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

Much attention is focused on characterizing the contribution of bone marrow (BM)-derived cells to regenerating skeletal muscle,fuelled by hopes for stem cell-mediated therapy of muscle degenerative diseases. Though physical integration of BM stem cells has been well documented,little evidence of functional commitment to myotube phenotype has been reported. This is due to the innate difficulty in distinguishing gene products derived from donor versus host nuclei. Here,we demonstrate that BM-derived stem cells contribute via gene expression following incorporation to skeletal myotubes. By co-culturing human BM-derived mesenchymal stem cells (MSC) with mouse skeletal myoblasts,physical incorporation was observed by genetic lineage tracing and species-specific immunofluorescence. We used a human-specific antibody against the intermediate filament protein nestin,a marker of regenerating skeletal muscle,to identify functional contribution of MSC to myotube formation. Although nestin expression was never detected in MSC,human-specific expression was detected in myotubes that also contained MSC-derived nuclei. This induction of gene expression following myotube integration suggests that bone marrow-derived stem cells can reprogram and functionally contribute to the muscle cell phenotype. We propose that this model of myogenic commitment may provide the means to further characterize functional reprogramming of MSC to skeletal muscle. View Publication

To better characterize biological responses to atmospheric organic aerosols,the efficient delivery of aerosol to in vitro lung cells is necessary. In this study,chamber generated secondary organic aerosol (SOA) entered the commercialized exposure chamber (CULTEX® Radial Flow System Compact) where it interfaced with an electrostatic precipitator (ESP) (CULTEX® Electrical Deposition Device) and then deposited on a particle collection plate. This plate contained human lung cells (BEAS-2B) that were cultured on a membrane insert to produce an air-liquid interface (ALI). To augment in vitro assessment using the ESP exposure device,the particle dose was predicted for various sampling parameters such as particle size,ESP deposition voltage,and sampling flowrate. The dose model was evaluated against the experimental measured mass of collected airborne particles. The high flowrate used in this study increased aerosol dose but failed to achieve cell stability. For example,RNA in the ALI BEAS-2B cells in vitro was stable at 0.15 L/minute but decayed at high flowrates. The ESP device and the resulting model were applied to in vitro studies (i.e.,viability and IL-8 expression) of toluene SOA using ALI BEAS-2B cells with a flowrate of 0.15 L/minute,and no cellular RNA decay occurred. View Publication

The mechanisms by which sex differences in the mammalian brain arise are poorly understood,but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development,we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR%=%0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes,causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts,there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes,a transmissible effect that was maintained in cellular progeny. Additionally,we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS,and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained. View Publication

BACKGROUND Transplanting autologous patient-derived enteric neuronal stem/progenitor cells (ENSCs) is an innovative approach to replacing missing enteric neurons in patients with Hirschsprung disease (HSCR). Using autologous cells eliminates immunologic and ethical concerns raised by other cell sources. However,whether postnatal aganglionic bowel is permissive for transplanted ENSCs and whether ENSCs from HSCR patients can be successfully isolated,cultured,and transplanted in vivo remains unknown. METHODS ENSCs isolated from the ganglionic intestine of Ednrb(-/-) mice (HSCR-ENSCs) were characterized immunohistochemically and evaluated for their capacity to proliferate and differentiate in vitro. Fluorescently labeled ENSCs were co-cultured ex vivo with aganglionic Ednrb(-/-) colon. For in vivo transplantation,HSCR-ENSCs were labeled with lentivirus expressing green fluorescent protein (GFP) and implanted into aganglionic embryonic chick gut in ovo and postnatal aganglionic Ednrb(-/-) rectum in vivo. KEY RESULTS HSCR-ENSCs maintain normal capacity self-renewal and neuronal differentiation. Moreover,the Ednrb(-/-) aganglionic environment is permissive to engraftment by wild-type ENSCs ex vivo and supports migratrion and neuroglial differentiation of these cells following transplantation in vivo. Lentiviral GFP-labeled HSCR-ENSCs populated embryonic chick hindgut and postnatal colon of Ednrb(-/-) HSCR,with cells populating the intermuscular layer and forming enteric neurons and glia. CONCLUSIONS & INFERENCES ENSCs can be isolated and cultured from mice with HSCR,and transplanted into the aganglionic bowel of HSCR littermates to generate enteric neuronal networks. These results in an isogenic model establish the potential of using autologous-derived stem cells to treat HSCR and other intestinal neuropathies. View Publication

The molecular mechanisms underlying the differentiation of neural progenitor cells (NPCs) remain poorly understood. In this study we investigated the role of Ca(2+) and cAMP (cyclic adenosine monophosphate) in the differentiation of NPCs extracted from the subventricular zone of E14.5 rat embryos. Patch clamp recordings revealed that increasing cAMP-signaling with Forskolin or IBMX (3-isobutyl-1-methylxantine) significantly facilitated neuronal functional maturation. A continuous application of IBMX to the differentiation medium substantially increased the functional expression of voltage-gated Na(+) and K(+) channels,as well as neuronal firing frequency. Furthermore,we observed an increase in the frequency of spontaneous synaptic currents and in the amplitude of evoked glutamatergic and GABAergic synaptic currents. The most prominent acute effect of applying IBMX was an increase in L-type Ca(2+)currents. Conversely,blocking L-type channels strongly inhibited dendritic outgrowth and synapse formation even in the presence of IBMX,indicating that voltage-gated Ca(2+) influx plays a major role in neuronal differentiation. Finally,we found that nifedipine completely blocks IBMX-induced CREB phosphorylation (cAMP-response-element-binding protein),indicating that the activity of this important transcription factor equally depends on both enhanced cAMP and voltage-gated Ca(2+)-signaling. Taken together,these data indicate that the up-regulation of voltage-gated L-type Ca(2+)-channels and early electrical excitability are critical steps in the cAMP-dependent differentiation of SVZ-derived NPCs into functional neurons. To our knowledge,this is the first demonstration of the acute effects of cAMP on voltage-gated Ca(+2)channels in NPC-derived developing neurons. View Publication

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons,which degenerate in Parkinson's disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells,followed by ventralizing,patterning,continued exposure to the TGFβ receptor inhibitor,SB431542,and at later stages of differentiation the presence of the WNT inhibitor,IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification,with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type,hEPI-NCSC. View Publication

Background Proteoglycans are found on the cell surface and in the extracellular matrix,and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate,and therefore represent an excellent tool to manipulate these pathways. Despite their importance,there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Methods Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages,and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis,immunoblotting,immunofluorescence and disaccharide analysis. Results Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1,HS6ST2 and HS6ST3,three heparan sulfate biosynthetic enzymes,within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Conclusions Differentiation of embryonic stem cells markedly changes the proteoglycanome. General significance The glycosaminoglycan biosynthetic pathway is complex and highly regulated,and therefore,understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. ?? 2014 Elsevier B.V. All rights reserved. View Publication

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however,loss of potency and inconsistent quality severely limit their use. To overcome these issues,we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking,scraping,and sorting that other hESC-MSC derivation methods require. Moreover,unlike previous reports on hESC-MSCs,we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected,they dynamically secrete a range of bioactive factors,display enzymatic activity,and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However,they also display unique immunophenotypic properties,as well as a smaller size and textgreater30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition,this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models,including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources. View Publication

There is an urgent need for new and advanced approaches to modeling the pathological mechanisms of complex human neurological disorders. This is underscored by the decline in pharmaceutical research and development efficiency resulting in a relative decrease in new drug launches in the last several decades. Induced pluripotent stem cells represent a new tool to overcome many of the shortcomings of conventional methods,enabling live human neural cell modeling of complex conditions relating to aberrant neurodevelopment,such as schizophrenia,epilepsy and autism as well as age-associated neurodegeneration. This review considers the current status of induced pluripotent stem cell-based modeling of neurological disorders,canvassing proven and putative advantages,current constraints,and future prospects of next-generation culture systems for biomedical research and translation. View Publication

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work,we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation,after four days of culture,3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm,which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up. View Publication

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10,IFN-γ and IL-17A in FOXP3(+) cells. Further,CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together,these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus. View Publication

The intracellular expression of the programmed death receptor 1 (PD1) identifies a subset of naive T(reg) cells with enhanced suppressive ability; antigen stimulation results in the surface expression of PD1. Because the role of T(reg) impairments in multiple sclerosis (MS) is still contradictory,we analyzed naive PD1- and PD1+ T(reg) cells in peripheral blood and cerebrospinal fluid (CSF) of relapsing-remitting multiple sclerosis (RR-MS) patients and of healthy control subjects. Results showed that 1) CSF PD1- T(reg) cells were significantly augmented in MS patients; 2) PD1- T(reg) cells were significantly increased in the peripheral blood of patients with stable disease (SMS) compared to those with acute (AMS) disease,and in patients responding to glatiramer acetate (COPA) compared to AMS- and COPA-unresponsive patients; and 3) PD1+ T(reg) cells were similar in CSF and peripheral blood of all groups analyzed. PD1- T(reg) cells were not increased in the peripheral blood of interferon-beta (IFNbeta) -responsive patients,but the suppressive ability of T(reg) cells was significantly higher in SMS and in COPA- or IFNbeta-responsive compared to AMS- and COPA-unresponsive individuals. The data herein suggest that PD1- T(reg) cells play a pivotal role in MS and offer a biological explanation for disease relapse and for the mechanism associated with response to COPA and IFNbeta. View Publication