Optically diffracting hydrogels for screening kinase activity in vitro and in cell lysate: impact of material and solution properties.

Optically diffracting films based on hydrogel-encapsulated crystalline colloidal arrays have considerable utility as sensors for detecting enzymaticphosphorylation and, thus, in screening small molecule modulators of kinases. In this work, we have investigated the impact of hydrogel properties, as well as the role of the ionic character of the surrounding environment, on the optical sensitivity of kinase responsive crystalline colloidal array-containing hydrogels. In agreement with a model of hydrogel swelling, the optical sensitivity of such materials increased as the shear modulus and the Flory-Huggins interaction parameter between polymer and solvent decreased. Additionally, elimination of extraneous charges in the polymer backbone by exploiting azide-alkyne click chemistry to functionalize the hydrogels with a peptide substrate for protein kinase A further enhanced the sensitivity of the optically diffracting films. Increasing peptide concentration and, in turn, immobilized charge within the hydrogel network was shown to increase the optical response over a range of ionic strength conditions. Ultimately, we showed that, by tuning the hydrogel and solution properties, as little as 0.1 U/µL protein kinase A could be detected in short reaction times (i.e., 2 h), which is comparable to conventional biochemical kinase assays. We further showed that this approach can be used to detect protein kinase A activity in lysate from HEK293 cells. The sensitivity of the resulting films, coupled with the advantages of photonic crystal based sensors (e.g., label free detection), makes this approach highly attractive for screening enzymatic phosphorylation.

Label-free detection of missense mutations and methylation differences in the p53 gene using optically diffracting hydrogels.

We have developed a novel approach for DNA detection as well as genetic screening of mutations by uniquely combining DNA-responsive and optically diffracting materials. This approach entails the polymerization of a photonic crystal within a hydrogel network that alters the diffraction of light in response to a target DNA strand. The utility of this approach, which permits label-free sensing, was demonstrated via the detection of a target sequence from the DNA binding domain of the major tumor suppressor protein p53. Using a complementary capture probe strand, we were able to detect down to picomole concentrations of the target p53 sequence. Moreover, we demonstrated that this approach could readily detect a single base pair mutation in the target strand, which corresponds to the hotspot cancer mutation R175H in p53. The sensitivity of detection was increased by lowering the rate of annealing of the target strand and adjusting the solution ionic strength during optical characterization. Changes in ionic strength during characterization impact the melting temperature of the bound target DNA and the Donnan potential between the hydrogel and solution, which influence detection. We further showed that this approach is sensitive to epigenetic changes via the detection of a fully methylated form of the target p53 sequence. Ultimately, this approach represents a new paradigm for DNA detection and specifically genetic screening of p53 as well as other disease markers and nucleotide modifications that alter the properties of DNA (e.g., epigenetic alterations and adducts with chemical carcinogens).

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

Publisher Correction: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Full Factorial Microfluidic Designs and Devices for Parallelizing Human Pluripotent Stem Cell Differentiation.

Human pluripotent stem cells (hPSCs) are promising therapeutic tools for regenerative therapies and disease modeling. Differentiation of cultured hPSCs is influenced by both exogenous factors added to the cultures and endogenously secreted molecules. Optimization of protocols for the differentiation of hPSCs into different cell types is difficult because of the many variables that can influence cell fate. We present microfluidic devices designed to perform three- and four-factor, two-level full factorial experiments in parallel for investigating and directly optimizing hPSC differentiation. These devices feature diffusion-isolated, independent culture wells that allow for control of both exogenous and endogenous cellular signals and that allow for immunocytochemistry (ICC) and confocal microscopy in situ. These devices are fabricated by soft lithography in conjunction with 3D-printed molds and are operable with a single syringe pump, eliminating the need for specialized equipment or cleanroom facilities. Their utility was demonstrated by on-chip differentiation of hPSCs into the auditory neuron lineage. More broadly, these devices enable multiplexing for experimentation with any adherent cell type or even multiple cell types, allowing efficient investigation of the effects of medium conditions, pharmaceuticals, or other soluble reagents.

Developmental profiling of microRNAs in the human embryonic inner ear.

Due to the extreme inaccessibility of fetal human inner ear tissue, defining of the microRNAs (miRNAs) that regulate development of the inner ear has relied on animal tissue. In the present study, we performed the first miRNA sequencing of otic precursors in human specimens. Using HTG miRNA Whole Transcriptome assays, we examined miRNA expression in the cochleovestibular ganglion (CVG), neural crest (NC), and otic vesicle (OV) from paraffin embedded (FFPE) human specimens in the Carnegie developmental stages 13-15. We found that in human embryonic tissues, there are different patterns of miRNA expression in the CVG, NC and OV. In particular, members of the miR-183 family (miR-96, miR-182, and miR-183) are differentially expressed in the CVG compared to NC and OV at Carnegie developmental stage 13. We further identified transcription factors that are differentially targeted in the CVG compared to the other tissues from stages 13-15, and we performed gene set enrichment analyses to determine differentially regulated pathways that are relevant to CVG development in humans. These findings not only provide insight into the mechanisms governing the development of the human inner ear, but also identify potential signaling pathways for promoting regeneration of the spiral ganglion and other components of the inner ear.

Directed Differentiation of Human Embryonic Stem Cells Toward Placode-Derived Spiral Ganglion-Like Sensory Neurons.

The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923-936.

Creating a stem cell niche in the inner ear using self-assembling peptide amphiphiles.

The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.