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

Long non-coding RNAs (lncRNAs) are a numerous class of newly discovered genes in the human genome,which have been proposed to be key regulators of biological processes,including stem cell pluripotency and neurogenesis. However,at present very little functional characterization of lncRNAs in human differentiation has been carried out. In the present study,we address this using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. With a newly developed method,hESCs were robustly and efficiently differentiated into neurons,and we profiled the expression of thousands of lncRNAs using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified,and shown to physically interact with SOX2,and PRC2 complex component,SUZ12. Using a similar approach,we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis,and immunoprecipitation studies revealed physical association with REST and SUZ12. This study indicates that lncRNAs are important regulators of pluripotency and neurogenesis,and represents important evidence for an indispensable role of lncRNAs in human brain development. View Publication

Blood consists of different cell populations with distinct functions and correspondingly,distinct gene expression profiles. In this study,global miRNA expression profiling was performed across a panel of nine human immune cell subsets (neutrophils,eosinophils,monocytes,B cells,NK cells,CD4 T cells,CD8 T cells,mDCs and pDCs) to identify cell-type specific miRNAs. mRNA expression profiling was performed on the same samples to determine if miRNAs specific to certain cell types down-regulated expression levels of their target genes. Six cell-type specific miRNAs (miR-143; neutrophil specific,miR-125; T cells and neutrophil specific,miR-500; monocyte and pDC specific,miR-150; lymphoid cell specific,miR-652 and miR-223; both myeloid cell specific) were negatively correlated with expression of their predicted target genes. These results were further validated using an independent cohort where similar immune cell subsets were isolated and profiled for both miRNA and mRNA expression. miRNAs which negatively correlated with target gene expression in both cohorts were identified as candidates for miRNA/mRNA regulatory pairs and were used to construct a cell-type specific regulatory network. miRNA/mRNA pairs formed two distinct clusters in the network corresponding to myeloid (nine miRNAs) and lymphoid lineages (two miRNAs). Several myeloid specific miRNAs targeted common genes including ABL2,EIF4A2,EPC1 and INO80D; these common targets were enriched for genes involved in the regulation of gene expression (ptextless9.0E-7). Those miRNA might therefore have significant further effect on gene expression by repressing the expression of genes involved in transcriptional regulation. The miRNA and mRNA expression profiles reported in this study form a comprehensive transcriptome database of various human blood cells and serve as a valuable resource for elucidating the role of miRNA mediated regulation in the establishment of immune cell identity. View Publication

Neural differentiation of human embryonic (ES) and induced pluripotent (iPS) stem cell lines has been used for research in early human development,drug discovery,and cell replacement therapies. It is critical to establish generic differentiation protocols to compare the neural specification potential of each individually derived pluripotent stem cell line and identify the efficacious lines for research and therapeutic use. Here,we describe a reproducible and quantitative protocol to assess the neural progenitor (NP) generation of human pluripotent stem cell lines. This method includes a robust and well-defined neural inducing platform for Pax6(+) neural rosette (neuroectodermal cells) generation,propagation,and subsequent differentiation into nestin(+) NPs. A side-by-side comparison under common culture conditions among three human ES cell lines,TE03,TE06,and BG01V,and one iPS cell line,HD02,showed highly variable efficiency in their differentiation into NPs. View Publication

In pluripotent stem cell differentiation,embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here,we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 $$m diameter microarray wells by use of single cells,2D clusters,or 3D early aggregates were compared. We observe that 200 $$m EBs are monocystic versus 500 $$m multicystic EBs that contain macro,meso and microsized cysts. In adherent differentiation of 500 $$m EBs,the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation. View Publication

Human induced pluripotent stem cells (hiPSCs),like embryonic stem cells,are under intense investigation for novel approaches to model disease and for regenerative therapies. Here,we describe the derivation and characterization of hiPSCs from a variety of sources and show that,irrespective of origin or method of reprogramming,hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium,CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage,we were able to demonstrate by single cell analysis (flow cytometry),that hiPSC-derived erythroblasts express alpha globin as previously described,and that a sub-population of these erythroblasts also express haemoglobin F (HbF),indicative of fetal definitive erythropoiesis. More notably however,we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner,but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover,the HbA expressing erythroblast population could be greatly enhanced (44textperiodcentered0 ± 6textperiodcentered04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine. View Publication

Mammary alveologenesis is abrogated in the absence of the transcription factors STAT5A/5B,which mediate cytokine signaling. To reveal the underlying causes for this developmental block,we studied mammary stem and progenitor cells. While loss of STAT5A/5B did not affect the stem cell population and its ability to form mammary ducts,luminal progenitors were greatly reduced and unable to form alveoli during pregnancy. Temporally controlled expression of transgenic STAT5A in mammary epithelium lacking STAT5A/5B restored the luminal progenitor population and rescued alveologenesis in a reversible fashion in vivo. Thus,STAT5A is necessary and sufficient for the establishment of luminal progenitor cells. View Publication

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells,presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study,20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02),number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02),tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01),and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis. View Publication

High maternal blood glucose levels caused by diabetes mellitus can irreversibly lead to maldevelopment of the growing fetus with specific effects on the skeleton. To date,it remains controversial at which stage embryonic development is affected. Specifically during embryonic bone development,it is unclear whether diminished bone mineral density is caused by reduced osteoblast or rather enhanced osteoclast function. Therefore,the aim of this study was to characterize the growth as well as the skeletal differentiation capability of pluripotent embryonic stem cells (ESCs),which may serve as an in vitro model for all stages of embryonic development,when cultured in diabetic levels of D-glucose (4.5 g/L) versus physiological levels (1.0 g/L). Results showed that cells cultivated in physiological glucose gave rise to a higher number of colonies with an undifferentiated character as compared to cells grown in diabetic glucose concentrations. In contrast,these cultures were characterized by slightly decreased expression of proteins associated with the stem cell state. Furthermore,differentiation of ESCs into osteoblasts and osteoclasts was favored in physiological glucose concentrations,demonstrated by an increased matrix calcification,enhanced expression of cell-type-specific mRNAs,as well as activity of the cell-type-specific enzymes,alkaline,and tartrate resistant acidic phosphatase. In fact,this pattern was noted in murine as well as in primate ESCs. Our study suggests that an interplay between both the osteoblast and the osteoclast lineage is needed for proper skeletal development to occur,which seems impaired in hyperglycemic conditions. View Publication

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium,we generated a diverse,comprehensive,and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities,β-globin gene (HBB) haplotypes,and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development,model disease,and perhaps eventually,treat patients. Here,we describe this unique resource for the study of sickle cell disease,including novel haplotype-specific polymorphisms that affect disease severity,as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library,and as proof of principle for future cell- and gene-based therapies,we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation. View Publication

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors,but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue,we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct,which inhibits signaling through all Notch receptors,and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease,but not a complete abrogation,of these cells in dnMAML-expressing cells. Interestingly,when assessed in secondary assays in vitro or in vivo,there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool,which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population. View Publication

Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation therapy for various diseases with impaired organs. However,the low efficiency of iPSC derived from somatic cells (0.01-0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal,proliferation,and maintenance of embryonic stem cells (ESCs),but the contribution of this pathway or its well-known negative regulator,phosphatase,and tensin homolog deleted on chromosome ten (Pten),to somatic cell reprogramming remains largely unknown. Here,we show that activation of the PI3K pathway by the Pten inhibitor,dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate,improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs. View Publication