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

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media,attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component,as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces,we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives,and should be applicable to other reprogramming methods. View Publication

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors,where it initiates critical cell survival and proliferation signals after stimulation by its ligand,thrombopoietin (TPO). As a result,a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore,they show very low-level expression of Mpl on platelets and megakaryocytes,and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However,impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels,which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis. View Publication

More recently,reprogramming of somatic cells to an embryonic stem cell-like state presents a milestone in the realm of stem cells,making it possible to derive all cell types from any patients bearing specific genetic mutations. With the development of induced pluripotent stem (iPS) cells,we are now able to use the derivatives of iPS cells to study the mechanisms of disease and to perform drug screening and toxicology testing. In addition,differentiated iPS cells are now close to be used in clinical practice. Here we review the progress of iPS technique and the possible application in the area of Parkinson's disease treatment. View Publication

It is likely that arrhythmias should be avoided for therapies based on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CM) to be effective. Towards achieving this goal,we introduced light-activated channelrhodopsin-2 (ChR2),a cation channel activated with 480 nm light,into human embryonic stem cells (hESC). By using in vitro approaches,hESC-CM are able to be activated with light. ChR2 is stably transduced into undifferentiated hESC via a lentiviral vector. Via directed differentiation,hESCChR2-CM are produced and subjected to optical stimulation. hESCChR2-CM respond to traditional electrical stimulation and produce similar contractility features as their wild-type counterparts but only hESCChR2-CM can be activated by optical stimulation. Here it is shown that a light sensitive protein can enable in vitro optical control of hESC-CM and that this activation occurs optimally above specific light stimulation intensity and pulse width thresholds. For future therapy,in vivo optical stimulation along with optical inhibition could allow for acute synchronization of implanted hPSC-CM with patient cardiac rhythms. View Publication

We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However,a small number of regenerated myofibers were detected,suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally,we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore,we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells. View Publication

PURPOSE: Genz-644282 [8,9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one] has emerged as a promising candidate for antitumor agents. This report describes the bone marrow colony-forming unit,granulocyte macrophage (CFU-GM) and tumor cell CFU activity of topoisomerase I (Top1) inhibitors,such as Genz-644282,topotecan,irinotecan/SN-38,and ARC-111,and examines their activity in several human tumor xenograft models. EXPERIMENTAL DESIGN: Colony-forming assays were conducted with mouse and human bone marrow and eight human tumor cell lines. In addition,29 human tumor cell lines representing a range of histology and potential resistance mechanisms were assayed for sensitivity to Genz-644282 in a 72-hour exposure assay. The efficacy of Genz-644282 was compared with standard anticancer drugs (i.e.,irinotecan,docetaxel,and dacarbazine) in human tumor xenografts of colon cancer,renal cell carcinoma,non-small cell lung cancer,and melanoma. RESULTS: Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than was mouse bone marrow CFU-GM. The ratio of mouse to human IC(90) values was more than 10 for the camptothecins and less than 10 for Genz-644282,which had more potency as a cytotoxic agent toward human tumor cells in culture than the camptothecins in the colony-forming and 72-hour proliferation assays. Genz-644282 has superior or equal antitumor activity in the human tumor xenografts than the standard drug comparators. CONCLUSIONS: On the basis of preclinical activity and safety,Genz-644282 was selected for development and is currently undergoing phase 1 clinical trial. View Publication

The laminin receptor integrin alpha6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. We have studied its role for homing of stem and progenitor cells to mouse hematopoietic tissues in vivo. A function-blocking anti-integrin alpha6 antibody significantly reduced progenitor cell homing to bone marrow (BM) of lethally irradiated mice,with a corresponding retention of progenitors in blood. Remarkably,the anti-integrin alpha6 antibody profoundly inhibited BM homing of long-term multilineage engrafting stem cells,studied by competitive repopulation assay and analysis of donor-derived lymphocytes and myeloid cells in blood 16 weeks after transplantation. A similar profound inhibition of long-term stem cell homing was obtained by using a function-blocking antibody against alpha4 integrin,studied in parallel. Furthermore,the anti-integrin alpha6 and alpha4 antibodies synergistically inhibited homing of short-term repopulating stem cells. Intravenous injection of anti-integrin alpha6 antibodies,in contrast to antibodies against alpha4 integrin,did not mobilize progenitors or enhance cytokine-induced mobilization by G-CSF. Our results provide the first evidence for a distinct functional role of integrin alpha6 receptor during hematopoietic stem and progenitor cell homing and collaboration of alpha6 integrin with alpha4 integrin receptors during homing of short-term stem cells. View Publication

Erythropoiesis,the essential process of hematopoietic stem cell development into erythrocytes,is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver,neonatal spleen,and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen,short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype,demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation,including hemoglobinization,cell condensation,and enucleation,and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells. View Publication