搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

PURPOSE This study aimed to develop a feasible and efficient method for generating embryonic stem cell (ESC)-like induced pluripotent stem (iPS) cells from human Tenon's capsule fibroblasts (HTFs) through the expression of a defined set of transcription factors,which will have significant application value for ophthalmic personalized regenerative medicine. METHODS HTFs were harvested from fresh samples,and reprogramming was induced by the exogenous expression of the four classic transcription factors,OCT-3/4,SOX-2,KLF-4,and C-MYC. The HTF-derived iPS (TiPS) cells were analyzed with phase contrast microscopy,real-time PCR,immunofluorescence,FACS analysis,alkaline phosphatase activity analysis,and a teratoma formation assay. Human ESC colonies were used as the positive control. RESULTS The resulting HTF-derived iPS cell colonies were indistinguishable from human ESC colonies regarding morphology,gene expression levels,pluripotent gene expression,alkaline phosphatase activity,and the ability to generate all three embryonic germ layers. CONCLUSIONS This study presents a simple,efficient,practical procedure for generating patient-tailored iPS cells from HTFs. These cells will serve as a valuable and preferred candidate donor cell population for ophthalmological regenerative medicine. View Publication

Many solid cancers display cellular hierarchies with self-renewing,tumorigenic stemlike cells,or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies,they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs,we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays,but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines,including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion,and targeting IL-17A signaling impaired CIC growth. Notably,IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer. View Publication

Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse,and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically,following interaction with Pin1,Notch1 and Notch4,key regulators of cell fate,escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7$$. Functionally,we show that Fbxw7$$ acts as an essential negative regulator of breast CSCs' expansion by restraining Notch activity,but the establishment of a Notch/Pin1 active circuitry opposes this effect,thus promoting breast CSCs self-renewal,tumor growth and metastasis in vivo. In human breast cancers,despite Fbxw7$$ expression,high levels of Pin1 sustain Notch signaling,which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes,through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers. View Publication

Ring chromosomes are structural aberrations commonly associated with birth defects,mental disabilities and growth retardation. Rings form after fusion of the long and short arms of a chromosome,and are sometimes associated with large terminal deletions. Owing to the severity of these large aberrations that can affect multiple contiguous genes,no possible therapeutic strategies for ring chromosome disorders have been proposed. During cell division,ring chromosomes can exhibit unstable behaviour leading to continuous production of aneuploid progeny with low viability and high cellular death rate. The overall consequences of this chromosomal instability have been largely unexplored in experimental model systems. Here we generated human induced pluripotent stem cells (iPSCs) from patient fibroblasts containing ring chromosomes with large deletions and found that reprogrammed cells lost the abnormal chromosome and duplicated the wild-type homologue through the compensatory uniparental disomy (UPD) mechanism. The karyotypically normal iPSCs with isodisomy for the corrected chromosome outgrew co-existing aneuploid populations,enabling rapid and efficient isolation of patient-derived iPSCs devoid of the original chromosomal aberration. Our results suggest a fundamentally different function for cellular reprogramming as a means of /`chromosome therapy/' to reverse combined loss-of-function across many genes in cells with large-scale aberrations involving ring structures. In addition,our work provides an experimentally tractable human cellular system for studying mechanisms of chromosomal number control,which is of critical relevance to human development and disease. View Publication

GANT61 is a small-molecule inhibitor of glioma-associated oncogene 1 (GLI1)- and GLI2-mediated transcription at the nuclear level that exerts its effect by preventing DNA binding. It has been demonstrated to induce cell death against Ewing's sarcoma family tumor (ESFT) cell lines in a dose-dependent manner. The most sensitive cell line was SK-N-LO,which expresses the EWS-FLI1 fusion gene. SK-N-LO cells treated with GANT61 showed cellular and nuclear morphological changes,including cell shrinkage,chromatin condensation and nuclear fragmentation,in a concentration-dependent manner,as visualized by Hoechst 33342 staining. Furthermore,annexin V-propidium iodide (PI) double-staining revealed a significant increase in the number of late apoptotic cells. GANT61 induced a significant decrease in the proportion of cells in the S phase. Significant decrease of the protein levels of GLI2,survivin,cyclin A and claspin,and significant increase of p21 expression was also observed in the cells treated with GANT61. Moreover,poly (ADP-ribose) polymerase (PARP) cleavage was observed,but no cleavage of caspase-3 or -7,or any change in the expressions of Bcl-2 or p53 were observed. These findings suggest that GANT61 induces cell death of SK-N-LO cells in a caspase-independent manner,by inhibiting DNA replication in the S phase. View Publication

Exosomes are small extracellular vesicles (sEVs),playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here,we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions,including cytostatic,heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance,melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study,we concluded that (i) molecular patterns of tumour-derived sEVs,dictated by the microenvironmental conditions,resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response,with a potential influence on treatment efficacy. View Publication

SummaryKnowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete,yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress,we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens,aggrecan,perlecan,proteoglycans,and elastic fibers. We identified two populations of chondroprogenitor cells,mesenchyme cells and nascent chondrocytes,and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage,but also play a pivotal autocrine cell signaling role in determining chondrocyte fate. Graphical abstract Highlights•Craniofacial cartilage organoids were grown from human neural crest stem cells•These organoids exhibited elastic cartilage architecture and characteristic markers•Paracrine signaling drove chondrogenesis in mesenchyme cells and nascent chondrocytes•ECM components cemented chondrocyte cell fate through autocrine signaling Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Specialized functions of cells View Publication

Background: Human pluripotent stem cells (hPSCs),including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs),can undergo erythroid differentiation,offering a potentially invaluable resource for generating large quantities of erythroid cells. However,the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors,with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. Methods: In this study,we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies,including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs,morphological analysis,quantitative real-time PCR (qRT-PCR),western blotting,flow cytometry,karyotyping,cytospin,Benzidine-Giemsa staining,immunofluorescence assay,and high-speed cell imaging analysis,were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation,as well as their differentiated erythroid cells. Results: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells,whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. Conclusions: VIM disappear during the normal maturation of erythroid cells,whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform,we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization. View Publication

Cell signaling plays a critical role in neurodevelopment,regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing,scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq,an integrated approach that aims to quantify cytoplasmic and nuclear proteins,including those with post-translational modifications,and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom neuro-focused antibody panels for simultaneous protein and scATAC-seq profiling on whole cells,alongside both experimental and computational strategies to incorporate transcriptomic measurements. We apply our workflow to cell lines,induced pluripotent stem cells,and months-old retinal and brain organoids to demonstrate its broad applicability. We show that Phospho-seq can provide insights into cellular states and trajectories,shed light on gene regulatory relationships,and help explore the causes and effects of diverse cell signaling in neurodevelopment. Here,the authors demonstrate Phospho-seq,a single-cell multiomics method capable of quantifying chromatin accessibility alongside intracellular proteins,including post-translationally modified proteins. Then,they apply Phospho-seq to organoid models of neurodevelopment. View Publication

The generation of iPSC-derived hepatocyte-like cells (HLCs) is a powerful tool for studying liver diseases,their therapy as well as drug development. iPSC-derived disease models benefit from their diverse origin of patients,enabling the study of disease-associated mutations and,when considering more than one iPSC line to reflect a more diverse genetic background compared to immortalized cell lines. Unfortunately,the use of iPSC-derived HLCs is limited due to their lack of maturity and a rather fetal phenotype. Commercial kits and complicated 3D-protocols are cost- and time-intensive and hardly useable for smaller working groups. In this study,we optimized our previously published protocol by fine-tuning the initial cell number,exchanging antibiotics and basal medium composition and introducing the small molecule forskolin during the HLC maturation step. We thereby contribute to the liver research field by providing a simple,cost- and time-effective 2D differentiation protocol. We generate functional HLCs with significantly increased HLC hallmark gene (ALB,HNF4?,and CYP3A4) and protein (ALB) expression,as well as significantly elevated inducible CYP3A4 activity. Graphical Abstract View Publication

BackgroundDendritic cell (DC)-mediated antigen presentation is essential for the priming and activation of tumor-specific T cells. However,few drugs that specifically manipulate DC functions are available. The identification of drugs targeting DC holds great promise for cancer immunotherapy.MethodsWe observed that type 1 conventional DCs (cDC1s) initiated a distinct transcriptional program during antigen presentation. We used a network-based approach to screen for cDC1-targeting therapeutics. The antitumor potency and underlying mechanisms of the candidate drug were investigated in vitro and in vivo.ResultsSitagliptin,an oral gliptin widely used for type 2 diabetes,was identified as a drug that targets DCs. In mouse models,sitagliptin inhibited tumor growth by enhancing cDC1-mediated antigen presentation,leading to better T-cell activation. Mechanistically,inhibition of dipeptidyl peptidase 4 (DPP4) by sitagliptin prevented the truncation and degradation of chemokines/cytokines that are important for DC activation. Sitagliptin enhanced cancer immunotherapy by facilitating the priming of antigen-specific T cells by DCs. In humans,the use of sitagliptin correlated with a lower risk of tumor recurrence in patients with colorectal cancer undergoing curative surgery.ConclusionsOur findings indicate that sitagliptin-mediated DPP4 inhibition promotes antitumor immune response by augmenting cDC1 functions. These data suggest that sitagliptin can be repurposed as an antitumor drug targeting DC,which provides a potential strategy for cancer immunotherapy. View Publication