技术资料

Polycomb group protein BMI1 plays an important role in cellular homeostasis by maintaining a balance between proliferation and senescence. It is often overexpressed in cancer cells and is required for self-renewal of stem cells. At present,very little is known about the signaling pathways that regulate the expression of BMI1. Here,we report that BMI1 autoactivates its own promoter via an E-box present in its promoter. We show that BMI1 acts as an activator of the WNT pathway by repressing Dickkopf (DKK) family of WNT inhibitors. BMI1 mediated repression of DKK proteins; in particular,DKK1 led to up-regulation of WNT target c-Myc,which in turn further led to transcriptional autoactivation of BMI1. Thus,a positive feedback loop connected by the WNT signaling pathway regulates BMI1 expression. This positive feedback loop regulating BMI1 expression may be relevant to the role of BMI1 in promoting cancer and maintaining stem cell phenotype. View Publication

Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus,many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study,we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells,which are shown by multiple marker or phenotypes of CSCs. Thus,these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus,further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer. View Publication

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36),an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly,TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand,inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. View Publication

A practical synthesis of the Rho-Kinase inhibitor Y-27632 and two new fluoro derivatives was achieved in seven steps and with a good overall yield of 45% starting from commercially available (R)-1-phenylethylamine. Compared to Y-27632 the new fluoro derivatives showed reduced or no effect on hPSC vitality and expansion after dissociation in human pluripotent stem cells. View Publication

Temporal manipulation of the in vitro environment and growth factors can direct differentiation of human pluripotent stem cells into organoids - aggregates with multiple tissue-specific cell types and three-dimensional structure mimicking native organs. A mechanistic understanding of early organoid formation is essential for improving the robustness of these methods,which is necessary prior to use in drug development and regenerative medicine. We investigated intestinal organoid emergence,focusing on measurable parameters of hindgut spheroids,the intermediate step between definitive endoderm and mature organoids. We found that 13{\%} of spheroids were pre-organoids that matured into intestinal organoids. Spheroids varied by several structural parameters: cell number,diameter and morphology. Hypothesizing that diameter and the morphological feature of an inner mass were key parameters for spheroid maturation,we sorted spheroids using an automated micropipette aspiration and release system and monitored the cultures for organoid formation. We discovered that populations of spheroids with a diameter greater than 75 $\mu$m and an inner mass are enriched 1.5- and 3.8-fold for pre-organoids,respectively,thus providing rational guidelines towards establishing a robust protocol for high quality intestinal organoids. View Publication

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid,we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3,Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells,whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. View Publication

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung,liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells,hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2,SOX17,HNF1B,members of the GATA family,and the surface receptor CXCR4. However,differentiation protocols are rarely 100% efficient. Here,we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations. View Publication

The development of targeted therapeutics for rare neurodevelopmental disorders (NDDs) faces significant challenges due to the scarcity of subjects and the difficulty of obtaining human neural cells. Here,we illustrate a rapid,simple protocol by which patient derived cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using an episomal vector and differentiated into neurons. Using this platform enables patient somatic cells to be converted to physiologically active neurons in less than two months with minimal labor. This platform includes a method to combine somatic cell reprogramming with CRISPR/Cas9 gene editing at single cell resolution,which enables the concurrent development of clonal knockout or knock-in models that can be used as isogenic control lines. This platform reduces the logistical barrier for using iPSC technology,allows for the development of appropriate control lines for use in rare neurodevelopmental disease research,and establishes a fundamental component to targeted therapeutics and precision medicine. Stem Cells Translational Medicine 2017;6:886-896. View Publication

Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible,cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05[x02013]2x105 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy. View Publication

We have identified a rare coding mutation,T835M (rs137875858),in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15,Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C,and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore,neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli,including $\beta$-amyloid (A$\beta$),glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system,we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death,particularly in vulnerable regions of the Alzheimer's disease brain. View Publication