技术资料

Plp1 gene expression occurs very early in development,well before the onset of myelination,creating a conundrum with regard to the function of myelin proteolipid protein (PLP),one of the major proteins in compact myelin. Using PLP-EGFP mice to investigate Plp1 promoter activity,we found that,at very early time points,PLP-EGFP was expressed in Sox2+ undifferentiated precursors in the spinal cord ventricular zone (VZ),as well as in the progenitors of both neuronal and glial lineages. As development progressed,most PLP-EGFP-expressing cells gave rise to oligodendrocyte progenitor cells (OPCs). The expression of PLP-EGFP in the spinal cord was quite dynamic during development. PLP-EGFP was highly expressed as cells delaminated from the VZ. Expression was downregulated as cells moved laterally through the cord,and then robustly upregulated as OPCs differentiated into mature myelinating oligodendrocytes. The presence of PLP-EGFP expression in OPCs raises the question of its role in this migratory population. We crossed PLP-EGFP reporter mice into a Plp1-null background to investigate the role of PLP in early OPC development. In the absence of PLP,normal numbers of OPCs were generated and their distribution throughout the spinal cord was unaffected. However,the orientation and length of OPC processes during migration was abnormal in Plp1-null mice,suggesting that PLP plays a role either in the structural integrity of OPC processes or in their response to extracellular cues that orient process outgrowth. View Publication

MED13L is a component subunit of the Mediator complex,an important regulator of transcription that is highly conserved across eukaryotes. Here we report MED13L disruption in a translocation t(12;19) breakpoint of a patient with Pierre-Robin syndrome,moderate intellectual disability (ID),craniofacial anomalies,and muscular defects. The phenotype is similar to previously described patients with MED13L haploinsufficiency. Knockdown of MED13L orthologue in zebrafish,med13b,showed early defective migration of cranial neural crest cells (NCCs) that contributed into cartilage structure deformities in the later stage,recapitulating craniofacial anomalies seen in human patients. Notably,we observed abnormal distribution of developing neurons in different brain regions of med13b morphant embryos,which could be rescued upon introduction of full-length human MED13L mRNA. To compare with mammalian system,we suppressed MED13L expression by short-hairpin RNA in ES-derived human neural progenitors,and differentiated them into neurons. Transcriptome analysis revealed differential expression of components of Wnt and FGF signalling pathways in MED13L-deficient neurons. Our finding provides a novel insight into the mechanism of overlapping phenotypic outcome targeting NCCs derivatives organs in patients with MED13L haploinsufficiency,and emphasizes a clinically recognizable syndromic phenotype in these patients. This article is protected by copyright. All rights reserved. View Publication

Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics,but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge,we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs),pericytes,astrocytes and neurons derived from renewable cell sources. First,retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs,particularly through adherens junction,tight junction,and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (˜5,000 $\$(2)). Overall,this scalable human BBB model may enable a wide range of neuroscience studies. 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

The quantification of cellular mechanical properties is of tremendous interest in biology and medicine. Recent microfluidic technologies that infer cellular mechanical properties based on analysis of cellular deformations during microchannel traversal have dramatically improved throughput over traditional single-cell rheological tools,yet the extraction of material parameters from these measurements remains quite complex due to challenges such as confinement by channel walls and the domination of complex inertial forces. Here,we describe a simple microfluidic platform that uses hydrodynamic forces at low Reynolds number and low confinement to elongate single cells near the stagnation point of a planar extensional flow. In tandem,we present,to our knowledge,a novel analytical framework that enables determination of cellular viscoelastic properties (stiffness and fluidity) from these measurements. We validated our system and analysis by measuring the stiffness of cross-linked dextran microparticles,which yielded reasonable agreement with previously reported values and our micropipette aspiration measurements. We then measured viscoelastic properties of 3T3 fibroblasts and glioblastoma tumor initiating cells. Our system captures the expected changes in elastic modulus induced in 3T3 fibroblasts and tumor initiating cells in response to agents that soften (cytochalasin D) or stiffen (paraformaldehyde) the cytoskeleton. The simplicity of the device coupled with our analytical model allows straightforward measurement of the viscoelastic properties of cells and soft,spherical objects. View Publication

Studies have shown that neural stem/progenitor cell (NPC) transplantation is beneficial in experimental autoimmune encephalomyelitis (EAE),an established animal model of multiple sclerosis (MS). It is unclear whether NPCs have the ability to integrate into the host CNS to replace lost cells or if their main mechanism of action is via bystander immunomodulation. Understanding the mechanisms by which NPCs exert their beneficial effects as well as exploring methods to increase post-transplantation survival and differentiation is critical to advancing this treatment strategy. Using the EAE model and Fas-deficient (lpr) NPCs,we investigated the effects of altering the Fas system in NPC transplantation therapy. We show that transplantation of NPCs into EAE mice ameliorates clinical symptoms with greater efficacy than sham treatments regardless of cell type (wt or lpr). NPC transplantation via retro-orbital injections significantly decreased inflammatory infiltrates at the acute time point,with a similar trend at the chronic time point. Both wt and lpr NPCs injected into mice with EAE were able to home to sites of CNS inflammation in the periventricular brain and lumbar spinal cord. Both wt and lpr NPCs have the same capacity for inducing apoptosis of Th1 and Th17 cells,and minimal numbers of NPCs entered the CNS. These cells did not express terminal differentiation markers,suggesting that NPCs exert their effects mainly via bystander peripheral immunomodulation. View Publication

Differentiation of pluripotent and lineage restricted stem cells such as neural stem cells (NSCs) was studied on conducting substrates of various nature without perturbation of the genome with exogenous genetic material or chemical stimuli. Primary mouse adult neural stem cells (NSCs) and P19 pluripotent embryonal (P19 EC) carcinoma cells were used. Expression levels of neuronal markers β-III-tubulin and neurofilament were evaluated by immunochemistry and flow cytometry. It was shown that the ability of the substrate to induce differentiation directly correlated with its conductivity. Conducting substrates (conducting oxides or doped pi-conjugated organic polymers) with different morphology,structure,and conductivity mechanisms all promoted differentiation of NSC and P19 cells into neuronal lineage to a similar degree without use of additional factors such as poly-L-ornithine coating or retinoic acid,as verified by their morphology and upregulation of the neuronal markers but not astrocyte marker GFAP. However,substrates with low conductance below ca. 10(-4) S cm(-2) did not show this ability. Morphology of differentiating cells was visualized by atomic force microscopy. NSCs cells increased β-III-tubulin expression by 95% and P19 cells by over 30%. Our results suggest that the substrate conductivity is a key factor governing the cell fate. Differentiation of P19 cells into neuronal lineage on conducting substrates was attributed to downregualtion of Akt signaling pathway and increase in expression of dual oxidase 1 (DUOX 1). View Publication