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

Integrating viruses represent robust tools for cellular reprogramming; however,the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here,we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage,we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple,rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling,tissue engineering and cell replacement therapies. View Publication

BACKGROUND Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver,lungs,thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1,2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells. MATERIALS AND METHODS Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry,real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG),definitive endoderm (SOX17,CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures. RESULTS Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG,accompanied by an increase expression of the DE genes SOX17,CXCR4 and GATA4. Importantly,the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment. View Publication

Umbilical cord blood (CB) is a valuable source of stem cells for transplantation,but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)-derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this,we cultured CB- and PB-CD34(+) cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo,and the addition of CM inhibited their maturation. In contrast,CB-MKs reached highest ploidy in CM without rTpo,and high rTpo concentrations (textgreater 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines. View Publication

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that,in most transplantation patients,variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype,as well as through phenotypic and functional analyses of NK cells,both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells,including patient leukemia blasts. Differently,KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly,this was due to recognition of C2 by KIR2DL2/3,as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably,however,C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether,these results may have important clinical implications for the selection of optimal donors for haplo-HSCT. View Publication

Abnormal trophoblast self-renewal and differentiation during early gestation is the major cause of miscarriage,yet the underlying regulatory mechanisms remain elusive. Here,we show that trophoblast specific deletion of Kat8,a MYST family histone acetyltransferase,leads to extraembryonic ectoderm abnormalities and embryonic lethality. Employing RNA-seq and CUT&Tag analyses on trophoblast stem cells (TSCs),we further discover that KAT8 regulates the transcriptional activation of the trophoblast stemness marker,CDX2,via acetylating H4K16. Remarkably,CDX2 overexpression partially rescues the defects arising from Kat8 knockout. Moreover,increasing H4K16ac via using deacetylase SIRT1 inhibitor,EX527,restores CDX2 levels and promoted placental development. Clinical analysis shows reduced KAT8,CDX2 and H4K16ac expression are associated with recurrent pregnancy loss (RPL). Trophoblast organoids derived from these patients exhibit impaired TSC self-renewal and growth,which are significantly ameliorated with EX527 treatment. These findings suggest the therapeutic potential of targeting the KAT8-H4K16ac-CDX2 axis for mitigating RPL,shedding light on early gestational abnormalities. Embryo implantation failure is a leading cause of miscarriage,though the mechanisms underlying trophoblast defects are not well understood. Here they show that the histone acetyltransferase KAT8 is essential for proper activation of the trophoblast stemness gene CDX2,and that placental development can be partially rescued by inhibiting histone deacetylase activity. View Publication

Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP,or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk,thus potentially offering a new target for breast cancer treatment. We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration,invasion,and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice,Balb/c mice,and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity,we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. Herein,we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone,named 12G2,which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth,was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions,16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases. The online version contains supplementary material available at 10.1186/s13058-024-01873-y. View Publication

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important,given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here,we present an in-depth quantitative mass spectrometry-based analysis of hESCs,two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless,a small group of 58 proteins,mainly related to metabolism,antigen processing and cell adhesion,was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap,highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. View Publication

Human embryonic stem cell (hESC)-based assay systems and genetically modified hESCs are very useful tools for screening drugs that regulate stemness and differentiation and for studying the molecular mechanisms involved in hESC fate determination. For these types of studies,feeder cell-dependent cultures of hESCs are often problematic because the physiology of the feeder cells is perturbed by the drug treatments or genetic modifications,which potentially obscures research outcomes. In this study,we evaluated three commonly used feeder-free culture conditions to determine whether they supported the undifferentiated growth of hESCs and to determine whether the hESCs grown in these conditions displayed gene expression patterns that were similar to the expression patterns of feeder cell-dependent hESCs. Our results demonstrate that hESCs grown in the three feeder-free conditions expressed undifferentiation marker genes as strongly as hESCs that were grown in the feeder-dependent cultures. Furthermore,genome-wide gene expression profiles indicated that the gene expression patterns of hESCs that were grown under feeder-free or feeder-dependent culture conditions were highly similar. These results indicate that the feeder-free culture conditions support the undifferentiated growth of hESCs as effectively as the feeder-dependent culture conditions. Therefore,feeder-free culture conditions are potentially suitable for drug screening and for the genetic manipulation of hESCs in basic research. View Publication

Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation,microcephaly,and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4),Artemis,and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders,we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity,but not Artemis deficiency,were associated with markedly reduced reprogramming efficiency,which could be partially rescued by genetic complementation. Moreover,we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest,particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-,but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming,with a major role for LIG4 and DNA-PKcs and a minor,if any,for Artemis. View Publication

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture,the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study,we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells,including cardiomyocytes and fibroblasts,using a methionine-free culture condition. When cultured in the methionine-free condition,human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition,gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore,in fabricated cardiac cell sheets,spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination. View Publication

The recent development of induced pluripotent stem cells (iPSCs) by ectopic expression of defined reprogramming factors offers enormous therapeutic opportunity. To deliver these factors,murine leukemia virus (MLV)-based vectors have been broadly used in the setting of hematopoietic stem cell transplantation. However,MLV vectors have been implicated in malignancy induced by insertional mutagenesis,whereas lentiviral vectors have not. Furthermore,the infectivity of MLV vectors is limited to dividing cells,whereas lentiviral vectors can also transduce nondividing cells. One important characteristic of MLV vectors is a self-silencing property of the promoter element in pluripotent stem cells,allowing temporal transgene expression in a nonpluripotent state before iPSC derivation. Here we test iPSC generation using a novel chimeric vector carrying a mutant MLV promoter internal to a lentiviral vector backbone,thereby containing the useful properties of both types of vectors. Transgene expression of this chimeric vector was highly efficient compared with that of MLV vectors and was silenced specifically in human embryonic stem cells. Human fetal fibroblasts transduced with the vector encoding each factor were efficiently reprogrammed into a pluripotent state,and these iPSCs had potential to differentiate into a variety of cell types. To explore the possibility of iPSCs for gene therapy,we established iPSC clones expressing a short hairpin RNA (shRNA) targeting chemokine receptor 5 (CCR5),the main coreceptor for HIV-1. Using a reporter construct for CCR5 expression,we confirmed that CCR5 shRNA was expressed and specifically knocked down the reporter expression in iPSCs. These data indicate that our chimeric lentiviral vector is a valuable tool for generation of iPSCs and the combination with vectors encoding transgenes allows for rapid establishment of desired genetically engineered iPSC lines. View Publication