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

Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study,we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles,globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally,these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably,we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype,which reactivates fetal hemoglobin levels and rescues the disease phenotypes,thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether,we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling,drug screenings and cell and gene therapy-based applications. Sickle cell disease (SCD) and β-thalassemia (BT) are globally prevalent inherited blood disorders but,despite extensive research,no ex vivo system exists for SCD and BT. Here,the authors generate pathophysiologically relevant erythroid progenitor models of SCD and BT. View Publication

Siglec-6 is a lectin receptor with restricted expression in the placenta,mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL),its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8,a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand,sTn,results in Cdc42 activation,WASP protein recruitment and F-actin polymerization,which are all associated with cell migration. Therapeutically,a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL,which can be therapeutically targeted using a Siglec-6 specific T-biAb. Siglec-6 is often overexpressed in chronic lymphocytic leukaemia (CLL),but its role is unclear. Here,the author report that Siglec-6 regulates the migration and adhesion of CLL B cells via interaction with sialyl Tn on bone marrow stromal cells driving invasion which could be therapeutically targeted using a Siglec-6/CD3-bispecfiic antibody. View Publication

We demonstrated previously that mouse hepatic stellate cells (HSCs) suppress T cells via programmed death-ligand 1 (PD-L1),but it remains unknown whether they exert any effects on B cells,the other component of the adaptive immune system. In this study,we found that mouse HSCs directly inhibited B cells and that PD-L1 was also integrally involved. We found that HSCs inhibited the upregulation of activation markers on activated B cells,as well as the proliferation of activated B cells and their cytokine/Ig production in vitro,and that pharmaceutically or genetically blocking the interaction of PD-L1 with programmed cell death protein 1 impaired the ability of HSCs to inhibit B cells. To test the newly discovered B cell-inhibitory activity of HSCs in vivo,we developed a protocol of intrasplenic artery injection to directly deliver HSCs into the spleen. We found that local delivery of wild-type HSCs into the spleens of mice that had been immunized with 4-hydroxy-3-nitrophenylacetyl-Ficoll,a T cell-independent Ag,significantly suppressed Ag-specific IgM and IgG production in vivo,whereas splenic artery delivery of PD-L1-deficient HSCs failed to do so. In conclusion,in addition to inhibiting T cells,mouse HSCs concurrently inhibit B cells via PD-L1. This direct B cell-inhibitory activity of HSCs should contribute to the mechanism by which HSCs maintain the liver's immune homeostasis. View Publication

Pluripotent stem cells can be induced from somatic cells,providing an unlimited cell resource,with potential for studying disease and use in regenerative medicine. However,genetic manipulation and technically challenging strategies such as nuclear transfer used in reprogramming limit their clinical applications. Here,we show that pluripotent stem cells can be generated from mouse somatic cells at a frequency up to 0.2% using a combination of seven small-molecule compounds. The chemically induced pluripotent stem cells resemble embryonic stem cells in terms of their gene expression profiles,epigenetic status,and potential for differentiation and germline transmission. By using small molecules,exogenous master genes" are dispensable for cell fate reprogramming. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications." View Publication

The prevalence of hearing loss after damage to the mammalian cochlea has been thought to be due to a lack of spontaneous regeneration of hair cells,the primary receptor cells for sound. Here,we show that supporting cells,which surround hair cells in the normal cochlear epithelium,differentiate into new hair cells in the neonatal mouse following ototoxic damage. Using lineage tracing,we show that new hair cells,predominantly outer hair cells,arise from Lgr5-expressing inner pillar and third Deiters cells and that new hair cell generation is increased by pharmacological inhibition of Notch. These data suggest that the neonatal mammalian cochlea has some capacity for hair cell regeneration following damage alone and that Lgr5-positive cells act as hair cell progenitors in the cochlea. View Publication

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However,whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues,such as umbilical cord mesenchymal cells (UMCs),are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report,we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs),we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly,we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay,reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system,in T cell co-culture system as well. Furthermore,through whole genome expression microarray analysis,we showed that over 70 immune genes,including all members of HLA-I,were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation,thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine. View Publication

Although it is well established that women with germ-line mutations in the BRCA1 gene have a greatly increased lifetime incidence of breast and ovarian cancer,the molecular mechanisms responsible for this tissue-specific carcinogenesis remain undefined. The majority of these breast cancers are of the basal-like phenotype characterized by lack of expression of ER,PR,and ERBB2. Because this phenotype has been proposed to resemble that of normal breast stem cells,we examined the role of BRCA1 in human mammary stem cell fate. Using both in vitro systems and a humanized NOD/SCID mouse model,we demonstrate that BRCA1 expression is required for the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells displaying the stem/progenitor cell marker ALDH1 and a decrease in cells expressing luminal epithelial markers and estrogen receptor. In breast tissues from women with germ-line BRCA1 mutations,but not normal controls,we detect entire lobules that,although histologically normal,are positive for ALDH1 expression but are negative for the expression of ER. Loss of heterozygosity for BRCA1 was documented in these ALDH1-positive lobules but not in adjacent ALDH1-negative lobules. Taken together,these studies demonstrate that BRCA1 plays a critical role in the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Because BRCA1 also plays a role in DNA repair,our work suggests that loss of BRCA1 may result in the accumulation of genetically unstable breast stem cells,providing prime targets for further carcinogenic events. View Publication

Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease,such as Parkinson's disease (PD),in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis,release,and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2,henceforth referred to as GIRK2),representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (textless10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites,and in the soma. Finally,neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models. View Publication

A human invitro cardiac tissue model would be a significant advancement for understanding,studying,and developing new strategies for treating cardiac arrhythmias and related cardiovascular diseases. We developed an invitro model of three-dimensional (3D) human cardiac tissue by populating synthetic filamentous matrices with cardiomyocytes derived from healthy wild-type volunteer (WT) and patient-specific long QT syndrome type 3 (LQT3) induced pluripotent stem cells (iPS-CMs) to mimic the condensed and aligned human ventricular myocardium. Using such a highly controllable cardiac model,we studied the contractility malfunctions associated with the electrophysiological consequences of LQT3 and their response to a panel of drugs. By varying the stiffness of filamentous matrices,LQT3 iPS-CMs exhibited different level of contractility abnormality and susceptibility to drug-induced cardiotoxicity. textcopyright 2013 Elsevier Ltd. View Publication