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

BACKGROUND As one of the least studied bone morphogenetic proteins (BMPs),BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development,differentiation and bone metabolism. In this study,we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs). METHODOLOGY/PRINCIPAL FINDINGS Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9,RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker,such as alkaline phosphatase (ALP),and late osteogenic markers,such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants,we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation. CONCLUSION/SIGNIFICANCE Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore,a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects,non-union fracture,and/or osteoporotic fracture. View Publication

Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive,undifferentiated cells,capable of self-renewal and with the ability to give rise to different cell lineages,including adipocytes,osteocytes,fibroblasts,chondrocytes,and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies,including some from our own group,suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However,in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules,thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study,we have focused on the biological characterization of MSC from MDS. As a first approach,we have quantified their numbers in bone marrow,and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology,as well as the expression of certain cell markers,no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases,MSC expressed CD29,CD90,CD105 and Prolyl-4-hydroxylase; in contrast,they did not express CD14,CD34,CD68,or alkaline phosphatase. Interestingly,in five out of nine MDS patients,MSC developed in culture showed cytogenetic abnormalities,usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly,in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge,the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients,MSC are cytogenetically abnormal. Although the reason of this is still unclear,such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC. View Publication

CONTEXT: Endothelial dysfunction seems to be the first step of the atherosclerotic process. In the past few years,it has been demonstrated that injured endothelial monolayer is restored by a premature pool of circulating progenitor cells (PCs) and a more mature one of circulating endothelial PCs (EPCs). Even though there is increasing evidence that estrogens play a beneficial role on EPCs and,even if debated,on the cardiovascular system,less is known about androgens. OBJECTIVE: Our objective was to evaluate the levels of circulating PCs and EPCs in men with hypogonadotropic hypogonadism (HH) and the effect of prolonged testosterone (T) replacement therapy on these cells. DESIGN AND SETTING: We conducted a prospective study on males with HH at a university andrological center. PATIENTS: The study included 10 young HH patients (28.6 +/- 3.1 yr) and 25 age-matched controls. INTERVENTIONS: Idiopathic HH patients were treated with T gel therapy,50 mg/d for 6 months. MAIN OUTCOME MEASURES: We assessed circulating PC and EPC concentrations and immunocytochemistry for androgen receptor expression on cultured EPCs. RESULTS: At baseline,HH patients showed a significant reduction of both PCs and EPCs with respect to controls. T replacement therapy induced a significant increase of these cells with respect to baseline. Immunocytochemistry on cultured EPCs showed strong expression of the androgen receptor. CONCLUSIONS: Hypotestosteronemia is associated with a low number of circulating PCs and EPCs in young HH subjects. T treatment is able to induce an increase in these cells through a possible direct effect on the bone marrow. View Publication

BACKGROUND Although studies have shown that Oct4,Sox2,Klf4,and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs,the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors,including us,have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein,we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells. METHODS Among three miRNAs (miR-200c,miR-302s,and miR-369s) that were previously presented for miRNA-mediated reprogramming,miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302,the cells were incubated in ES medium for 3 weeks and then characterized. RESULTS iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression,low proliferation,high apoptosis,low epithelial-mesenchymal transition marker expression (N-cadherin,TGFBR2),and sensitization to drugs. Several miRNAs were changed (e.g.,low oncomiR miR-21,high miR-29b). cMyc was decreased,and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP,FABP4,and ALB). AOF2 (also known as LSD1 or KDM1),one of the targets for miR-302,was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres,including cMyc down-regulation and H3K4 methylation. In drug-resistant cells,sensitization was achieved through miR-302-mediated reprogramming. CONCLUSIONS miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation,which caused H3K4 methylation and c-Myc repression. View Publication

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S),production of granulocyte precursor cells (CFU-C),and extensive granulopoiesis can be maintained in vetro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells,epithelial" cells� View Publication

BACKGROUND: Conventional regulatory T cells (Tregs) can suppress human immunodeficiency virus type 1 (HIV-1)-specific immune responses but cannot control immune activation in primary HIV infection. Here,we characterized Treg subsets,using recently defined phenotypic delineation,and analyzed the relative contribution of cell subsets to the production of immunosuppressive cytokines in primary HIV infection. METHODS: In a longitudinal prospective study,ex vivo phenotyping of fresh peripheral blood mononuclear cells from patients with primary HIV infection was performed at baseline and month 6 of follow-up to characterize Treg subsets,immune activation,and cytokine production in isolated CD4(+) T cells. RESULTS: The frequency of CD4(+)CD25(+)CD127(low) Tregs and the distribution between the naive,memory,and activated/memory Treg subsets was similar in patients and healthy donors. However,Tregs from patients with primary HIV infection showed peculiar phenotypic profiles,such as elevated FoxP3,ICOS,and CTLA-4 expression,with CTLA-4 expression strikingly increased in all Treg subsets both at baseline and month 6 of follow-up. The great majority of interleukin 10 (IL-10)-producing CD4(+) T cells were FoxP3(neg) (ie,Tr1-like cells). In contrast to conventional Tregs,Tr1-like cells were inversely correlated with immune activation and not associated with lower effector T-cell responses. CONCLUSION: FoxP3(neg) Tr1-like cells-major contributors to IL-10 production-may have a beneficial role by controlling immune activation in early HIV infection. View Publication

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans,with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models,indicating that an appropriate adaptive immune response is required for the control of the disease. However,cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients,the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points,downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice. View Publication

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here,we show that three members of the Ccr4-Not complex,Cnot1,Cnot2,and Cnot3,play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts,these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs,Cnot1,Cnot2,and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead,they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly,these Cnot genes are also necessary for the maintenance of human ESCs,and silencing them mainly lead to TE and primitive endoderm differentiation. Together,our results indicate that Cnot1,Cnot2,and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs. View Publication

BACKGROUND Brucellosis is a persistent health problem in many developing countries throughout the world,and the search for simple and effective treatment continues to be of great importance. METHODS AND FINDINGS A search was conducted in MEDLINE and in the Cochrane Central Register of Controlled Trials (CENTRAL). Clinical trials published from 1985 to present that assess different antimicrobial regimens in cases of documented acute uncomplicated human brucellosis were included. The primary outcomes were relapse,therapeutic failure,combined variable of relapse and therapeutic failure,and adverse effect rates. A meta-analysis with a fixed effect model was performed and odds ratio with 95% confidence intervals were calculated. A random effect model was used when significant heterogeneity between studies was verified. Comparison of combined doxycycline and rifampicin with a combination of doxycycline and streptomycin favors the latter regimen (OR = 3.17; CI95% = 2.05-4.91). There were no significant differences between combined doxycycline-streptomycin and combined doxycycline-gentamicin (OR = 1.89; CI95% = 0.81-4.39). Treatment with rifampicin and quinolones was similar to combined doxycycline-rifampicin (OR = 1.23; CI95% = 0.63-2.40). Only one study assessed triple therapy with aminoglycoside-doxycycline-rifampicin and only included patients with uncomplicated brucellosis. Thus this approach cannot be considered the therapy of choice until further studies have been performed. Combined doxycycline/co-trimoxazole or doxycycline monotherapy could represent a cost-effective alternative in certain patient groups,and further studies are needed in the future. CONCLUSIONS Although the preferred treatment in uncomplicated human brucellosis is doxycycline-aminoglycoside combination,other treatments based on oral regimens or monotherapy should not be rejected until they are better studied. Triple therapy should not be considered the current treatment of choice. View Publication