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

Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells,the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease - dyslipidemia,insulin resistance,hypoglycemia,lipodystrophy,motor-neuron death,and hepatitis C infection. We found little evidence of TALEN off-target effects,but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines,we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease. textcopyright 2013 Elsevier Inc. View Publication

Realization of the full potential of human induced pluripotent stem cells (hiPSCs) in clinical applications requires development of well-defined conditions for their growth and differentiation. A novel fully defined polyvinyl alcohol/hyaluronan (PVA/HA) polysaccharide nanofiber was developed for hiPSCs culture in commercially available xeno-free,chemically defined medium. Vitronectin peptide (VP) was immobilized to PVA/HA nanofibers through NHS/EDC chemistry. The hiPSCs successfully grew and proliferated on the VP-decorated PVA/HA nanofibers,similar to those on MatrigelTM. Such well-defined,xeno-free and safe nanofiber substrate that supports culture of hiPSCs will not only help to accelerate the translational perspectives of hiPSCs,but also provide a platform to investigate the cell-nanofiber interaction mechanisms that regulate stem cell proliferation and differentiation. ?? 2013 Elsevier Ltd. All rights reserved. View Publication

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation,and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently,Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively,and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein,and by using oligonucleotides as probes,have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore,Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells. View Publication

AbstractThe landscape of cancer treatment has been transformed by immune checkpoint inhibitors; however,the failure to benefit a large number of patients with cancer has underlined the need to identify promising targets for more effective interventions. In this study,we leverage 23andMe,Inc.’s large-scale human germline genetic and health database to uncover the previously unknown role of UL16-binding protein 6 (ULBP6),a high-affinity NK group 2D (NKG2D) ligand,in cancer and its promise as an immuno-oncology therapeutic target. We confirm ULBP6 expression in human tumors and demonstrate that soluble ULBP6 shed from tumors circumvents NKG2D activation provided by membrane-anchored NKG2D ligands to inhibit immune cell activation and tumor cell killing. Based on these findings,we developed 23ME-01473,a humanized Fc effector–enhanced antibody that binds to ULBP6 and its closely related family members,ULBP2 and ULBP5. 23ME-01473 effectively blocks soluble ULBP6-mediated immunosuppression to restore the NKG2D axis on NK and T cells to elicit tumor growth control. Moreover,the Fc effector–enhanced design of 23ME-01473 increases its binding affinity to fragment crystallizable gamma receptor IIIa,which,together with 23ME-01473’s binding to membrane-anchored ULBP6/2/5 on cancer cells,allows for augmented antibody-dependent cellular cytotoxicity induction,providing a second activation node for NK cells. Our studies demonstrate the therapeutic potential of an Fc effector–enhanced anti-ULBP6/2/5 antibody to reinvigorate NK cell and T-cell activation and cytotoxicity for the treatment of cancer.Significance:This study emphasizes the utility of population-based genome-wide assessments for discovering naturally occurring genetic variants associated with lifetime risks for cancer or immune diseases as novel drug targets. We identify ULBP6 as a potential keystone member of the NKG2D pathway,which is important for antitumor immunity. Targeting ULBP6 may hold therapeutic promise for patients with cancer. View Publication

Acute myelogenous leukemia 1 (AML1; runt-related transcription factor 1 [Runx1]) is a member of Runx transcription factors and is essential for definitive hematopoiesis. Although AML1 possesses several subdomains of defined biochemical functions,the physiologic relevance of each subdomain to hematopoietic development has been poorly understood. Recently,the consequence of carboxy-terminal truncation in AML1 was analyzed by the hematopoietic rescue assay of AML1-deficient mouse embryonic stem cells using the gene knock-in approach. Nonetheless,a role for specific internal domains,as well as for mutations found in a human disease,of AML1 remains to be elucidated. In this study,we established an experimental system to efficiently evaluate the hematopoietic potential of AML1 using a coculture system of the murine embryonic para-aortic splanchnopleural (P-Sp) region with a stromal cell line,OP9. In this system,the hematopoietic defect of AML1-deficient P-Sp can be rescued by expressing AML1 with retroviral infection. By analysis of AML1 mutants,we demonstrated that the hematopoietic potential of AML1 was closely related to its transcriptional activity. Furthermore,we showed that other Runx transcription factors,Runx2/AML3 or Runx3/AML2,could rescue the hematopoietic defect of AML1-deficient P-Sp. Thus,this experimental system will become a valuable tool to analyze the physiologic function and domain contribution of Runx proteins in hematopoiesis. View Publication

Chimerism testing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represents a promising tool for predicting disease relapse,although its precise role in this setting remains unclear. We investigated the predictive value of T lymphocyte chimerism analysis at 90 to 120 days after allo-HSCT in 378 patients with AML/MDS who underwent busulfan/fludarabine-based myeloablative preparative regimens. Of 265 (70%) patients with available T lymphocyte chimerism data,43% of patients in first or second complete remission (CR1/CR2) at the time of transplantation had complete (100%) donor T lymphocytes at day +90 to +120 compared with 60% of patients in the non-CR1/CR2 cohort (P = .005). In CR1/CR2 patients,donor T lymphocyte chimerism ?85% at day +90 to +120 was associated with a higher frequency of 3-year disease progression (29%; 95% confidence interval [CI],18% to 46% versus 15%; 95% CI,9% to 23%; hazard ratio [HR],2.1; P = .04). However,in the more advanced,non-CR1/CR2 cohort,mixed T lymphocyte chimerism was not associated with relapse (37%; 95% CI,20% to 66% versus 34%; 95% CI,25% to 47%; HR,1.3; P = .60). These findings demonstrate that early T lymphocyte chimerism testing at day +90 to +120 is a useful approach for predicting AML/MDS disease recurrence in patients in CR1/CR2 at the time of transplantation. View Publication

Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work,the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa,cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected,the easy phenotypical identification of colonies in stained gels,and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures. View Publication

To investigate the influence of antibody structure and specificity on antibody efficacy against Streptococcus pneumoniae,human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures,epitope specificities,and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7,1A2,and 7C5) revealed that they use two different V(H)3 genes (A7 and 1A2 both use V3-15) and three different V(kappa) gene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the V(H)-D junction,whereas 1A2 had a threonine. Challenge experiments with serotype 3 S. pneumoniae in BALB/c mice revealed that both 10- and 1- micro g doses of A7 and 7C5 were protective,while only a 10- micro g dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB(-/-)) or classical (C4(-/-)) complement pathway,but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure,specificity,and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy. View Publication

Type 1 interferon-producing cells (IPCs),also known as plasmacytoid dendritic cell (DC) precursors,represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection,autoimmune SLE,and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3),IL-7,stem cell factor (SCF),macrophage-colony-stimulating factor (M-CSF),and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture,they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L,inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 x 10(6) IPCs per 10(6) CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-alpha (IFN-alpha) and to differentiate into mature DCs. This culture system,combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF-mobilized donors,permits the generation of more than 10(9) IPCs from a single blood donor. View Publication

OBJECTIVE: We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS: After single cell deposition onto 96-well plates,cells were cocultured for 10 days with the stem cell supporting cell line AFT024,fibronectin (FN),or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS: Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times,a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS: For the first time,we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC),this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential. View Publication

Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However,optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs,including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20,they remain highly useful. In particular,CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus,108 CA1S cells can be generated within one week from 106 cells to seed 106 wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm,a finding that was confirmed with H1 hESCs,although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines,these cells should ease the development of optimized hESC growth and differentiation protocols. In particular,they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses. Biotechnol. Bioeng. 2013;110: 2706–2716. textcopyright 2013 Wiley Periodicals,Inc. View Publication