技术资料

Spinal muscular atrophy (SMA),an autosomal recessive neuromuscular disease,is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product,survival of motor neuron (SMN),is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention,particularly of minor U12 introns,in the spinal cord of mice 30 d after SMA induction,which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response,manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns,high in GC content,served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures,leading to motor neuron death. View Publication

Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis,we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably,we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media,up to 80% of cells expressed the neural stem cell marker nestin,and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation,we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together,our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein. View Publication

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods,it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous,nonsense or splice variants,and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies,whereas the rest occurred during or after reprogramming. Thus,hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use. View Publication

High purity chromosome sorting can be performed on instruments such as MoFlo MLS and BD influx,which are stream-in-air sorters equipped with water-cooled high power lasers. The FACSAria is a true fixed alignment,low laser powered instrument with a quartz flow cell gel-coupled to the collection optics. However,whether high purity mouse and human chromosomes can be obtained by sorting on the BD FACSAria(TM) Special Order Research Product (FACSAria SORP) remains to be determined. Here,we report that the high resolution flow karyotype of mouse lymphocytes and normal male human peripheral blood mononuclear cells (hPBMCs) can be obtained on the FACSAria SORP using laser power settings of 50 mW for 355 nm and 20 mW for 444 nm excitation. Furthermore,the use of Fluorescence in situ hybridization (FISH) confirmed that chromosome paints prepared from the sorted chromosomes demonstrated high purity and signal specificity. Notably,human chromosome 12 was separated from the chromosome 9-12 cluster in the flow karyotype,and its identity was confirmed using FISH in trisomy 12 human ES cell lines B2-C7 and B2-B8. In addition,multicolor FISH (mFISH) with human chromosome painting probes to 13,18,21,and sex chromosomes X and Y showed high signal specificity in hPBMCs. Taken together,our findings demonstrated that high resolution flow karyotype can be obtained using FACSAria SORP. Moreover,a FISH analysis confirmed high purity of the sorted chromosomes. Additionally,in contrast to centromeric satellite probes,chromosome painting probes with high specificity are more suitable for detection of chromosome aberrations,such as deletions and translocations,in prenatal diagnosis. textcopyright 2016 International Society for Advancement of Cytometry. View Publication

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs,but Sox4,Sox5,Sox6,Sox8,Sox9,Sox11,Sox12,Sox13,and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover,the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming,albeit at low efficiency. By molecular dissection of the C-terminus of Sox17,we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK,we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2. View Publication

In this study,we sought to establish a novel method to prospectively and dynamically identify live human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells from brain dissociates and pluripotent stem cell culture. We selected a highly conserved enhancer element of the Sox10 gene,known as MCS5,which directs reporter expression to oligodendrocyte lineage cells in mouse and zebrafish. We demonstrate that lentiviral Sox10-MCS5 induced expression of GFP at high levels in a subpopulation of human CD140a/PDGF??R-sorted OPCs as well as their immature oligodendrocyte progeny. Furthermore,we show that almost all Sox10-MCS5:GFPhigh cells expressed OPC antigen CD140a and human OPCs expressing SOX10,OLIG2,and PDGFRA mRNAs could be prospectively identified using GFP based fluorescence activated cells sorting alone. Additionally,we established a human induced pluripotent cell (iPSC) line transduced with the Sox10-MCS5:GFP reporter using a Rex-Neo cassette. Similar to human primary cells,GFP expression was restricted to embryoid bodies containing both oligodendrocyte progenitor and oligodendrocyte cells and co-localized with NG2 and O4-positive cells respectively. As such,we have developed a novel reporter system that can track oligodendrocyte commitment in human cells,establishing a valuable tool to improve our understanding and efficiency of human oligodendrocyte derivation. ?? 2013 Elsevier Inc. View Publication

The neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems,that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFβ inhibitor SB431542 and GSK3 inhibitor CHIR99021. We found that all SOX10-NL-positive cells expressed an early neural crest marker NGFR,however SOX10-NL-positive cells purified from differentiated hiPS cells progressively attenuate their NL-expression under proliferation. We therefore attempted to maintain SOX10-NL-positive cells with additional signaling on the plane and sphere culture conditions. These SOX10-NL cells provide us to investigate mass culture with neural crest cells for stem cell research. View Publication

Despite advances in detection and therapy,castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways,and their regulation by the Androgen Receptor (AR),has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced,castrate-resistant prostate cancers. To this end,we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate,Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative,with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1,endogenous expression of Sox2 was repressed by AR signaling,and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise,in normal prostate epithelial cells and human embryonic stem cells,increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines,and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration,and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways. View Publication

Transcription factors NANOG,OCT4,and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however,their expression profiles during early differentiation of hES cells are unclear. In this study,we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG,OCT4,and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells,characterized by specific expression patterns of NANOG,OCT4,and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time,indicating a gradual mode of developmental transition in these cells. Notably,distinct regulation of SOX2 during early differentiation events was detected,highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation. View Publication

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work,we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation,after four days of culture,3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm,which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up. View Publication

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here,we show dynamic recruitment of GATA-1,TFIIB,Mediator,and RNA polymerase II to the β-globin locus in induced mouse erythroid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice,we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development,highlighted by the complete absence of β-globin gene expression. Thus,Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation. View Publication

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease primarily characterized by degeneration of spinal motor neurons,and caused by reduced levels of the SMN protein. Previous studies to understand the proteomic consequences of reduced SMN have mostly utilized patient fibroblasts and animal models. We have derived human motor neurons from type I SMA and healthy controls by creating their induced pluripotent stem cells (iPSCs). Quantitative mass spectrometry of these cells revealed increased expression of 63 proteins in control motor neurons compared to respective fibroblasts,whereas 30 proteins were increased in SMA motor neurons vs. their fibroblasts. Notably,UBA1 was significantly decreased in SMA motor neurons,supporting evidence for ubiquitin pathway defects. Subcellular distribution of UBA1 was predominantly cytoplasmic in SMA motor neurons in contrast to nuclear in control motor neurons; suggestive of neurodevelopmental abnormalities. Many of the proteins that were decreased in SMA motor neurons,including beta III-tubulin and UCHL1,were associated with neurodevelopment and differentiation. These neuron-specific consequences of SMN depletion were not evident in fibroblasts,highlighting the importance of iPSC technology. The proteomic profiles identified here provide a useful resource to explore the molecular consequences of reduced SMN in motor neurons,and for the identification of novel biomarker and therapeutic targets for SMA. View Publication