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

Atezolizumab plus bevacizumab and lenvatinib are standard treatments for unresectable hepatocellular carcinoma; however,tumor markers such as alpha-fetoprotein and des-gamma-carboxy prothrombin have a limited ability to reflect treatment responses. Circulating tumor cells are non-invasive biomarkers associated with cancer stemness and treatment resistance. We assessed circulating tumor cell subsets expressing cancer stem cell markers (CD90,epithelial cell adhesion molecule,CD133,vimentin) using multiparametric flow cytometry at early and maximal response phases in patients receiving atezolizumab plus bevacizumab or lenvatinib. Early decreases in CD90-positive circulating tumor cells after therapy initiation were associated with tumor shrinkage and longer progression-free survival in both groups,as well as prolonged overall survival in the atezolizumab plus bevacizumab group. At maximal response,changes in CD90-positive circulating tumor cells reflected tumor burden more accurately than alpha-fetoprotein or des-gamma-carboxy prothrombin. These findings highlight the potential of CD90-positive circulating tumor cells to become dynamic biomarkers in systemic therapy for unresectable hepatocellular carcinoma. View Publication

Ataxia with oculomotor apraxia type 1 (AOA1) is a rare,autosomal recessive,early-onset,progressive cerebellar ataxia caused by mutations in the APTX gene,which encodes aprataxin,a DNA-adenylate hydrolase involved in DNA damage repair. The pathogenesis of AOA1 remains unclear. The purpose of this study was to investigate the pathogenesis of a novel mutation,p.H201P/H201R,carried by our AOA1 patient and the mechanism of AOA1 in an induced pluripotent stem cells (iPSCs) model. We edited iPSCs derived from a healthy individual to carry the APTX homozygous mutation p.H201P (H201P-iPSCs) or p.H201R (H201R-iPSCs) via CRISPR/Cas9. We found that aprataxin expression was absent in both H201P- and H201R-iPSCs. The capacity of these APTX-mutant iPSCs to differentiate into neural progenitor cells (NPCs) and mature neurons was diminished. We observed an increase in DNA single-strand breaks (SSB) via a comet assay and poly(ADP-ribose) staining,and an increase in the ratio of cleaved PARP-1/total PARP-1 in APTX-mutant NPCs and early immature neurons (EiNs),in addition of a heightened sensitivity to tert-butyl hydroperoxide in APTX-mutant EiNs. Moreover,a decrease of APE1 expression was observed in APTX-mutant NPCs and H201R-EiNs during neural differentiation. Our study established a practical iPSCs model to investigate AOA1 disease. We found that mutant aprataxin leads to defective neural differentiation,accompanied by the accumulation of DNA SSBs with increased cleaved PARP-1 and reduced APE1 expression of the base excision repair pathway. View Publication

The ST6Gal-I sialyltransferase adds an $$2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer,but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here,we show upregulated ST6Gal-I protein in several epithelial cancers,including many colon carcinomas. In normal colon,ST6Gal-I localized selectively to the base of crypts,where stem/progenitor cells are found,and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly,ST6Gal-I expression was restricted to basal epidermal layers in skin,another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells,with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations,we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells,as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely,short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively,our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations. View Publication

Acute myeloid leukemia (AML) is characterized by the block of differentiation,deregulated apoptosis,and an increased self-renewal of hematopoietic precursors. It is unclear whether the self-renewal of leukemic blasts results from the cumulative effects of blocked differentiation and impaired apoptosis or whether there are mechanisms directly increasing self-renewal. The AML-associated translocation products (AATPs) promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha),promyelocytic leukemia zinc finger (PLZF)/RAR alpha (X-RAR alpha),and AML-1/ETO block hematopoietic differentiation. The AATPs activate the Wnt signaling by up-regulating gamma-catenin. Activation of the Wnt signaling augments self-renewal of hematopoietic stem cells (HSCs). Therefore,we investigated how AATPs influence self-renewal of HSCs and evaluated the role of gamma-catenin in the determination of the phenotype of HSCs expressing AATPs. Here we show that the AATPs directly activate the gamma-catenin promoter. The crucial role of gamma-catenin in increasing the self-renewal of HSCs upon expression of AATPs is demonstrated by (i) the abrogation of replating efficiency upon hindrance of gamma-catenin expression through RNA interference,and (ii) the augmentation of replating efficiency of HSCs upon overexpression of gamma-catenin itself. In addition,the inoculation of gamma-catenin-transduced HSCs into irradiated recipient mice establishes the clinical picture of AML. These data provide the first evidence that the aberrant activation of Wnt signaling by the AATP decisively contributes to the pathogenesis of AML. View Publication

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes,respectively. Using patient-derived induced pluripotent stem cells (iPSC),we corrected the dysferlin nonsense mutation c.5713CtextgreaterT; p.R1905X and the most common alpha-sarcoglycan mutation,missense c.229CtextgreaterT; p.R77C,by single-stranded oligonucleotide-mediated gene editing,using the CRISPR/Cas9 gene editing system to enhance the frequency of homology-directed repair. We demonstrated seamless,allele-specific correction at efficiencies of 0.7-1.5%. As an alternative,we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22,using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination,and DICE also utilized site-specific recombinases. With DICE and THRIP,we obtained targeting efficiencies after selection of ˜20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization,as shown by immunoblot and immunocytochemistry. In summary,we demonstrate for the first time precise correction of LGMD iPSC and validation of expression,opening the possibility of cell therapy utilizing these corrected iPSC.Molecular Therapy (2016); doi:10.1038/mt.2016.40. View Publication

Autologous platelet rich plasma (PRP) is clinically used to induce repair of different tissues through the release of bioactive molecules. In some patients,the production of an efficient autologous PRP is unfeasible due to their compromised health. We developed an allogeneic PRP mismatched for AB0 and Rh antigens. To broadcast its clinical applications avoiding side effects the outcome of allogeneic PRP on immune response should be defined. Thus,we investigated whether PRP affected the differentiation of peripheral blood monocytes to dendritic cells upon stimulation with granulocyte monocyte colony stimulating factor and interleukin-4. Indeed,these cells are the main players of immune response and tissue repair. PRP inhibited the differentiation of monocytes to CD1a(+) dendritic cells and favored the expansion of phagocytic CD163(+) CD206(+) fibrocyte-like cells. These cells produced inteleukin-10 and prostaglandin-E2,but not interferon-γ,upon stimulation with lipopolysaccharides. Moreover,they promoted the expansion of regulatory CD4(+) CD25(+) FoxP3(+) T cells upon allostimulation or antigen specific priming. Finally,the conditioned medium harvested from monocytes differentiated with PRP triggered a strong chemotactic effect on mesenchymal cells in both scratch and transwell migration assays. These results strongly suggest that allogeneic PRP can foster the differentiation of monocytes to a regulatory anti-inflammatory population possibly favoring wound healing. View Publication

Cancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor 1 (ANTXR1) has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f(+)/EpCAM(-)/Lin(-)) expressed higher levels of ANTXR1 compared with mature luminal cells. CD49f(+)/EpCAM(-),CD44(+)/EpCAM(-),CD44(+)/CD24(-),or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44(+)/CD24(-)/ANTXR1(+) cells displayed enhanced self-renewal as measured by mammosphere assay compared with CD44(+)/CD24(-)/ANTXR1(-) cells. Activation of ANTXR1 by its natural ligand C5A,a fragment of collagen VI $$3,increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor-lipoprotein receptor-related protein 6 (LRP6),phosphorylation of GSK3$$/$$,and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression,self-renewal,invasion,tumorigenicity,and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A,which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Furthermore,ANTXR1 is among the 10% most overexpressed genes in breast cancer and is coexpressed with collagen VI. Thus,ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment,linking it to a stemness signaling network that drives metastatic progression. View Publication

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However,residual undifferentiated iPS derivates (iPSD),which are not fully eliminated by cell differentiation or purification protocols,may form tumors after transplantation,thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells,which share many features with iPSD. Here,we tested the effects of PluriSin1,a small-molecule inhibitor of SCD,on iPS-derived CM. We found that plurisin1 treatment significantly decreased the mRNA and protein level of Nanog,a marker for both cell pluripotency and tumor progression; importantly,we provide evidence that PluriSin1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition,PluriSin1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin1 treatment prevents tumorigenicity of iPSD after cell transplantation,we intramyocardially injected PluriSin1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection,which was prevented by treatment with PluriSin1. Moreover,treatment with PluriSin1 did not change the expression of cTnI,α-MHC,or MLC-2v,markers of cardiac differentiation (Ptextgreater0.05,n = 4). Importantly,pluriSin1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration. View Publication

The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosa-associated lymphoid tissue (MALT)-type lymphomas,and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence,constitutive nuclear factor-kappaB (NF-kappaB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis,Emu-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless,API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IkappaB kinase gamma (IKKgamma),indicative for enhanced NF-kappaB activation,was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition,we show that the API2-MALT1 fusion resided in the cholesterol- and sphingolipid-enriched membrane microdomains,termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts,which is mediated by the API2 portion,is sufficient to trigger NF-kappaB activation via enhanced polyubiquitination of IKKgamma. Taken together,these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-kappaB activation. View Publication

Recently,a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT,we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs,whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study,we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions,such as cytotoxic activity and cytokine production. Moreover,we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30,NKp44,and NKG2D. Finally,we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells. View Publication

Studies have shown that neural stem/progenitor cell (NPC) transplantation is beneficial in experimental autoimmune encephalomyelitis (EAE),an established animal model of multiple sclerosis (MS). It is unclear whether NPCs have the ability to integrate into the host CNS to replace lost cells or if their main mechanism of action is via bystander immunomodulation. Understanding the mechanisms by which NPCs exert their beneficial effects as well as exploring methods to increase post-transplantation survival and differentiation is critical to advancing this treatment strategy. Using the EAE model and Fas-deficient (lpr) NPCs,we investigated the effects of altering the Fas system in NPC transplantation therapy. We show that transplantation of NPCs into EAE mice ameliorates clinical symptoms with greater efficacy than sham treatments regardless of cell type (wt or lpr). NPC transplantation via retro-orbital injections significantly decreased inflammatory infiltrates at the acute time point,with a similar trend at the chronic time point. Both wt and lpr NPCs injected into mice with EAE were able to home to sites of CNS inflammation in the periventricular brain and lumbar spinal cord. Both wt and lpr NPCs have the same capacity for inducing apoptosis of Th1 and Th17 cells,and minimal numbers of NPCs entered the CNS. These cells did not express terminal differentiation markers,suggesting that NPCs exert their effects mainly via bystander peripheral immunomodulation. View Publication

Objective Dendritic cells (DCs) are key orchestrators of immune function. To date,rheumatoid arthritis (RA) researchers have predominantly focused on a potential pathogenic role for CD1c+ DCs. In contrast,CD141+ DCs and plasmacytoid DCs (pDCs) have not been systematically examined,at least in early RA. In established RA,the role of pDCs is ambiguous and,since disease duration and treatment both impact RA pathophysiology,we examined pDCs,and CD1c+ and CD141+ conventional DCs (cDCs),in early,drug-na{\{i}}ve RA (eRA) patients. Methods We analyzed the frequency and phenotype of pDCs View Publication