技术资料

Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore,this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model. To evaluate their antigenicity and neutralization profile,indirect enzyme-linked immunosorbent assay (ELISA),an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated,and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate,CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay,3E7b blocks CHIKV attachment to permissive cells,possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection,3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively,these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design. View Publication

Multiple myeloma (MM) is a malignancy of differentiated B lymphocytes and has remained an incurable disease. Chromosomal abnormalities are among the most important prognostic parameters for MM. Cytoplasm immunoglobulin-enhanced interphase fluorescent in situ hybridization (FISH) has been a standard cell-targeting method for identifying genomic aberrations in MM. We have developed another cell-targeting approach by using CD138 magnetic microbeads to sort plasma cells for FISH analysis. The FISH panel consisted of four probes targeting RB-1,D13S319,immunoglobulin H,and p53 loci. We reviewed the FISH and conventional cytogenetic results of 60 patients with MM. The present cell-targeting approach in conjunction with the FISH probe panel was more sensitive than FISH performed on untargeted cells in detecting prognostically significant genomic aberrations (72 versus 24%,P = 0.0016). The frequencies of genomic abnormalities identified were similar to previously reported data obtained with the standard cell-targeting method. Therefore,our cell-targeting approach and FISH panel reliably detect prognostically important genomic abnormalities in MM and are potentially suitable for widespread use. 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

BACKGROUND/OBJECTIVE A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion,the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media,and markers such as NG2 and $\alpha$SMA in mesenchymal media. However,analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However,nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease. View Publication

Neural–tumor interactions drive glioma growth as evidenced in preclinical models,but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients’ survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma,primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like,astrocyte-like and oligodendrocyte-progenitor-like,alongside oligodendrocytes and excitatory neurons. In line with these findings,high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients,a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients’ plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes. Subject terms: Translational research,CNS cancer,DNA methylation 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

Here,we present a protocol for isolating and culturing mouse photoreceptors in a minimal,chemically defined medium free from serum. We describe steps for retina dissection,enzymatic dissociation,photoreceptor enrichment,cell culture,extracellular vesicles (EVs) enrichment,and EV ultrastructural analysis. This protocol,which has been verified for cultured cells derived from multiple murine strains,allows for the study of several aspects of photoreceptor biology,including EV isolation and nanotube formation. For complete details on the use and execution of this protocol,please refer to Kalargyrou et al. (2021). 1 Subject areas: Cell Biology,Molecular Biology,Neuroscience View Publication

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is,therefore,crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterization. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript,we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDH(dim) cells expressed significantly higher levels of DeltaNp63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDH(bright) cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker,RHAMM/HMMR,which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR- limbal epithelial cells are smaller in size,express negligible CK3,have higher levels of DeltaNp63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/ HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterization. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Pygopus 2 (Pygo2) is a coactivator of Wnt/$\beta$-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer,we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller,differentiated,and less metastatic tumors,due,in part,to decreased canonical Wnt/$\beta$-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGF$\beta$ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically,the Pygo2-histone interaction potentiated Wnt/$\beta$-catenin signaling,in part,by repressing the expression of Wnt signaling antagonists. Furthermore,Pygo2 and $\beta$-catenin regulated the expression of miR-29 family members,which,in turn,repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively,these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors,and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer,as its histone-binding capability promotes $\beta$-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation,EMT,and metastasis. 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

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)-keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial,patients received either control (KLH only) or Id-KLH vaccine,autologous transplantation,vaccine-specific costimulated T cells expanded ex vivo,and 2 booster doses of assigned vaccine. In 36 patients (KLH,n = 20; Id-KLH,n = 16),no dose-limiting toxicity was seen. At last evaluation,6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms,respectively (P = .22),and no difference in 3-year progression-free survival was observed (59% and 56%,respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR),compared with patients receiving KLH only,there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically,upregulation of genes associated with activation,effector function induction,and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly,in responding patients across both arms,upregulation of genes associated with T-cell activation was seen. At baseline,all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion,in this combination immunotherapy approach,we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm,supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828. View Publication