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

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However,the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p,miR-199a-3p,and miR-214 in the microRNA gene cluster. Meanwhile,the expression levels of their targets related to regulation of hypoxia-stimulated cell migration,such as HIF1A,MET,and MAPK1,were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy. View Publication

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription,CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG,OCT4,and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG,OCT4,and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal,endodermal,and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells. View Publication

Memory T-cells are essential to maintain long-term immunological memory. It is widely thought that the bone marrow (BM) plays an important role in the long-term maintenance of memory T-cells. There is controversy however on the longevity and recirculating kinetics of BM memory T-cells. While some have proposed that the BM is a reservoir for long-lived,non-circulating memory T-cells,it has also been suggested to be the preferential site for memory T-cell self-renewal. In this study,we used in vivo deuterium labeling in goats to simultaneously quantify the average turnover rates-and thereby expected lifespans-of memory T-cells from BM,blood and lymph nodes (LN). While the fraction of Ki-67 positive cells,a snapshot marker for recent cell division,was higher in memory T-cells from blood compared to BM and LN,in vivo deuterium labeling revealed no substantial differences in the expected lifespans of memory T-cells between these compartments. Our results support the view that the majority of memory T-cells in the BM are self-renewing as fast as those in the periphery,and are continuously recirculating between the blood,BM,and LN. View Publication

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10),which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however,there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore,the involvement of resident airway immune cells,predominantly bronchoalveolar macrophages,in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard,our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release,which occurred in a time- and dose-dependent manner. Furthermore,HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus,this work supports a model,wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo. View Publication

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with one of the worst outcomes among all cancers. PDA often recurs after initial treatment to result in patient death despite the use of chemotherapy or radiation therapy. PDA contains a subset of tumor-initiating cells capable of extensive self-renewal known as cancer stem cells (CSC),which may contribute to therapeutic resistance and metastasis. At present,conventional chemotherapy and radiotherapy are largely ineffective in depleting CSC pool,suggesting the need for novel therapies that specifically target the cancer-sustaining stem cells for tumor eradication and to improve the poor prognosis of PDA patients. In this study,we report that death receptor 5 (DR5) is enriched in pancreatic CSCs compared with the bulk of the tumor cells. Treating a collection of freshly generated patient-derived PDA xenografts with gemcitabine,the first-line chemotherapeutic agent for PDA,is initially effective in reducing tumor size,but largely ineffective in diminishing the CSC populations,and eventually culminated in tumor relapse. However,a combination of tigatuzumab,a fully humanized DR5 agonist monoclonal antibody,with gemcitabine proved to be more efficacious by providing a double hit to kill both CSCs and bulk tumor cells. The combination therapy produced remarkable reduction in pancreatic CSCs,tumor remissions,and significant improvements in time to tumor progression in a model that is considered more difficult to treat. These data provide the rationale to explore the DR5-directed therapies in combination with chemotherapy as a therapeutic option to improve the current standard of care for pancreatic cancer patients. View Publication

UNLABELLED Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development,as well as the state of differentiation of susceptible cells at the time of infection,affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs,infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection,we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1,and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally,we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence,and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion,our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. IMPORTANCE Human cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study,we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells,which are similar to those present in the developing neural tube,do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS. View Publication

All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When exposed to the pluripotent human embryonal carcinoma (EC) stem cell line,TERA2.cl.SP12,ATRA induces ectoderm differentiation and the formation of neuronal cell types. We have previously generated synthetic analogues of retinoic acid (EC23 and EC19) which also induce the differentiation of EC cells. Even though EC23 and EC19 have similar chemical structures,they have differing biochemical effects in terms of EC cell differentiation. EC23 induces neuronal differentiation in a manner similar to ATRA,whereas EC19 directs the cells to form epithelial-like derivatives. Previous MALDI-TOF MS analysis examined the response of TERA2.cl.SP12 cells after exposure to ATRA,EC23 and EC19 and further demonstrated the similarly in the effect of ATRA and EC23 activity whilst responses to EC19 were very different. In this study,we show that Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS) coupled with appropriate scatter correction and multivariate analysis can be used as an effective tool to further investigate the differentiation of human pluripotent stem cells and monitor the alternative affects different retinoid compounds have on the induction of differentiation. FT-IRMS detected differences between cell populations as early as 3 days of compound treatment. Populations of cells treated with different retinoid compounds could easily be distinguished from one another during the early stages of cell differentiation. These data demonstrate that FT-IRMS technology can be used as a sensitive screening technique to monitor the status of the stem cell phenotype and progression of differentiation along alternative pathways in response to different compounds. View Publication

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors,including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC),have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study,which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis,we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC,often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly,both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+,as previously documented for LTC-IC in normal marrow. Thus,neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless,an association of these phenotypes with LTC-IC function did allow highly enriched (textgreater 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph-,respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally,they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells. View Publication

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless,detection of spurious reactivities remains a recognized assay limitation. In addition,the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique,which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed,with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly,negligible reactivity was detected in the last wash control,confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly,antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis,AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity. View Publication

Chromatin structure affects the extent of DNA damage and repair. Thus,it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here,we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC,heterochromatin is confined to distinct regions,while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives,normal human fibroblasts; and one cancer cell line. At the same time,the number of DNA repair foci were not statistically different among these cells. We showed that in hESC,DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells,and to a lesser extent in IMR90 normal fibroblasts,but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC. View Publication

Microenvironmental factors such as oxygen concentration mediate key effects on the biology of mesenchymal stromal cells (MSCs). Herein,we performed an in-depth characterization of the metabolic behavior of MSCs derived from the placenta,umbilical cord,and adipose tissue (termed hPMSCs,UC-MSCs,and AD-MSCs,respectively) at physiological (hypoxic; 5{\%} oxygen [O2]) and standardized (normoxic; 21{\%} O2) O2 concentrations using chemical isotope labeling liquid chromatography-mass spectrometry. 12C- and 13C-isotope dansylation (Dns) labeling was used to analyze the amine/phenol submetabolome,and 2574 peak pairs or metabolites were detected and quantified,from which 52 metabolites were positively identified using a library of 275 Dns-metabolite standards; 2189 metabolites were putatively identified. Next,we identified six metabolites using the Dns library,as well as 14 hypoxic biomarkers from the human metabolome database out of 96 altered metabolites. Ultimately,metabolic pathway analyses were performed to evaluate the associated pathways. Based on pathways identified using the Kyoto Encyclopedia of Genes and Genomes,we identified significant changes in the metabolic profiles of MSCs in response to different O2 concentrations. These results collectively suggest that O2 concentration has the strongest influence on hPMSCs metabolic characteristics,and that 5{\%} O2 promotes arginine and proline metabolism in hPMSCs and UC-MSCs but decreases gluconeogenesis (alanine-glucose) rates in hPMSCs and AD-MSCs. These changes indicate that MSCs derived from different sources exhibit distinct metabolic profiles. View Publication

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder resulting from deficiency of iduronate-2-sulphatase activity. The disease manifests almost exclusively in males; only 16 symptomatic heterozygote girls have been reported so far. We describe the results of X-chromosome inactivation analysis in a 5-year-old girl with clinically severe disease and heterozygous mutation p.Arg468Gln in the IDS gene. X inactivation analysed at three X-chromosome loci showed extreme skewing (96/4 to 99/1) in two patient's cell types. This finding correlated with exclusive expression of the mutated allele. Induced pluripotent stem cells (iPSC) generated from the patient's peripheral blood demonstrated characteristic pluripotency markers,deficiency of enzyme activity,and mutation in the IDS gene. These cells were capable of differentiation into other cell types (cardiomyocytes,neurons). In MPS II iPSC clones,the X inactivation ratio remained highly skewed in culture conditions that led to partial X inactivation reset in Fabry disease iPSC clones. Our data,in accordance with the literature,suggest that extremely skewed X inactivation favouring the mutated allele is a crucial condition for manifestation of MPS II in females. This suggests that the X inactivation status and enzyme activity have a prognostic value and should be used to evaluate MPS II in females. For the first time,we show generation of iPSC from a symptomatic MPS II female patient that can serve as a cellular model for further research of the pathogenesis and treatment of this disease. View Publication