技术资料

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly,which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However,mechanisms ofNPCs maintenance remain unclear. Here,we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC),which includes Nde1,Aurora A,andOFD1,recruited to the ciliary base for timely cilium disassembly. In contrast,mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment,long cilia,retarded cilium disassembly,and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus,our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control. View Publication

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD,and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs,using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected,cell lines harboring the PSEN2 N141I mutation displayed an increase in the A$\beta$42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit,restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased A$\beta$42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in A$\beta$42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology. View Publication

The embryonic neuroepithelium gives rise to the entire central nervous system in vivo,making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here,we report that hPSCs maintained under chemically defined,feeder-independent,and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6(+)/N-cadherin(+) with widespread rosette formation) within 6 days under adherent conditions,without small molecule inhibitors,and using only minimalistic medium consisting of Dulbecco's modified Eagle's medium/F-12,sodium bicarbonate,selenium,ascorbic acid,transferrin,and insulin (i.e.,E6 medium). Furthermore,we provide evidence that the defined culture conditions enable this high level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts (MEFs). In addition,hPSCs previously maintained on MEFs could be rapidly converted to a neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall,this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. View Publication

Aging and oxidative stress are two prominent pathological mechanisms for Parkinson's disease (PD) that are strongly associated with the degeneration of dopamine (DA) neurons in the midbrain. DA and other catechols readily oxidize into highly reactive o-quinone species that are precursors of neuromelanin (NM) pigment and under pathological conditions can modify and damage macromolecules. The role of DA oxidation in PD pathogenesis remains unclear in part due to the lack of appropriate disease models and the absence of a simple method for the quantification of DA-derived oxidants. Here,we describe a rapid,simple,and reproducible method for the quantification of o-quinones in cells and tissues that relies on the near-infrared fluorescent properties of these species. Importantly,we demonstrate that catechol-derived oxidants can be quantified in human neuroblastoma cells and midbrain dopamine neurons derived from induced pluripotent stem cells,providing a novel model to study the downstream actions of o-quinones. This method should facilitate further study of oxidative stress and DA oxidation in PD and related diseases that affect the dopaminergic system. View Publication

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication

Somatic cell reprogramming may become a powerful approach to generate specific human cell types for cell-fate determination studies and potential transplantation therapies of neurological diseases. Here we report a reprogramming methodology with which human adipose stem cells (hADSCs) can be differentiated into neural cells. After being reprogrammed with polycistronic plasmid carrying defined factor OCT3/4,SOX2,KLF4 and c-MYC,and further treated with neural induce medium,the hADSCs switched to differentiate toward neural cell lineages. The generated cells had normal karyotypes and exogenous vector sequences were not inserted in the genomes. Therefore,this cell lineage conversion methodology bypasses the risk of mutation and gene instability,and provides a novel strategy to obtain patient-specific neural cells for basic research and therapeutic application. 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

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson's disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development,A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here,we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons,which mimics embryonic DA neuron development. In our protocol,we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method,and then convert the FP cells to A9 DA neurons,which could be maintained in vitro for several months. This efficient,repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients,in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD. View Publication

Heterozygous loss-of-function mutations in GRIN2B,a subunit of the NMDA receptor,cause intellectual disability and language impairment. We developed clonal models of GRIN2B deletion and loss-of-function mutations in a region coding for the glutamate binding domain in human cells and generated neurons from a patient harboring a missense mutation in the same domain. Transcriptome analysis revealed extensive increases in genes associated with cell proliferation and decreases in genes associated with neuron differentiation,a result supported by extensive protein analyses. Using electrophysiology and calcium imaging,we demonstrate that NMDA receptors are present on neural progenitor cells and that human mutations in GRIN2B can impair calcium influx and membrane depolarization even in a presumed undifferentiated cell state,highlighting an important role for non-synaptic NMDA receptors. It may be this function,in part,which underlies the neurological disease observed in patients with GRIN2B mutations. View Publication

Incurable neurological disorders such as Parkinson's disease (PD),Huntington's disease (HD),and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases,we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor,valproic acid (VPA),and inhibitor of p160-Rho associated coiled-coil kinase (ROCK),Y-27632,after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology,cell surface antigens,pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542,inhibitors of the SMAD signaling pathway,HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction,neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture,which had the ability to differentiate further into NPCs and neurons,as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays. View Publication