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

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature,with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries,including planar 2-D films,macroporous 3-D sponges,and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However,synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion,viability,proliferation,self-renewal,and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover,the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment,where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus,synthetic substrates with specific 3-D microgeometries can support hESC colony formation,self-renewal,and directed differentiation to multiple lineages while obviating the stringent needs for complex,exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions. View Publication

We report an injectable hydrogel scaffold system with tunable stiffness for controlling the proliferation rate and differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) context in normal growth media. The hydrogels composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate were formed using the oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H(2)O(2)) and horseradish peroxidase (HRP). The stiffness of the hydrogels was readily tuned by varying the H(2)O(2) concentration without changing the concentration of polymer precursor. We found that the hydrogel stiffness strongly affected the cell proliferation rates. The rate of hMSC proliferation increased with the decrease in the stiffness of the hydrogel. Also,the neurogenesis of hMSCs was controlled by the hydrogel stiffness in a 3D context without the use of any additional biochemical signal. These cells which were cultured in hydrogels with lower stiffness for 3 weeks expressed much more neuronal protein markers compared to those cultured within stiffer hydrogels for the same period of time. View Publication

BACKGROUND We previously generated induced pluripotent stem cells by reprograming adipose stem cells through the introduction of microRNAs targeting four transcription factors (Oct3/4,Sox2,c-Myc,and Klf4). In this study,we aimed to reprogram cancer cells using microRNAs to explore their therapeutic potential. METHODS Mature microRNAs (mir-302a-d,369-3p and 5p,and mir-200c,as needed) were introduced into colon cancer cells (DLD-1,RKO,and HCT116) using lipofection. RESULTS The transfected cells exhibited an embryonic stem cell-like morphology and expressed the undifferentiated marker genes Nanog,Oct3/4,SOX2,and Klf4,as well as tumor-related antigen-1-60. These cells expressed neurogenic or adipogenic markers,indicating that reprogramming of the cancer cells was partially successful. Moreover,we found that miRNA-expressing DLD-1 cells showed low proliferative activity in vitro and in vivo,accompanied by increased expression of the tumor suppressor genes p16 (ink4a) and p21 (waf1) . miRNA-expressing DLD-1 cells also exhibited enhanced sensitivity to 5-fluorouracil,possibly through the downregulation of multidrug-resistant protein 8. The reprogrammed cells from DLD-1,RKO,and HCT116 cells exhibited reduced c-Myc expression,in contrast to the high c-Myc expression in the induced pluripotent cancer cells that were generated using four transcription factors. CONCLUSIONS Our cancer reprogramming method employing simple lipofection of mature microRNAs is safe and well suited for clinical application,because it avoids integration of exogenous genes into the host genome and allows escape from augmentation of c-Myc gene expression. View Publication

We asked this question: in normal humans,is either a normal dietary intake or normal serum concentration of phosphorus a determinant of the serum concentration of 1,25(OH)2D? In seven normal men whose dietary phosphorus was decreased from 2,300 to 625 mg/d,each intake for 8-9 d,under strictly controlled,normal metabolic conditions,we measured serum concentrations of 1,25(OH)2D daily,and concentrations of phosphorus hourly throughout a 24-h period,before and after restriction. Decreasing dietary phosphorus induced: (a) a 58% increase in serum levels of 1,25(OH)2D; (b) a 35% decrease in serum levels of phosphorus measured in the afternoon; (c) a 12% decrease in the 24-h mean serum level of phosphorus; but,(d) no decrease in morning fasting levels of phosphorus. Serum concentrations of 1,25(OH)2D varied inversely and significantly with 24-h mean concentrations of phosphorus (r = -0.77,P less than 0.001). When these data are combined with those of our prior study in which dietary phosphorus was varied over an extreme range,the relationship between serum levels of 1,25(OH)2D and 24-h mean serum levels of phosphorus is even stronger (r = -0.90,P less than 0.001). In the aggregate,the results demonstrate that in normal men,dietary phosphorus throughout a normal range and beyond,can finely regulate the renal production and serum concentration of 1,25(OH)2D,and provide evidence that this regulation is mediated by fine modulation of the serum concentration of phosphorus. View Publication

The response to programmed death-1 (PD-1) blockade varies in hepatocellular carcinoma (HCC). We utilize a panel of 16 serum factors to show that a circulating level of serum amyloid A (SAA) > 20.0 mg/L has the highest accuracy in predicting anti-PD-1 resistance in HCC. Further experiments show a correlation between peritumoral SAA expression and circulating SAA levels in patients with progressive disease after PD-1 inhibition. In vitro experiments demonstrate that SAA induces neutrophils to express PD-L1 through glycolytic activation via an LDHA/STAT3 pathway and to release oncostatin M,thereby attenuating cytotoxic T cell function. In vivo,genetic or pharmacological inhibition of STAT3 or SAA eliminates neutrophil-mediated immunosuppression and enhances antitumor efficacy of anti-PD-1 treatment. This study indicates that SAA may be a critical inflammatory cytokine implicated in anti-PD-1 resistance in HCC. Targeting SAA-induced PD-L1+ neutrophils through STAT3 or SAA inhibition may present a potential approach for overcoming anti-PD1 resistance. The reasons for why hepatocellular carcinoma (HCC) is unresponsive to anti-PD-1 inhibition in some patients is not fully understood. Here the authors use human samples and mice tumour models to implicate serum amyloid A and STAT3 signalling involvement in the resistance to anti-PD1 immunotherapy in HCC. View Publication

Targeted delivery of siRNA controlled by near-infrared light using hollow gold nanoshells has been demonstrated in cancer and stem cells models. Here,a universal surface module and several functionalization rules for the maximized delivery of short nucleic acids (here,siRNA) applicable for diverse gold nanocarriers are described. Streptavidin is devised as a handle to assemble biotinylated cell penetrating peptides (e.g.,transactivating transcriptional activator (TAT)),as well as an insulator between the positive charge of TAT and the dense negative charge of RNA. However,direct linking of streptavidin to functional siRNA inhibits its silencing activity. The approach then involves the orthogonal assembly of two types of RNA strands: one with biotin modification for cell targeting and penetration (scaffold RNA); the other without biotin as functional RNA (i.e.,siRNA). Initially,flexible single-stranded RNA is used for dense surface-packing,followed by hybridization with the complementary RNA strand to maximize the assembly of the targeting peptide for cellular uptake and siRNA delivery. This orthogonal approach for the delivery of short oligonucleotides,together with novel surface functionalization rules discovered here,should enable the use of these materials for nanomedicinal research and applications. View Publication

Although it has been shown that unfractionated bone marrow,hematopoietic stem cells,common myeloid progenitors,and bipotent megakaryocyteerythrocyte progenitors can give rise to megakaryocyte colonies in culture,monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here,we use a monoclonal antibody to the megakaryocyte-associated surface protein,CD9,to purify MKPs from the c-kit(+)Sca-1(-)IL7Ralpha(-)Thy1.1(-)Lin(-) fraction of adult C57BLKa-Thy1.1 bone marrow. The CD9(+) fraction contained a subset of CD41(+)FcgammaR(lo)CD34(+)CD38(+) cells that represent approximately 0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes,with a plating efficiency textgreater60% at the single-cell level. In vivo,MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for approximately 3 weeks. Common myeloid progenitors and megakaryocyteerythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures,indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns. View Publication

Recently,it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study,we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules,thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines,particularly IL-12,whereas the expression of costimulatory molecules remained unchanged. Consequently,synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore,we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However,noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally,we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment,suggesting an autocrine action of type I IFNs. View Publication

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research,and a potential source of different tissues for transplantation. However,one important challenge for the clinical use of these cells is the issue of immunocompatibility,which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population,named BR-1,in commercial defined medium. In contrast to the other hES cell lines established in defined medium,BR-1 maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge,this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-1 and another 22 hES cell lines established elsewhere with those of the Brazilian population,finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture. View Publication

Various preclinical models have been developed to clarify the pathophysiology of prostate cancer (PCa). Traditional PCa cell lines from clinical metastatic lesions,as exemplified by DU-145,PC-3,and LNCaP cells,are useful tools to define mechanisms underlying tumorigenesis and drug resistance. Cell line-based experiments,however,have limitations for preclinical studies because those cells are basically adapted to 2-dimensional monolayer culture conditions,in which the majority of primary PCa cells cannot survive. Recent tissue engineering enables generation of PCa patient-derived xenografts (PDXs) from both primary and metastatic lesions. Compared with fresh PCa tissue transplantation in athymic mice,co-injection of PCa tissues with extracellular matrix in highly immunodeficient mice has remarkably improved the success rate of PDX generation. PDX models have advantages to appropriately recapitulate the molecular diversity,cellular heterogeneity,and histology of original patient tumors. In contrast to PDX models,patient-derived organoid and spheroid PCa models in 3-dimensional culture are more feasible tools for in vitro studies for retaining the characteristics of patient tumors. In this article,we review PCa preclinical model cell lines and their sublines,PDXs,and patient-derived organoid and spheroid models. These PCa models will be applied to the development of new strategies for cancer precision medicine. View Publication

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics,and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is,however,traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer,and as a result,possible xenogeneic contamination,contribution of unlabeled amino acids by the feeders,interlaboratory variability of MEF preparation,and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used,chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison,WI) licensed to STEMCELL Technologies (Vancouver,Canada)]. This medium,together with adjustments to the culturing protocol,facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151. View Publication