Scientific Resources

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

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. textcopyright Stem Cells Translational Medicine 2017;6:923-936. View Publication

The essential functions of Polycomb Repressive Complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs),but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs which co-precipitate with PRC1 from chromatin and found candidates that impact Polycomb Group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen,CAT7,regulates expression and PcG binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain which shares high sequence similarity to a non-syntenic zebrafish analog,cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA,enhanced by interference of a PRC1 component,and suppressed by interference of a known PRC1 target gene,demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs,and that CAT7/cat7l represent convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci. View Publication

Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson's disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson's Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression,especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies,including in vitro modeling of PD. However,further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs. View Publication