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

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However,whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues,such as umbilical cord mesenchymal cells (UMCs),are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report,we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs),we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly,we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay,reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system,in T cell co-culture system as well. Furthermore,through whole genome expression microarray analysis,we showed that over 70 immune genes,including all members of HLA-I,were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation,thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine. View Publication

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However,it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells,designated tumor-evoked Bregs (tBreg),phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-$\beta$-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs,4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications,as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis. View Publication

Mutations at the steel locus (Sl) of the mouse affect the same cellular targets as mutations at the white spotting locus (W),which is allelic with the c-kit proto-oncogene. We show that KL,a hematopoietic growth factor obtained from conditioned medium of BALB/c 3T3 fibroblasts that stimulates the proliferation of mast cells and early erythroid progenitors,specifically binds to the c-kit receptor. The predicted amino acid sequence of isolated KL-specific cDNA clones suggests that KL is synthesized as an integral transmembrane protein. Linkage analysis maps the KL gene to the Sl locus on mouse chromosome 10,and KL sequences are deleted in the genome of the Sl mouse. These results indicate that the Sl locus encodes the ligand of the c-kit receptor,KL. View Publication

NK cells protect hosts against viral pathogens and transformed cells,and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma,despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast,IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition,blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally,presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming. View Publication

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters,miR-200c-141,miR-200b-200a-429,and miR-183-96-182 were downregulated in human BCSCs,normal human and murine mammary stem/progenitor cells,and embryonal carcinoma cells. Expression of BMI1,a known regulator of stem cell self-renewal,was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly,miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells. View Publication

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important,given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here,we present an in-depth quantitative mass spectrometry-based analysis of hESCs,two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless,a small group of 58 proteins,mainly related to metabolism,antigen processing and cell adhesion,was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap,highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. View Publication

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes,mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture,hES cells expressing cell-surface NGFR protein (CD271,p75NTR) were isolated by immunoaffinity adsorption,and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression,examined by quantitative RT-PCR,found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR,SNAI1,NTRK3,SOX9,and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer,mRNAs typifying adult stromal stem cells were detected,including BMI1,KIT,NES,NOTCH1,and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1,B3GNT7,PTDGS,and ALDH3A1 were upregulated. mRNA for keratocan (KERA),a cornea-specific proteoglycan,was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate,a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells,therefore,may provide a renewable source of material for development of treatment of corneal stromal opacities. View Publication

NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor,2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report,we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator beta-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor,Vav-1,and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition,the activity of MEK-2 (but not MEK-1) was up-regulated,and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally,inactivation of GSK-3 using a specific inhibitor of GSK-3beta increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and beta-catenin,possibly through a signal transduction pathway that involves Vav-1,Rac-1,Raf-1,MEK-2,and Erk1/2 and that this pathway is aberrant in individuals with XLP. View Publication

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes,humanization of rodent Abs,or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however,this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g.,affinity and titers) of mAb-producing cell lines,including hybridomas. We reasoned that this process,named morphogenics,could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover,these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing. View Publication

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA,followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample,whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles,we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP,CTCGAGCTGHHHHHAACTAC,where H is a mixture of base consisting of 50% A,25% T,and 25% C; and second 5' DOP,CTCGAGCTGDDDDDGTTTAG,where D is a mixture of base consisting of 50% T,25% G,and 25% A. Our results showed that bisulfite-modified DNAs from a cell line,cord blood cells,or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template. View Publication

Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML),we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues,and developed an algorithm to identify potential targets expressed in leukemia stem cells,but not in normal CD34+CD38- hematopoietic cells,T cells,or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19,we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML. View Publication

Store-operated calcium entry (SOCE) is a Calcium (Ca2+) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons,the presence and functions of SOCE are still in question. Here,we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase,which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs),being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions,but the underlying mechanism and,specifically,the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons,we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD,pointing out a key function of calcium and SOCE in synaptic plasticity. View Publication