No abstract

Voltage imaging with fluorescent dyes offers promise for interrogating the complex roles of membrane potential in coordinating the activity of neurons in the brain. Yet, low sensitivity often limits the broad applicability of optical voltage indicators. In this paper, we use molecular dynamics (MD) simulations to guide the design of new, ultra-sensitive fluorescent voltage indicators that use photoinduced electron transfer (PeT) as a voltage-sensing switch. MD simulations predict an approximately 16% increase in voltage sensitivity resulting purely from improved alignment of dye with the membrane. We confirm this theoretical finding by synthesizing 9 new voltage-sensitive (VoltageFluor, or VF) dyes and establishing that all of them display the expected improvement of approximately 19%. This synergistic outworking of theory and experiment enabled computational and theoretical estimation of VF dye orientation in lipid bilayers and has yielded the most sensitive PeT-based VF dye to date. We use this new voltage indicator to monitor voltage spikes in neurons from rat hippocampus and human pluripotent stem cell-derived dopaminergic neurons.

Human pluripotent stem cells (hPSCs) offer considerable potential for biomedical applications including drug screening and cell replacement therapies. Clinical translation of hPSCs requires large quantities of high quality cells, so scalable methods for cell culture are needed. However, current methods are limited by scalability, the use of animal-derived components, and/or low expansion rates. A thermoresponsive 3D hydrogel for scalable hPSC expansion and differentiation into several defined lineages is recently reported. This system would benefit from increased control over material properties to further tune hPSC behavior, and here a scalable 3D biomaterial with the capacity to tune both the chemical and the mechanical properties is demonstrated to promote hPSC expansion under defined conditions. This 3D biomaterial, comprised of hyaluronic acid and poly(N-isopropolyacrylamide), has thermoresponsive properties that readily enable mixing with cells at low temperatures, physical encapsulation within the hydrogel upon elevation at 37 °C, and cell recovery upon cooling and reliquefaction. After optimization, the resulting biomaterial supports hPSC expansion over long cell culture periods while maintaining cell pluripotency. The capacity to modulate the mechanical and chemical properties of the hydrogel provides a new avenue to expand hPSCs for future therapeutic application.

Huntington’s disease (HD) is a currently incurable and, ultimately, fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion within exon 1 of the huntingtin (HTT) gene, which results in the production of a mutant protein that forms inclusions and selectively destroys neurons in the striatum and other adjacent structures. The RNA-guided Cas9 endonuclease from CRISPR-Cas9 systems is a versatile technology for inducing DNA double-strand breaks that can stimulate the introduction of frameshift-inducing mutations and permanently disable mutant gene function. Here, we show that the Cas9 nuclease from Staphylococcus aureus , a small Cas9 ortholog that can be packaged alongside a single guide RNA into a single adeno-associated virus (AAV) vector, can be used to disrupt the expression of the mutant HTT gene in the R6/2 mouse model of HD following its in vivo delivery to the striatum. Specifically, we found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by a trinucleotide repeat expansion via in vivo genome editing.

SummaryHuntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy.