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Genetic variants in the X‐chromosomal gene coding for the calcium−/calmodulin‐dependent serine protein kinase (CASK) are associated with a neurodevelopmental disorder. CASK is a member of the membrane‐associated guanylate kinase (MAGUK) family of proteins. It acts as a scaffold at presynaptic sites,as a regulator of the transport of glutamate receptors,and as a transcriptional regulator. The PDZ domain of CASK has been reported to bind to presynaptic cell adhesion molecules such as Neurexin1‐3,CNTNAP2,SynCAM and SALM1. Structural analyses of related MAGUKs indicate that the canonical SH3 and GK domains combine with the PDZ domain to form the so‐called PSG supramodule. Conserved aromatic residues (Y723 and W914) flanking the GK domain contribute to the formation of a dimeric structure of two PSG modules,which is required for high‐affinity binding to the type 2 PDZ ligand motif of,for example,Neurexin. Here we identify previously uncharacterized patient variants in the SH3 domain of CASK (I672V; P673L),which alter the intermolecular binding pocket for Y723. Both variants interfere with the binding of Neurexin‐1β,in a manner similar to the previously reported Y723C variant. Intriguingly,binding to the type 1 PDZ ligand of the cell adhesion molecule SALM1 is not altered. Using a set of highly selective patient variants,we show that the binding of SALM1 to CASK is actually not mediated by the CASK PDZ domain or the PSG supramodule,but depends on other type 1 PDZ domain‐containing proteins such as SAP97 and Veli,which associate with CASK through its L27 domains. Our data underline the relevance of an intact PSG tandem of CASK for human health. View Publication

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens,or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here,to yield functional human haematopoietic stem cells,we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG,HOXA5,HOXA9,HOXA10,LCOR,RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid,B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. View Publication

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. They can maintain an undifferentiated state indefinitely and can differentiate into derivatives of all three germ layers,namely ectoderm,endoderm and mesoderm. Although much progress has been made in the propagation and differentiation of ES cells,induction of photoreceptors has generally required coculture with or transplantation into developing retinal tissue. Here,we describe a protocol for generating retinal cells from ES cells by stepwise treatment with defined factors. This method preferentially induces photoreceptor and retinal pigment epithelium (RPE) cells from mouse and human ES cells. In our protocol,differentiation of RPE and photoreceptors from mouse ES cells requires 28 d and the differentiation of human ES cells into mature RPE and photoreceptors requires 120 and 150 d,respectively. This differentiation system and the resulting pluripotent stem cell-derived retinal cells will facilitate the development of transplantation therapies for retinal diseases,drug testing and in vitro disease modeling. It will also improve our understanding of the development of the central nervous system,especially the eye. View Publication

Retroviral vectors with long terminal repeats (LTRs),which contain strong enhancer/promoter sequences at both ends of their genome,are widely used for stable gene transfer into hematopoietic cells. However,recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter,theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution,we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones,including those obtained after dose escalation of SIN vectors,showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number,we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis. 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

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening,potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics. View Publication

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of NAD(P)+-dependent enzymes,which catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding acids. Increased expression and/or activity of ALDHs,particularly ALDH1A1,have been reported to occur in human cancers. It is proposed that the metabolic function of ALDH1A1 confers the stemness" properties to normal and cancer stem cells. Nevertheless� View Publication

Disentangling the complex interactions that govern stem cell fate choices of self-renewal,differentiation,or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that improved cell survival or induced differentiation. The survival compounds Y-27632,HA-1077,and H-8 all strongly inhibit the kinases ROCK and PRK2,highlighting the important role of these kinases in EC cell survival. Two molecules,GF109203x and rottlerin,induced EC differentiation. The effects of rottlerin were also investigated in human ES cells. Rottlerin inhibited the self-renewal ability of ES cells,caused the cell cycle arrest,and repressed the expression of pluripotency-associated genes. View Publication

Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism,digestion,satiety and lipid absorption,yet their development remains poorly understood. Here we show that Arx,a homeodomain-containing transcription factor,is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice,removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-,glucagon/GLP-1-,CCK-,secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition,depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK,secretin and glucagon while expression of a pan-intestinal epithelial marker,CDX2,and other non-endocrine markers remained unchanged. Taken together,our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations. View Publication