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

We report that a single growth factor,NM23-H1,enables serial passaging of both human ES and iPS cells in the absence of feeder cells,their conditioned media or bFGF in a fully defined xeno-free media on a novel defined,xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1� View Publication

Cardiovascular progenitor cells (CVPCs) derived from human pluripotent stem cells (hPSCs),including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs),hold great promise for the study of cardiovascular development and cell-based therapy of heart diseases,but their applications are challenged by the difficulties in their efficient generation and stable maintenance. This study aims to develop chemically defined systems for robust generation and stable propagation of hPSC-derived CVPCs by modulating the key early developmental pathways involved in human cardiovascular specification and CVPC self-renewal. Herein we report that a combination of bone morphogenetic protein 4 (BMP4),glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 and ascorbic acid is sufficient to rapidly convert monolayer-cultured hPSCs,including hESCs and hiPSCs,into homogeneous CVPCs in a chemically defined medium under feeder- and serum-free culture conditions. These CVPCs stably self-renewed under feeder- and serum-free conditions and expanded over 10(7)-fold when the differentiation-inducing signals from BMP,GSK3 and Activin/Nodal pathways were simultaneously eliminated. Furthermore,these CVPCs exhibited expected genome-wide molecular features of CVPCs,retained potentials to generate major cardiovascular lineages including cardiomyocytes,smooth muscle cells and endothelial cells in vitro,and were non-tumorigenic in vivo. Altogether,the established systems reported here permit efficient generation and stable maintenance of hPSC-derived CVPCs,which represent a powerful tool to study early embryonic cardiovascular development and provide a potentially safe source of cells for myocardial regenerative medicine. View Publication

Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells,but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC₅₀ = ˜15 ng/mL) by inducing apoptosis,and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations,BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes,such as glucose-regulated protein 78 (GRP78),X-box binding protein 1 (XBP1),and C/EBP homologous protein (CHOP). Finally,we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer. View Publication

Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions,where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh),whose posttranslational lipid modifications and assembly into multimers enhance its biological potency,potentially through receptor clustering. Investigations of Shh typically utilize recombinant,monomeric protein,and thus the impact of multivalency on ligand potency is unexplored. Among its many activities,Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies,compared to the monomeric Shh,increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%,respectively. Thus,multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients. View Publication

Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance,but they can also play a detrimental role by preventing antitumor responses. Here,we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution,focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently,Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall,our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival,higher migratory capacity,and selective T-effector suppressive ability. View Publication

The novel immunosuppressant FTY720 activates sphingosine 1-phosphate receptors (S1PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor 1 (SDF-1),resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-1 and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs),we analyzed expression of S1PRs and the influence of FTY720 on SDF-1/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR),S1PRs were expressed in mobilized CD34+ HPCs,particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-1-induced calcium mobilization and actin polymerization,and substantially increased SDF-1-dependent in vitro transendothelial migration,without affecting VLA-4,VLA-5,and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice,the number of CD34+/CD38- cells that homed to the BM after 18 hours was significantly raised by pretreatment of animals and cells with FTY720,tending to result in improved engraftment. In addition,in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S1PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo,supporting homing and proliferation of HPCs. In the hematopoietic microenvironment,S1PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-1. View Publication

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically,hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and,in the long-term,stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and,more importantly,developmental stage-specific differentiation propensity. Here,we report synergistic inhibition of glycogen synthase kinase 3 (GSK3),transforming growth factor β (TGF-β),and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor,GSK3 inhibitor (CHIR99021),and TGF-β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs. View Publication

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (mushroom alcohol")� View Publication

Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions,but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here,we show that even in the presence of soluble pluripotency factors,compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors,the effective substrata produce neurons rapidly (2 wk) and more efficiently (textgreater75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type,independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound. View Publication

Terahertz (THz) radiation was proposed recently for use in various applications,including medical imaging and security scanners. However,there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli,and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes,which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure. View Publication

Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such,they hold promise for use in stem cell-based therapies. However,no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously,we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that $$2-6-sialylation is a marker of the differentiation potential of these cells. Nevertheless,no information was available about the structural details of these of $$2-6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs),human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of $$2-6-sialylated N-glycans was detected in early passage cells (24-28 % of sialylated N-glycans) compared with late passage cells (13-15 %). A major $$2-6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes,Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast,no significant differences were observed between early and late passage hMSCs with respect to $$2-6-sialylated O-glycan percentages. These results demonstrate that levels of $$2-6-sialylated N-glycans,but not O-glycans,could be used as markers of the differential potential of hMSCs. View Publication

Human embryonic stem (hES) cells have enormous potential for clinical applications. However,one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis,which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased,F-actin content and distribution is altered,and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However,exactly how the extrinsic pathway is activated is still unclear and deserves further investigation. View Publication