技术资料

Urine resource cells were collected from a 59-year-old female patient with multiple endocrine neoplasia type 1 syndrome (MEN1) for generating iPS cells with episomal plasmids carrying Oct4,Sox2,Klf4 and miR-302-367. The patient sustained a heterozygous GtextgreaterT transition mutation on the exon 9 of Men1 gene that was confirmed by sequencing analysis on the obtained iPSC lines. Karyotyping indicated the chromosomes with normal appearances and numbers. Their pluripotency was demonstrated by gene expression,as well as their abilities for differentiating into three germ layers. This cell line provides an ideal model for studying MEN1. View Publication

Peripheral blood was collected from a clinically diagnosed 72-year old male patient with later onset Alzheimer's disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for studying the pathological mechanism of Alzheimer's disease. View Publication

Peripheral blood was collected from a clinically diagnosed 64-year old male multiple schwannoma patient. Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the Yamanaka KMOS reprogramming factors using the Sendai-virus reprogramming system. The transgene-free iPSC line showed pluripotency verified by immunofluorescent staining for pluripotency markers,and the iPSC line was able to differentiate into the 3 germ layers in vivo. The iPSC line also showed normal karyotype. This in vitro cellular model will be useful for further pathological studies of multiple schwannoma. View Publication

Rett syndrome (RTT) is an X-linked,neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene,which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated,its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis,we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs,we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel,we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation,respectively,and ameliorated the observed alterations in neuronal differentiation. Moreover,overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together,our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.Molecular Psychiatry advance online publication,25 April 2017; doi:10.1038/mp.2017.86. View Publication

During pancreatic development,proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3),exit the cell cycle,and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183,which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle,NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G1/S cell-cycle checkpoint. Using models of mouse and human pancreas development,we show that lengthening of the G1 phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum,these studies demonstrate that progenitor cell-cycle G1 lengthening,through its actions on stabilization of NEUROG3,is an essential variable in normal endocrine cell genesis. View Publication

It has been postulated that during human fetal development,all cells of the lung epithelium derive from embryonic,endodermal,NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However,this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity,these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support,this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively,when recombined with fetal mouse lung mesenchyme,the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved,stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted,patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine. View Publication

Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia,spastic paraparesis,extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene,patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation,as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis,increased phospho-Tau,altered microtubule-associated transport and cell death. However,they failed to generate proteinase K-resistant prion. In this study we set out to test,for the first time,whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e,tauopathy) identified in the GSS patient. View Publication

Human pluripotent stem cells (HPSCs) cultured in conditions that maintain pluripotency via FGF and TGFβ signaling have been described as being in a primed state. These cells have been shown to exhibit characteristics more closely related to mouse epiblast-derived stem cells than to so called naïve mouse PSCs said to possess a more ground state pluripotency that mimics the early mouse embryo inner cell mass. Initial attempts to create culture conditions favorable for generation of naïve HPSCs from primed HPSCs has required the use of mouse embryonic fibroblasts as a feeder layer to support this transition. A protocol for the routine derivation and maintenance of naïve HPSCs in completely defined conditions is highly desirable for stem cell researchers to enhance the study and clinical translation of naïve HPSCs. Here we describe a standard protocol for transitioning primed HPSCs to a naïve state using commercial RSet media and xeno-free recombinant vitronectin. View Publication

Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist,intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression,IFN-λ responsiveness is restricted mainly to epithelial cells. Here,we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially,we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently,HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second,in contrast to the type I IFN system,polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally,we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity. View Publication

The human iPSC cell line,RP2-FiPS4F1 (RCPFi001-A),derived from dermal fibroblasts from the patient with retinitis pigmentosa caused by the mutation of the gene PRPF8,was generated by non-integrative reprogramming technology using OCT3/4,SOX2,CMYC and KLF4 reprogramming factors. View Publication

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S),production of granulocyte precursor cells (CFU-C),and extensive granulopoiesis can be maintained in vetro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells,epithelial" cells� View Publication

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation,and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently,Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively,and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein,and by using oligonucleotides as probes,have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore,Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells. View Publication