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

Cellular plasticity contributes to the regenerative capacity of plants,invertebrates,teleost fishes and amphibians. In vertebrates,differentiated cells are known to revert into replicating progenitors,but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells,we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast,direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming,the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states,notably cancer. View Publication

Neural tissue formation is induced by growth factors that activate networks of signal transduction cascades that ultimately lead to the expression of early neural genes,including transcription factors of the SoxB family. Here,we report that fibroblast growth factor (FGF)-induced Erk1/2 (Mapk3 and Mapk1,respectively) mitogen-activated protein kinase (MAPK),but not phosphatidylinositol 3'-OH kinase (PI3K,Pik3r1),signalling is required for neural specification in mouse embryonic stem (ES) cells and in the chick embryo. Further,blocking Erk1/2 inhibits the onset of key SoxB genes in both mouse ES cells (Sox1) and chick embryos (Sox2 and Sox3) and,in both contexts,Erk1/2 signalling is required during only a narrow time window,as neural specification takes place. In the absence of Erk1/2 signalling,differentiation of ES cells stalls following Fgf5 upregulation. Using differentiating ES cells as a model for neural specification,we demonstrate that sustained Erk1/2 activation controls the transition from an Fgf5-positive,primitive ectoderm-like cell state to a neural progenitor cell state without attenuating bone morphogenetic protein (BMP) signalling and we also define the minimum period of Erk1/2 activity required to mediate this key developmental step. Together,these findings identify a conserved,specific and stage-dependent requirement for Erk1/2 signalling downstream of FGF-induced neural specification in higher vertebrates and provide insight into the signalling dynamics governing this process. View Publication

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein,tandem dimer Tomato (tdTomato),are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons,evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. View Publication

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms,yet their clinical translation has been compromised by their biosafety concern. Here,we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/ progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immu-nodeficient mice. Moreover,removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplanta-tion,ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable,depending on the oncogenic load,with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus,transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:694–702 SIGNIFICANCE Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products,especially when reprogrammed with integrating vectors. Two major under-lying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzy-matic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in test-ing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature b cell phenotype would lead to safe islet replacement therapy for diabetes. View Publication

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However,the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1,the only known histone H3 lysine 79 (H3K79) methyltransferase,has been shown to interact with multiple MLL fusion partners including AF9,ENL,AF10,and AF17. In this study,we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9,we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis,suggesting the involvement of Dot1 in survival pathways. In summary,our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target. View Publication

Control of cellular (de)differentiation in a temporal,cell-specific,and exchangeable manner is of paramount importance in the field of reprogramming. Here,we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming,these factors can be replaced by genes of interest-for example,to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that,similar to ESCs,Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes. View Publication

Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless,a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process,including hypoxic stress,fibrosis,and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions,leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology,demonstrating that the phased combination of TGF? inhibitor SB431542,Rho kinase inhibitor Y27632,and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction,providing a crucial platform for discovering therapeutic targets for ischemic heart disease. Engineered human models of hearts are needed to study pathology and repair. Here,the authors develop a model which replicates the phased remodelling process. The model is then used to study signalling pathway modulators for their therapeutic potential in a mini-repair model. View Publication

SummaryThe BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS),a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits,ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes,little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/? induced pluripotent stem cells to model CNCC specification,we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT),a process necessary for CNCC delamination and migration from the neural tube. Furthermore,wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level,these EMT enhancers contain binding motifs for ZIC2,and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers,ZIC2 relocates to neuronal enhancers,triggering aberrant neuronal gene activation. In mice,deletion of Zic2 impairs NCC delamination,while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. Graphical abstract ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition,a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers,resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. View Publication

Using whole-cell patch-clamp,fluorescence microscopy and flow cytometry,we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD(+)CD27(+) memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca(2+)-activated intermediate-conductance IKCa1 channel when quiescent,and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast,quiescent class-switched memory B cells express high levels of Kv1.3 ( approximately 2000 channels/cell) and maintain their Kv1.3(high) expression after activation. Consistent with their channel phenotypes,proliferation of naive and IgD(+)CD27(+) memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin,whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore,specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders. View Publication

We investigated the expression status of periostin in breast cancer stem cells and its clinical implications in order to lay a foundation for managing breast cancer. CD44+/CD24-/line- tumor cells (CSC) from clinical specimens were sorted using flow cytometry. Periostin expression status was detected in CSC cells and 1,086 breast cancer specimens by Western blot and immunohistochemistry staining,with the CSC ratio determined by immunofluorescence double staining. The relationship between the periostin protein and clinico-pathological parameters and prognosis was subsequently determined. As a result,CSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. Periostin protein was expressed higher in CSC cells compared to the control cells and was found to be related to CSC chemotherapy resistance. Moreover,periostin expression was found to be related to the CSC ratio in 1,086 breast cancer specimens (P = 0.001). In total,334 (30.76%) of the 1,086 breast cases showed high periostin expression. After universal and Spearman regression correlation analysis,periostin was observed to be related to histological grade,CSC ratio,lymph node metastasis,tumor size,and triple-negative breast cancer (all Ptextless0.05). Furthermore,periostin was shown to attain a significantly more distant bone metastasis and worse disease-specific survival than those with none or low-expressed periostin protein (P = 0.001). In the Cox regression test,periostin protein was detected as an independent prognostic factor (P = 0.001). In conclusion,periostin was found to be related to the CSC and an independent prognostic factor for breast cancer. It is also perhaps a potential target to breast cancer. View Publication

Asthma is a complex and heterogeneous respiratory disorder characterized by chronic airway inflammation. It has generally been associated with allergic mechanisms related to type 2 airway inflammation. Nevertheless,between 10 and 33{\%} of asthmatic individuals have nonallergic asthma (NA). Several targeted treatments are in clinical development for patients with Th2 immune response,but few biomarkers are been defined for low or non-Th2-mediated inflammation asthma. We have recently defined by gene expression a set of genes as potential biomarkers of NA,mainly associated with disease severity: IL10,MSR1,PHLDA1,SERPINB2,CHI3L1,IL8,and PI3. Here,we analyzed their protein expression and specificity using sera and isolated peripheral blood mononuclear cells (PBMCs). First,protein quantification was carried out using ELISA (in sera) or Western blot (proteins extracted from PBMCs by Trizol procedure),depending on the biomarker in 30 healthy controls (C) subjects and 30 NA patients. A receiver operating characteristic curve analysis was performed by using the R program to study the specificity and sensitivity of the candidate biomarkers at a gene- and protein expression level. Four kinds of comparisons were performed: total NA group vs C group,severe NA patients vs C,moderate-mild NA patients vs C,and severe NA patients vs moderate-mild NA patients. We found that all the single genes showed good sensitivity vs specificity for some phenotypic discrimination,with CHI3L1 and PI3 exhibiting the best results for C vs NA: CHI3L1 area under the curve (AUC) (CI 95{\%}): 0.95 (0.84-1.00) and PI3 AUC: 0.99 (0.98-1.00); C vs severe NA: PI3 AUC: 1 (0.99-1.00); and C vs moderate-mild NA: CHI3L1 AUC: 1 (0.99-1.00) and PI3 AUC: 0.99 (0.96-1.00). However,the results for discriminating asthma disease and severity with protein expression were better when two or three biomarkers were combined. In conclusion,individual genes and combinations of proteins have been evaluated as reliable biomarkers for classifying NA subjects and their severity. These new panels could be good diagnostic tests. View Publication

Genetic variations in the Trpm8 gene that encodes the cold receptor TRPM8 have been linked to protection against polygenic migraine,a disabling condition primarily affecting women. Noteworthy,TRPM8 has been recently found in brain areas related to emotional processing,suggesting an unrecognized role in migraine comorbidities. Here,we use mouse behavioural models to investigate the role of Trpm8 in migraine-related phenotypes. Subsequently,we test the efficacy of rapamycin,a clinically relevant TRPM8 agonist,in these behavioural traits and in human induced pluripotent stem cell (iPSC)-derived sensory neurons. We report that Trpm8 null mice exhibited impulsive and depressive-like behaviours,while also showing frequent pain-like facial expressions detected by an artificial intelligence algorithm. In a nitroglycerin-induced migraine model,Trpm8 knockout mice of both sexes developed anxiety and mechanical hypersensitivity,whereas wild-type females also displayed depressive-like phenotype and hypernociception. Notably,rapamycin alleviated pain-related behaviour through both TRPM8-dependent and independent mechanisms but lacked antidepressant activity,consistent with a peripheral action. The macrolide ionotropically activated TRPM8 signalling in human sensory neurons,emerging as a new candidate for intervention. Together,our findings underscore the potential of TRPM8 for migraine relief and its involvement in affective comorbidities,emphasizing the importance of addressing emotional symptoms to improve clinical outcomes for migraine sufferers,especially in females. The online version contains supplementary material available at 10.1186/s10194-025-02082-4. View Publication