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

Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17),which are able to indirectly induce the recruitment of neutrophils. Recently,human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors,including CCR2 and CCR6. Herein,we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines,the known ligands of CCR2 and CCR6,respectively. Accordingly,supernatants from activated neutrophils induced chemotaxis of Th17 cells,which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment,neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally,we report that,although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor,tumor necrosis factor-alpha,and interferon-gamma release,these latter cells cannot be activated by IL-17A and IL-17F,because of their lack of IL-17RC expression. Collectively,our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells,which may represent a useful target for the treatment of chronic inflammatory diseases. View Publication

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However,the current conventional method leads to poor recovery of hPSCs after thawing. Here,we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze-thawing,we found that hPSCs that were freeze-thawed as colonies showed markedly decreased survival,whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover,the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs. View Publication

H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK),myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1),significantly increases cell survival and colony formation,and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. View Publication

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC),a characteristic of polycythemia vera and essential thrombocythemia,is not standardized. In this multicentric study,we tested four semisolid,serum-free,cytokine-free media based on either methylcellulose (M1,M2) or collagen (C1,C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26),essential thrombocythemia (19),secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without,or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific,as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia,nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria,respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo,the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay,now part of the new criteria of polycythemia vera. View Publication

Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells,its physiological function(s) remain unknown,and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations,followed by competitive bone marrow reconstitution assays,shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent,the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore,PrP is a marker for HSCs and supports their self-renewal. View Publication

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals,thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide,the use of human cell-teratogenicity assays has just started to be explored. Herein,a human pluripotent stem cell test (hPST) for identifying teratogens is described,benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically,71 drug-like compounds with known in vivo effects,including thalidomide,were examined in the hPST. A threshold of 5μM demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore,15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally,to assess the suitability of the hPST for high-throughput screens,a small library of 300 kinase inhibitors was tested,demonstrating the hPST platform's utility for interrogating teratogenic mechanisms and drug safety prediction. Thus,the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models. View Publication

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH,however,lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore,other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs),characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming,and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation,the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients. View Publication

The use of human pluripotent stem cells,including embryonic and induced pluripotent stem cells,in therapeutic applications will require the development of robust,scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs),but challenges specific to hESCs will also have to be addressed,including development of defined,humanized culture media and substrates,monitoring spontaneous differentiation and heterogeneity in the cultures,and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. View Publication

Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused. Anticipated novel therapeutic implementations of hematopoietic stem cells (HSCs) and their derivatives further increase interest in strategies to expand HSCs ex vivo. A fundamental limitation in all HSC-driven culture systems is the rapid generation of differentiating cells and their secreted inhibitory feedback signals. Herein we describe an integrated computational and experimental strategy that enables a tunable reduction in the global levels and impact of paracrine signaling factors in an automated closed-system process by employing a controlled fed-batch media dilution approach. Application of this system to human cord blood cells yielded a rapid (12-day) 11-fold increase of HSCs with self-renewing,multilineage repopulating ability. These results highlight the marked improvements that control of feedback signaling can offer primary stem cell culture and demonstrate a clinically relevant rapid and relatively low culture volume strategy for ex vivo HSC expansion. View Publication