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

Little is known on the embryonic origin and related heterogeneity of adult neural stem cells (aNSCs). We use conditional genetic tracing,activated in a global or mosaic fashion by cell type-specific promoters or focal laser uncaging,coupled with gene expression analyses and Notch invalidations,to address this issue in the zebrafish adult telencephalon. We report that the germinal zone of the adult pallium originates from two distinct subtypes of embryonic progenitors and integrates two modes of aNSC formation. Dorsomedial aNSCs derive from the amplification of actively neurogenic radial glia of the embryonic telencephalon. On the contrary,the lateral aNSC population is formed by stepwise addition at the pallial edge from a discrete neuroepithelial progenitor pool of the posterior telencephalic roof,activated at postembryonic stages and persisting lifelong. This dual origin of the pallial germinal zone allows the temporally organized building of pallial territories as a patchwork of juxtaposed compartments. View Publication

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties,yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME,we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway,possibly by modulating availability of hyaluronic acid (HA),the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships,and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions,respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction,whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo,targeting CD44 decreased PI3K-4EBP1-SOX2 signaling,tumor growth,and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally,a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes,respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44,TAM,and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery. View Publication

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that increases the expression of detoxifying enzymes upon ligand stimulation. Recent studies now suggest that novel endogenous roles of the AHR exist throughout development. In an effort to create an optimized model system for the study of AHR signaling in several cellular lineages,we have employed a CRISPR/CAS9 genome editing strategy in induced pluripotent stem cells (iPSCs) to incorporate a reporter cassette at the transcription start site of one of its canonical targets,cytochrome P450 1A1 (CYP1A1). This cell line faithfully reports on CYP1A1 expression,with luciferase levels as its functional readout,when treated with an endogenous AHR ligand (FICZ) at escalating doses. iPSC-derived fibroblast-like cells respond to acute exposure to environmental and endogenous AHR ligands,and iPSC-derived hepatocytes increase CYP1A1 in a similar manner to primary hepatocytes. This cell line is an important innovation that can be used to map AHR activity in discrete cellular subsets throughout developmental ontogeny. As further endogenous ligands are proposed,this line can be used to screen for safety and efficacy and can report on the ability of small molecules to regulate critical cellular processes by modulating the activity of the AHR. View Publication

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid,beta-glycerophosphate,and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after,but not before,14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair. View Publication

Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus,salivary glands,lung,thymus,parathyroid and thyroid. Despite its importance,reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1(+) lung and thyroid lineages. Here,we describe a novel protocol to derive a subdomain of AFE,identified by expression of Pax9,from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9(+) AFE cells,which when transplanted in vivo,can form several distinct complex AFE-derived epithelia,including mucosal glands and stratified squamous epithelium. Finally,we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus,this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders. View Publication

SummaryMutations in the DMD gene lead to Duchenne muscular dystrophy (DMD),a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2–4 years of age,but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients,which poses a serious challenge in current standards of care. Here,we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether,this work demonstrates that in vitro,dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development,which should be considered in future therapeutic strategies for DMD. Graphical abstract Highlights•Myogenic differentiation of DMD hiPSCs diverges at the somite stage•Cell junction formation is dysregulated in DMD somite cells•Somite cells from DMD hiPSCs have impaired epithelialization properties•Migration velocity of DMD-mutant somite progenitors is upregulated Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Developmental biology. View Publication

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine,a potent inhibitor of PNP,was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK),we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization,phosphorylation of p53 at Ser15,and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation,changes in mitochondrial membrane potential,and PARP cleavage. Based on these data,a phase 2 clinical trial of forodesine has been initiated for CLL patients. View Publication

Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. We found that DZNep effectively depleted cellular levels of PRC2 components EZH2,SUZ12,and EED and inhibited associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi),genome-wide expression analysis,and chromatin immunoprecipitation (ChIP) studies,we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep. We further demonstrate that the preferential reactivation of a set of these genes by DZNep,including a novel apoptosis affector,FBXO32,contributes to DZNep-induced apoptosis in breast cancer cells. Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy. View Publication

The identification of neural stem cells (NSCs) in situ has been prevented by the inability to identify a marker consistently expressed in all adult NSCs and is thus generally accomplished using the in vitro neurosphere-forming assay. The high-mobility group transcription factor Sox2 is expressed in embryonic neural epithelial stem cells; because these cells are thought to give rise to the adult NSC population,we hypothesized that Sox2 may continue to be expressed in adult NSCs. Using Sox2:EGFP transgenic mice,we show that Sox2 is expressed in neurogenic regions along the rostral-caudal axis of the central nervous system throughout life. Furthermore,all neurospheres derived from these neurogenic regions express Sox2,suggesting that Sox2 is indeed expressed in adult NSCs. We demonstrate that NSCs are heterogeneous within the adult brain,with differing capacities for cell production. In vitro,all neurospheres express Sox2,but the expression of markers common to early progenitor cells within individual neurospheres varies; this heterogeneity of NSCs is mirrored in vivo. For example,both glial fibrillary acidic protein and NG2 are expressed within individual neurospheres,but their expression is mutually exclusive; likewise,these two markers show distinct staining patterns within the Sox2+ regions of the brain's neurogenic regions. Thus,we propose that the expression of Sox2 is a unifying characteristic of NSCs in the adult brain,but that not all NSCs maintain the ability to form all neural cell types in vivo. View Publication

Summary Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture,raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV,we show that those containing this amplicon have higher population doubling rates,attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs,only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells,whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells,establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas,linking this mutation with malignant transformation. View Publication

Cerebral cavernous malformations (CCMs) are clusters of thin-walled enlarged blood vessels in the central nervous system that are prone to recurrent hemorrhage and can occur in both sporadic and familial forms. The familial form results from loss-of-function variants in the CCM1,CCM2,or CCM3 gene. Despite a better understanding of CCM pathogenesis in recent years,it is still unclear why CCM3 mutations often lead to a more aggressive phenotype than CCM1 or CCM2 variants. By combining high-throughput differentiation of blood vessel organoids from human induced pluripotent stem cells (hiPSCs) with a CCM1,CCM2,or CCM3 knockout,single-cell RNA sequencing,and high-content imaging,we uncovered both shared and distinct functions of the CCM proteins. While there was a significant overlap of differentially expressed genes in fibroblasts across all three knockout conditions,inactivation of CCM1,CCM2,or CCM3 also led to specific gene expression patterns in neuronal,mesenchymal,and endothelial cell populations,respectively. Taking advantage of the different fluorescent labels of the hiPSCs,we could also visualize the abnormal expansion of CCM1 and CCM3 knockout cells when differentiated together with wild-type cells into mosaic blood vessel organoids. In contrast,CCM2 knockout cells showed even reduced proliferation. These observations may help to explain the less severe clinical course in individuals with a pathogenic variant in CCM2 and to decode the molecular and cellular heterogeneity in CCM disease. Finally,the excellent scalability of blood vessel organoid differentiation in a 96-well format further supports their use in high-throughput drug discovery and other biomedical research studies. The online version contains supplementary material available at 10.1007/s10456-025-09985-5. View Publication

BACKGROUND: Transforming growth factor beta (TGF-beta) plays an important role in skeletal remodelling. However,few studies have examined its effects on cultured human osteoblasts. Our aim is to characterise the biological effects of TGF-beta1 on human osteoblasts and to examine the interaction between TGF-beta1 and calcitriol. DESIGN: In vitro study employing two models of normal human osteoblasts: human bone marrow stromal cells [hMS/(OB)] containing osteoprogenitor cells and trabecular bone osteoblasts (hOB),which are mature osteoblasts. A reverse-transcriptase-polymerase-chain-reaction assay was employed to measure steady state mRNA levels of TGF-beta(s) isoforms and receptors. Effects of short-term treatment of TGF-beta1 on osteoblast proliferation and differentiation markers were assessed. The effect of cotreatment of calcitriol (10-8 M) and TGF-beta1 on osteoblast differentiation was also determined. RESULTS: Both hMS(OB) and hOB cells expressed mRNA transcripts of TGF-beta1,TGF-beta2,TGF-beta 3,TGF-beta type I and type II receptors. TGF-beta 1 stimulated osteoblast proliferation in hMS(OB) and in hOB cultures. In hOB cultures,TGF-beta1 stimulated AP production and cotreatment with calcitriol induced a synergistic increase in AP levels to 250 +/- 61% of calcitriol-treated controls. Effects of TGF-beta1 and calcitriol were less pronounced in hMS(OB) cultures. TGF-beta1 inhibited collagen type I production in hMS(OB) cells and these effects were abolished in presence of calcitriol. In presence of calcitriol,TGF-beta1 increased collagen type I production in hOB cells. In both hOB and hMS(OB) cultures,TGF-beta1 inhibited osteocalcin production. CONCLUSIONS: TGF-beta increases osteoblastic cell proliferation irrespective of the differentiation state. In presence of calcitriol,it initiates osteoblast cell differentiation and matrix formation. As TGF-beta inhibits osteocalcin production,other factors are necessary for inducing terminal differentiation of osteoblasts. The observed effects of TGF-beta on human osteoblasts in vitro may represent important regulatory steps in controlling osteoblast cell proliferation and differentiation in vivo. View Publication