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

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population,broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study,we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach,we identified two endoderm-specific antibodies,HDE1 and HDE2,which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential,whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination,the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line. View Publication

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties,Sca-1 expression (Sca-1+),lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-),and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in textgreater or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients,respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor,interleukin-6 (IL-6),and erythropoietin (with or without IL-3),a large increase in total cell number,including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly,this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo,as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture,providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype. View Publication

Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers,yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development,we have examined the role of TGFbeta superfamily signaling in hESCs. We found that,in undifferentiated cells,the TGFbeta/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation,SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGFbeta/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling,which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly,we show that in ex vivo mouse blastocyst cultures,SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFbeta signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFbeta and WNT signaling in these contexts. View Publication

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched,full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell,HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units,CD56+CD3- placenta-derived NK cells,termed pNK cells,were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were textgreater80% CD56+CD3-,comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D,NKp46,and NKp44 (p textless 0.001,p textless 0.001,and p textless 0.05,respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile,immunophenotype,and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development,pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options. View Publication

Introduction: Human embryonic stem cells (hESC) provide an invaluable model for assessing the effects of environmental chemicals and drugs on human prenatal development. However,hESC are difficult to adapt to 96-well plate screening assays,because they survive best when plated as colonies,which are difficult to count and plate accurately. The purpose of this study is to present an experimental method and analysis procedure to accomplish reliable screening of toxicants using hESC. Methods: We present a method developed to rapidly and easily determine the number of cells in small colonies of hESC spectrophotometerically and then accurately dispense equivalent numbers of cells in 96-well plates. The MTT assay was used to evaluate plating accuracy,and the method was tested using known toxicants. Results: The quality of the plate set-up and analysis procedure was evaluated with NIH plate validation and assessment software. All statistical parameters measured by the software were acceptable,and no drift or edge effects were observed. The 96-well plate MTT assay with hESC was tested by performing a dose-response screen of commercial products,which contain a variety of chemicals. The screen was done using single wells/dose,and the reliability of this method was demonstrated in a subsequent screen of the same products repeated three times. The single and triple screens were in good agreement,and NOAELs and IC50s could be determined from the single screen. The effects of vapor from volatile chemicals were studied,and methods to monitor and avoid vapor effects were incorporated into the assay. Discussion: Our method overcomes the difficulty of using hESC for reliable quantitative 96-well plate assays. It enables rapid dose-response screening using equipment that is commonly available in laboratories that culture hESC. This method could have a broad application in studies of environmental chemicals and drugs using hESC as models of prenatal development. ?? 2012 Elsevier Inc. View Publication

Myocardial infarction is a leading cause of mortality and morbidity worldwide,and current treatments fail to address the underlying scarring and cell loss,which is a major cause of heart failure after infarction. The novel strategy,therapeutic angiogenesis and/or vasculogenesis with endothelial progenitor cells transplantation holds great promise to increase blood flow in ischemic areas,thus rebuild the injured heart and reverse the heart failure. Given the potential of self-renewal and differentiation into virtually all cell types,human embryonic stem cells (hESCs) may provide an alternate source of therapeutic cells by allowing the derivation of large numbers of endothelial cells for therapeutic angiogenesis and/or vasculogenesis of ischemic heart diseases. Moreover,to fully understand the fate of implanted hESCs or hESC derivatives,investigators need to monitor the motility of cells in living animals over time. In this chapter,we describe the application of bioluminescence reporter gene imaging to track the transplanted hESC-derived endothelial cells for treatment of myocardial infarction. The technology of inducing endothelial cells from hESCs will also be discussed. View Publication

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV,purified HPCs were exposed to KSHV,and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells,and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection,persistence,and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo. View Publication

The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However,previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs),but up-regulated as a consequence of their commitment and differentiation,suggesting that progenitors or differentiated blood cells,rather than HSCs,are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas,but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status. With the use of recently developed in vitro conditions promoting HSC self-renewing divisions,Fas was up-regulated on virtually all HSCs capable of multilineage reconstituting nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice in vivo,as well as on long-term culture-initiating cells (LTC-ICs). Similarly,in vivo cycling of NOD-SCID repopulating cells upon transplantation,resulted in up-regulation of Fas expression. However,repopulating HSCs expressing high levels of Fas remained highly resistant to Fas-mediated suppression,and HSC function was compromised only upon coactivation with tumor necrosis factor. Thus,reconstituting human HSCs up-regulate Fas expression upon active cycling,demonstrating that HSCs could be targets for Fas-mediated BM suppression. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. View Publication