrin-G plays a key role in the regulation of GABAergic synapses on the axon initial segment and somatodendritic domain of pyramidal neurons where it interacts directly with the GABAO_SCPCAPAC_SCPCAP receptor associated protein (GABARAP) to stabilize cell surface GABAO_SCPCAPAC_SCPCAP receptors. Here, we generated a knock-in mouse model expressing a mutation that abolishes the ankyrin-G/GABARAP interaction (Ank3 W1989R) to understand how ankyrin-G and GABARAP regulate GABAergic circuitry in vivo. We found that Ank3 W1989R mice exhibit a striking reduction in forebrain GABAergic synapses resulting in pyramidal cell hyperexcitability and disruptions in network synchronization. In addition, we identified changes in pyramidal cell dendritic spines and axon initial segments consistent with compensation for hyperexcitability. Finally, we identified the ANK3 W1989R variant in a family with bipolar disorder, suggesting a potential role of this variant in disease. Our results highlight the importance of ankyrin-G in regulating forebrain circuitry and provide novel insights into how ANK3 loss-of-function variants may contribute to human disease.

neuroscience↗