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

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here,we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes,including tal1/scl,gata1,gata2,hoxB4,and lhx2,into CM ESCs. By immunochemical and morphological analyses,we demonstrated that overexpression of tal1/scl,but not the remaining genes,dramatically increased hematopoiesis of CM ESCs,resulting in multiple blood-cell lineages. Furthermore,flow cytometric analysis demonstrated that CD34,a hematopoietic stem/progenitor cell marker,was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells. View Publication

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens,including live cells,without the need for chemi-selective stains. Using a microspectrometer,near-infrared Raman spectra throughout the range 663 cm(-1) to 1220 cm(-1) were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly,compared to proteins and lipids,the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus,we could identify intensity ratios of particular protein-related bands (e.g.,757 cm(-1) tryptophan) to nucleic acid bands (784 cm(-1) DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology,proliferation,or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid,noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the differentiation status of hESCs. View Publication

Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1-4),Neu1,Neu3,and Neu4 are expressed in human monocytes,but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes,but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir,a pharmacologic inhibitor of Neu3 but not Neu1,or DANA,an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6,IL-12p40,and TNF-α by mature DCs,demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1�?�/�?� and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production,thereby contributing to development of adaptive immune responses. View Publication

The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome,resulting in altered patterns of gene expression. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these,we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells,suggesting that their activation may promote the emergence of iPSCs. Supporting this,our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches,we found that one such lincRNA (lincRNA-RoR) modulates reprogramming,thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells. View Publication

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine,drug discovery,and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free,virus-free,serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency,offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study,we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research,facilitating future applications of human iPS cells. View Publication

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition,recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL,classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore,the murine WEHI-3B(D+) cells and human leukemic cells classified as M2,M3,and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600. View Publication

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication

Conventional methods for quantification of undifferentiated pluripotent stem cells such as fluorescence-activated cell sorting and real-time PCR analysis have technical limitations in terms of their sensitivity and recyclability. Herein,we designed a real-time in situ label-free monitoring system on the basis of a specific electrochemical signature of human pluripotent stem cells in vitro. The intensity of the signal of hPSCs highly corresponded to the cell number and remained consistent in a mixed population with differentiated cells. The electrical charge used for monitoring did not markedly affect the proliferation rate or molecular characteristics of differentiated human aortic smooth muscle cells. After YM155 treatment to ablate undifferentiated hPSCs,their specific signal was significantly reduced. This suggests that detection of the specific electrochemical signature of hPSCs would be a valid approach to monitor potential contamination of undifferentiated hPSCs,which can assess the risk of teratoma formation efficiently and economically. View Publication

BackgroundCHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders,including CHARGE (Coloboma,Heart defects,Atresia of the choanae,Retardation of growth and development,Genital hypoplasia,and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus,liver,pancreas,and intestine.MethodsIn this study,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7?/?),heterozygous mutant (CHD7+/?),and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers,mesoderm and ectoderm in vitro. We also compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq).ResultsWe found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. Loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.ConclusionsCHD7 is critical for DE and mesodermal development from hESCs. Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04437-9. View Publication

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However,we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis,particularly in the late stage,where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus,we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge,the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this,we generated inducible TRIB3 overexpression hESCs,named TRIB3tet-on OE H9,based on a Tet-On system. Then,we analyzed hemoglobin expression,condensed chromosomes,organelle clearance,and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry,enhanced hemoglobin and related protein expression in Western blot,decreased nuclear area size,promoted enucleation rate,decreased lysosome and mitochondria number,more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 ?g/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore,our study delineates the role of TRIB3 in terminal erythropoiesis,and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin. Highlights•TRIB3 boosts erythroid cell maturation.•Key insights into erythropoiesis from hESCs.•Enhanced ubiquitin-proteasome system and downstream mitophagy in erythroid differentiation. View Publication

Chromatin regulation is critical for differentiation and disease. However,features linking the chromatin environment of stem cells with disease remain largely unknown. We explored chromatin accessibility in embryonic and multipotent stem cells and unexpectedly identified widespread chromatin accessibility at repetitive elements. Integrating genomic and biochemical approaches,we demonstrate that these sites of increased accessibility are associated with well-positioned nucleosomes marked by distinct histone modifications. Differentiation is accompanied by chromatin remodeling at repetitive elements associated with altered expression of genes in relevant developmental pathways. Remarkably,we found that the chromatin environment of Ewing sarcoma,a mesenchymally derived tumor,is shared with primary mesenchymal stem cells (MSCs). Accessibility at repetitive elements in MSCs offers a permissive environment that is exploited by the critical oncogene responsible for this cancer. Our data demonstrate that stem cells harbor a unique chromatin landscape characterized by accessibility at repetitive elements,a feature associated with differentiation and oncogenesis. View Publication