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

Cytosolic aldehyde dehydrogenase (ALDH),an enzyme responsible for oxidizing intracellular aldehydes,has an important role in ethanol,vitamin A,and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues,including bone marrow and intestine,appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However,although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH,isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde,dansyl aminoacetaldehyde (DAAA),could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover,DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore,marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication

BACKGROUND The Rhinovirus C (RV-C),first identified in 2006,produce high symptom burdens in children and asthmatics,however,their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs),and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor,cadherin related family member 3 (CDHR3). METHODS RV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry. RESULTS C15 produced a scattered pattern of infection,and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat],p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC,p = ns). CDHR3 expression was increased on ciliated epithelial cells,but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs,CDHR3 expression progressively increased and correlated with both RV-C binding and replication. CONCLUSIONS The RV-C only replicate in ciliated AECs in vitro,leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication,and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity. View Publication

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset,which express na{\{i}}ve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of na{\"{i}}ve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy." View Publication

Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte-like cells provides a platform to study the molecular basis of human hepatocyte differentiation,to develop cell culture models of liver disease,and to potentially provide hepatocytes for treatment of end-stage liver disease. Additionally,hepatocyte-like cells generated from human pluripotent stem cells could serve as platforms for drug discovery,determination of pharmaceutical-induced hepatotoxicity,and evaluation of idiosyncratic drug-drug interactions. Here,we describe a step-wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte-like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. View Publication

Radiotherapy represents the most effective nonsurgical treatments for gliomas. However,gliomas are highly radioresistant and recurrence is nearly universal. Results from our laboratory and other groups suggest that cancer stem cells contribute to radioresistance in gliomas and breast cancers. The Notch pathway is critically implicated in stem cell fate determination and cancer. In this study,we show that inhibition of Notch pathway with gamma-secretase inhibitors (GSIs) renders the glioma stem cells more sensitive to radiation at clinically relevant doses. GSIs enhance radiation-induced cell death and impair clonogenic survival of glioma stem cells but not non-stem glioma cells. Expression of the constitutively active intracellular domains of Notch1 or Notch2 protect glioma stem cells against radiation. Notch inhibition with GSIs does not alter the DNA damage response of glioma stem cells after radiation but rather reduces Akt activity and Mcl-1 levels. Finally,knockdown of Notch1 or Notch2 sensitizes glioma stem cells to radiation and impairs xenograft tumor formation. Taken together,our results suggest a critical role of Notch signaling to regulate radioresistance of glioma stem cells. Inhibition of Notch signaling holds promise to improve the efficiency of current radiotherapy in glioma treatment. View Publication

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state,and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics,these two reprogramming methods reset genomic methylation,an epigenetic modification of DNA that influences gene expression,leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin,which favours their differentiation along lineages related to the donor cell,while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming,or by treatment of iPSCs with chromatin-modifying drugs. In contrast,the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming,which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment. View Publication

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process,cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes,driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire,it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ,and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly,postrevision cells do not proliferate in response to the tolerizing superantigen,implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host. View Publication