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

BACKGROUND: Despite increasing demand,current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time,labor,and cost intensive. Additionally,absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days. METHODS: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained,cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy,polarized VEGF expression,establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes,RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture. RESULTS: Using our 2D cytokine scarce protocol,hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless,at this stage,most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured,DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers. CONCLUSIONS: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable,and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications. View Publication

OBJECTIVE: +HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However,recent studies demonstrated,by RT-PCR,that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation. MATERIALS AND METHODS: We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM,BFU-E,and CFU-GEMM) and early progenitors (LTC-IC) was evaluated. RESULTS: The HOXC4 protein is indeed expressed in human CD34(+) cells,and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p textless 0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold,range 2.5-35; p textless 0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13,onefold; range 4.1-102.1,p textless 0.0001). CONCLUSION: We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p textless 0.009) in the number of erythroid colonies compared with CFU-GM,although without perturbing,at least in vitro,the maturation program of the cells. On the other hand,the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage. View Publication

The active movement of cells from subendothelial compartments into the bloodstream (intravasation) has been recognized for several decades by histologic and physiologic studies,yet the molecular effectors of this process are relatively uncharacterized. For extravasation,studies based predominantly on static transwell assays support a general model,whereby transendothelial migration (TEM) occurs via chemoattraction toward increasing chemokine concentrations. However,this model of chemotaxis cannot readily reconcile how chemokines influence intravasation,as shear forces of blood flow would likely abrogate luminal chemokine gradient(s). Thus,to analyze how T cells integrate perivascular chemokine signals under physiologic flow,we developed a novel transwell-based flow chamber allowing for real-time modulation of chemokine levels above (luminal/apical compartment) and below (abluminal/subendothelial compartment) HUVEC monolayers. We routinely observed human T cell TEM across HUVEC monolayers with the combination of luminal CXCL12 and abluminal CCL5. With increasing concentrations of CXCL12 in the luminal compartment,transmigrated T cells did not undergo retrograde transendothelial migration (retro-TEM). However,when exposedto abluminal CXCL12,transmigrated T cells underwent striking retro-TEM and re-entered the flow stream [corrected]. This CXCL12 fugetactic (chemorepellant) effect was concentration-dependent,augmented by apical flow,blocked by antibodies to integrins,and reduced by AMD3100 in a dose-dependent manner. Moreover,CXCL12-induced retro-TEM was inhibited by PI3K antagonism and cAMP agonism. These findings broaden our understanding of chemokine biology and support a novel paradigm by which temporospatial modulations in subendothelial chemokine display drive cell migration from interstitial compartments into the bloodstream. View Publication

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma,graft-versus-host disease,and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway,within a single day,without added cytokines,as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized,as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature,common to ECP-processed monocytes from normal subjects,and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway,this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances,ECP may potentially offer a general source of therapeutic DCs. View Publication

Bone marrow-derived endothelial precursor cells incorporate into neovasculature and have been successfully used as vehicles for gene delivery to brain tumors. To determine whether systemically administered Sca1+ bone marrow cells labeled with superparamagnetic iron oxide nanoparticles can be detected by in vivo magnetic resonance imaging in a mouse brain tumor model,mouse Sca1+ cells were labeled in vitro with ferumoxides-poly-L-lysine complexes. Labeled or control cells were administered intravenously to glioma-bearing severe combined immunodeficient (SCID) mice. Magnetic resonance imaging (MRI) was performed during tumor growth. Mice that received labeled cells demonstrated hypointense regions within the tumor that evolved over time and developed a continuous dark hypointense ring at a consistent time point. This effect was not cleared by administration of a gadolinium contrast agent. Histology showed iron-labeled cells around the tumor rim in labeled mice,which expressed CD31 and von Willebrand factor,indicating the transplanted cells detected in the tumor have differentiated into endothelial-like cells. These results demonstrate that MRI can detect the incorporation of magnetically labeled bone marrow-derived precursor cells into tumor vasculature as part of ongoing angiogenesis and neovascularization. This technique can be used to directly identify neovasculature in vivo and to facilitate gene therapy by noninvasively monitoring these cells as gene delivery vectors. View Publication

OBJECTIVE: Various preparative protocols have been proposed for the acquisition and cultivation of mesenchymal stem cells (MSC). Whereas surface antigen markers have failed to precisely define this population,microarray analysis might provide a better tool for characterization of MSC. METHODS: In this study,we have analyzed global gene expression profiles of human MSC isolated from adipose tissue (AT),from umbilical cord blood (CB),and from bone marrow (BM) under two growth conditions and have compared them to terminally differentiated human fibroblasts (HS68). Profiles were compared using our Human Genome Microarray representing 51.144 different cDNA clones. RESULTS: Cultured with the appropriate conditions,osteogenic and adipogenic differentiation could be confirmed in all MSC preparations but not in fibroblasts. No phenotypic differences were observed by flow cytometry using a panel of 22 surface antigen markers. Whereas MSC derived from different donors using the same culture procedure yielded a consistent and reproducible gene expression profile,many genes were differentially expressed in MSC from different ontogenetic sources or from different culture conditions. Twenty-five genes were overlapping and upregulated in all MSC preparations from AT,CB,and BM as compared to HS68 fibroblasts. These genes included fibronectin,ECM2,glypican-4,ID1,NF1B,HOXA5,and HOXB6. Many genes upregulated in MSC are involved in extracellular matrix,morphogenesis,and development,whereas several inhibitors of the Wnt pathway (DKK1,DKK3,SFRP1) were highly expressed in fibroblasts. CONCLUSION: Our results have provided a foundation for a more reproducible and reliable quality control using genotypic analysis for defining MSC. View Publication

Two monoclonal antibodies,BA16 and BA17,have been developed using a detergent-insoluble extract of human mammary epithelial organoids as immunogen. Indirect immunofluorescent staining of cultured cells showed that the component reacting with the antibodies was filamentous and the intensity of staining was stronger in mitotic cells. Immunoblotting of cell extracts showed that both antibodies react with only one band of 40 X 10(3) molecular weight,which was present in keratin-enriched extracts of cells or organoids. Furthermore,the tissue distribution of the component reacting with the antibodies was that predicted for human keratin 19. The antibodies showed differences in the intensity of staining of cells or tissue sections fixed and prepared in different ways indicating that they reacted with different epitopes. The pattern of expression of the 40 X 10(3) Mr keratin by normal mammary epithelial cells was investigated by immunoperoxidase staining of tissue sections,cultured milk cells,and organoids of different sizes cultured in collagen gels. It was found that basal or myoepithelial cells did not express this keratin. Some heterogeneity of expression of this component was seen in luminal epithelial cells,found almost exclusively in the smaller structures. These cells did,however,express other keratins characteristic of luminal cells. The distribution in the mammary tree of the luminal cells that did not express the 40 X 10(3) Mr keratin appears to be similar to that expected for cells with the proliferative potential to produce new terminal ductal lobular units or an increase in branching of existing terminal ductal lobular units. It is shown that these cells have considerable proliferative potential by the fact that they form large colonies in milk cell cultures. View Publication

Pluripotent stem cells display significant heterogeneity in gene expression,but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states,even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity,consistently high transcripts for all pluripotency-related genes studied,and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states,which ultimately become primed for lineage specification. ?? 2014 The Authors. View Publication

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover,we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining,transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling. View Publication

The Mesenchymal-to-Epithelial Transition (MET) has been recognized as a crucial step for successful reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Thus,it has been demonstrated,that the efficiency of reprogramming can be enhanced by promoting an epithelial expression program in cells,with a concomitant repression of key mesenchymal genes. However,a detailed characterization of the epithelial transition associated with the acquisition of a pluripotent phenotype is still lacking to this date. Here,we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming,as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6-12. days after electroporation,in synchrony with the upregulation of early pluripotency markers,and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT) which takes place during mammalian gastrulation. View Publication

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors,but the functional capacity of DC must be assessed in detail,especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant,providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol,resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors,or TLRs,do not show these functional features. Therefore,SeV-infected DC have the potential for DC-directed immunotherapy. View Publication