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

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however,differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4,colonies reproducibly differentiated to an outer trophectoderm-like ring,an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development. View Publication

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings,however,requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study,we show that once specified to neuro-epithelial lineage,hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However,our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes,namely,migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes,a new potential source for therapeutic use for brain tumors and other neurological diseases. View Publication

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical,environmental,and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part,previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation,which are highly nonphysiologic,or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair,we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs,compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus,the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny. View Publication

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general,but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies,however,is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is,however,limited and thereby further optimization in terms of time,efficiency,and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts,at least in part,in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1,Inhba and Grem1. Hence,we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. View Publication

Human embryonic stem cell-derived dendritic cells (hESC-DCs) may potentially provide a platform to generate off-the-shelf" therapeutic cancer vaccines. To apply hESC-DCs for cancer immunotherapy in a semiallogeneic setting� View Publication

IL-17-producing CD4+ T helper cells (TH17) have been extensively investigated in mouse models of autoimmunity. However,the requirements for differentiation and the properties of pathogen-induced human TH17 cells remain poorly defined. Using an approach that combines the in vitro priming of naive T cells with the ex vivo analysis of memory T cells,we describe here two types of human TH17 cells with distinct effector function and differentiation requirements. Candida albicans-specific TH17 cells produced IL-17 and IFN-γ,but no IL-10,whereas Staphylococcus aureus-specific TH17 cells produced IL-17 and could produce IL-10 upon restimulation. IL-6,IL-23 and IL-1β contributed to TH17 differentiation induced by both pathogens,but IL-1β was essential in C. albicans-induced TH17 differentiation to counteract the inhibitory activity of IL-12 and to prime IL-17/IFN-γ double-producing cells. In addition,IL-1β inhibited IL-10 production in differentiating and in memory TH17 cells,whereas blockade of IL-1β in vivo led to increased IL-10 production by memory TH17 cells. We also show that,after restimulation,TH17 cells transiently downregulated IL-17 production through a mechanism that involved IL-2-induced activation of STAT5 and decreased expression of ROR-γt. Taken together these findings demonstrate that by eliciting different cytokines C. albicans and S. aureus prime TH17 cells that produce either IFN-γ or IL-10,and identify IL-1β and IL-2 as pro- and anti-inflammatory regulators of TH17 cells both at priming and in the effector phase. View Publication

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures,where long single-stranded DNA is folded into a compact nanostructure,present an attractive approach to package genes; however,effective delivery of genetic material into cell nuclei has remained a critical challenge. Here,we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore,our study validates virus-like particles as an efficient method of DNA nanostructure delivery,opening the possibility of delivering nanostructures in vivo to specific cell types. Together,these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates,exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions,such as biosensing,into cell nuclei. View Publication

BackgroundUnderstanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models,lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate,and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule.MethodsTo establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells,we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways,and construct cell lines carrying an inducible NKX3-1 expressing cassette,together with three-dimensional culture system. Unpaired t test was applied for statistical analyses.ResultsWe first successfully generate the definitive endoderm,hindgut,and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1,but fail to express NKX3-1. Therefore,we construct NKX3-1-inducible cell line by homologous recombination,which is eventually able to yield AR,FOXA1,and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally,combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations.ConclusionsThis study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line,as well as provides a stepwise differentiation protocol to generate human prostate-like organoids,which should facilitate the studies on prostate development and disease pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03886-y. View Publication

Differentiation of induced pluripotent stem cells (iPSCs) into specialized cell types is essential for uncovering cell-type specific molecular mechanisms and interrogating cellular function. Transcription factor screens have enabled efficient production of a few cell types; however,engineering cell types that require complex transcription factor combinations remains challenging. Here,we report an iterative,high-throughput single-cell transcription factor screening method that enables the identification of transcription factor combinations for specialized cell differentiation,which we validated by differentiating human microglia-like cells. We found that the expression of six transcription factors,SPI1,CEBPA,FLI1,MEF2C,CEBPB,and IRF8,is sufficient to differentiate human iPSC into cells with transcriptional and functional similarity to primary human microglia within 4 days. Through this screening method,we also describe a novel computational method allowing the exploration of single-cell RNA sequencing data derived from transcription factor perturbation assays to construct causal gene regulatory networks for future cell fate engineering. Liu et al. developed a platform to identify transcription factors (TFs) that turn stem cells into desired cell types. They discovered six key TFs that produce microglia efficiently,enhancing cell differentiation methods. View Publication

BackgroundStreptococcus pyogenes (group A streptococcus,GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections,such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections.MethodsHuman primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle,cytokine release by human primary monocytes and macrophages was determined.ResultsIn vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast,moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates,28 harbored mutations resulting in dysfunctional CovR/S. However,analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations.ConclusionsOur data demonstrate that strains,which harbor covR/S mutations,interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-024-01014-9. View Publication

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment,often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however,the effects of melatonin on CSCs remain unclear,and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers,even in a strongly chemoprotective environment,as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover,melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality,including CSCs,stromal component,and a biomimetic matrix,providing a new valuable research tool in precision oncology. View Publication

Addiction is known to occur through the consumption of substances such as pharmaceuticals,illicit drugs,food,alcohol and tobacco. These addictions can be viewed as drug addiction,resulting from the ingestion of chemical substances contained in them. Multiple neural networks,including the reward system,anti‐reward/stress system and central immune system in the brain,are believed to be involved in the onset of drug addiction. Although various compound evaluations using microelectrode array (MEA) as an in vitro testing methods to evaluate neural activities have been conducted,methods for assessing addiction have not been established. In this study,we aimed to develop an in vitro method for assessing the addiction of compounds,as an alternative to animal experiments,using human iPS cell‐derived dopaminergic neurons with MEA measurements. MEA data before and after chronic exposure revealed specific changes in addictive compounds compared to non‐addictive compounds,demonstrating the ability to estimate addiction of compound. Additionally,conducting gene expression analysis on cultured samples after the tests revealed changes in the expression levels of various receptors (nicotine,dopamine and GABA) due to chronic administration of addictive compounds,suggesting the potential interpretation of these expression changes as addiction‐like responses in MEA measurements. The addiction assessment method using MEA measurements in human iPS cell‐derived dopaminergic neurons conducted in this study proves effective in evaluating addiction of compounds on human neural networks. View Publication