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

IntroductionImmune resilience is the capacity of the immune system to recover from sepsis-induced organ injury and reestablish host defense. While sepsis survivors are often viewed as immunocompromised,recent studies suggest that some may acquire adaptive immune traits that enhance resistance to secondary infections.MethodsWe employed a murine cecal ligation and puncture (CLP) model to study polymicrobial sepsis and subsequent immune responses. Listeria monocytogenes was used as a model intracellular pathogen to assess immune protection. We analyzed myeloid cell phenotypes using single-cell RNA sequencing and evaluated lipid metabolic changes through quantitative RT-PCR,immunohistochemistry,serum cytokine assays,and plasma lipidomics.ResultsSepsis-surviving mice showed enhanced resistance to Listeria infection. Single-cell RNA sequencing revealed transcriptional reprogramming in splenic CD11b+Ly6Chigh myeloid cells,including downregulation of lipoprotein lipase and lipid efflux genes. CD11b+ myeloid cells exhibited increased lipid droplet accumulation,accompanied by elevated serum interferon-gamma (IFN-γ) levels. Plasma lipidomics identified systemic lipid remodeling,with increased phosphatidylserine and decreased phosphatidylinositol and phosphatidylglycerol levels.DiscussionOur findings suggest that sepsis survival induces lipid metabolic reprogramming in myeloid cells,contributing to enhanced immunity against intracellular pathogens. These insights reveal potential therapeutic targets within lipid metabolic pathways to improve host defense in sepsis survivors. View Publication

In this study,we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells,in serum-free medium,supplemented only with fibroblast growth factor (FGF)-1,FGF-2,or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays,high levels of stem cell activity were detectable at 1,3,and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However,cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant,showing that HSC activity originated from LSK cells. Subsequently,we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules,stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response,inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly,although HSC activity is typically rapidly lost after short-term culture in vitro,our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks. View Publication

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation,and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%,but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single,larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture,or aggregates of ES cells in hanging drop culture,they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus,initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however,hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension,whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient,scalable bioprocesses for ES cell differentiation,and inform novel methods for the production of hematopoietic tissues. View Publication

Both extrinsic and intrinsic mechanisms tightly govern hematopoietic stem cell (HSC) decisions of self-renewal and differentiation. However,transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions using serial functional assays. Here,we show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore,Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore,we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery,thus providing therapeutic benefits for patients after clinical myelosuppressive treatment. View Publication

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples,as well as in wild-type versus Hoxa9-deficient marrow,using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors,being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells,indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1,an important regulator of myelopoiesis,was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein,HOXA9 did not appear to modulate Pu.1 expression in murine BM cells. View Publication

BackgroundPhotoreceptor death leads to inherited blinding retinal diseases,such as retinitis pigmentosa (RP). As disease progression often outpaces therapeutic advances,developing effective treatments is urgent. This study evaluates the efficacy of small peptides derived from pigment epithelium-derived factor (PEDF),which are known to restrict common cell death pathways associated with retinal diseases.MethodsWe tested chemically synthesized peptides (17-mer and H105A) with affinity for the PEDF receptor,PEDF-R,delivered as eye drops to two RP mouse models: rd10 (phosphodiesterase 6b mutation) and RhoP23H/+ (rhodopsin P23H mutation). Additionally,we engineered AAV-H105A vectors for intravitreal delivery in RhoP23H/+ mice. To assess peptide effects in human tissue,we used retinal organoids exposed to cigarette smoke extract,a model of oxidative stress. Photoreceptor survival,morphology and function were evaluated.ResultsHere we show that peptides 17-mer and H105A delivered via eye drops successfully reach the retina,promote photoreceptor survival,and improve retinal function in both RP mouse models. Intravitreal delivery of a AAV-H105A vector delays photoreceptor degeneration in RhoP23H/+ mice up to six months. In human retinal organoids,peptide H105A specifically prevents photoreceptor death induced by oxidative stress,a contributing factor to RP progression.ConclusionsPEDF peptide-based eye drops offer a promising,minimally invasive therapy to prevent photoreceptor degeneration in retinal disorders,with a favorable safety profile. Plain language summaryRetinitis pigmentosa (RP) is a rare inherited condition that causes the gradual death of photoreceptors (light-sensing cells) in the eye,leading to vision loss. There is currently no cure. This study tested a potential treatment using small protein fragments (peptides) from PEDF,a protective protein naturally found in the eye. Researchers delivered these peptides through eye drops or gene therapy in mouse models of RP and to human retinal organoids (lab-grown retina tissue). Mice treated early maintained healthy vision cells,while untreated mice experienced rapid cell loss and vision decline. These results suggest that peptide-based eye drops could be a simple,safe,and effective way to slow vision loss in patients with RP. Bernardo-Colón et al. evaluate small peptides derived from the neurotrophic region of pigment epithelium-derived factor (PEDF) as potential therapeutics for retinitis pigmentosa using mouse models and human retinal organoids. A significant delay in photoreceptor death with eye drop or gene therapy delivery is seen. View Publication

A careful prognostic evaluation of patients referred for high-dose therapy (HDT) is warranted to identify those who maximally benefit from HDT as well as those who clearly fail current HDT and are candidates for more innovative treatments. In a series of 110 patients with myeloma who received HDT as first-line therapy,times to event (disease progression and death) were studied through proportional hazard models,in relation to different prognostic factors,including a chromosome 13 fluorescence in situ hybridization (FISH) analysis using a D13S319 probe. Delta13 was detected in 42 patients (38%). Follow-up time among surviving patients and survival time were 48 +/- 3 and 51 +/- 7 months,respectively (median +/- SE). In the univariate analysis,Delta13 was the most powerful adverse prognostic factor for all times to event,especially for the survival time (P textless.0001) and was followed by beta2-microglobulin (beta2m) levels 2.5 mg/L or higher (P =.0001). The comparison of survival prognostic models including beta2m 2.5 mg/L or greater and another factor favored the Delta13/beta2m combination. In 22 patients (20%) with no unfavorable factor,the median survival time was not reached at 111 months. In contrast,among 55 patients (50%) with one unfavorable factor and 33 patients (30%) with 2 unfavorable factors,median survival times were 47.3 +/- 4.6 months and 25.3 +/- 3.2 months,respectively (P textless.0001). We conclude that delta13,adequately detected by FISH analysis,is a very strong factor related to poor survival,especially when associated with a beta2m level of 2.5 mg/L or higher. Routine FISH Delta13 assessment is strongly recommended for patients considered for HDT. View Publication

Natural killer T (NKT) cells are innate-like lymphocytes that recognize lipid antigens and have been shown to enhance B-cell activation and antibody production. B cells typically recruit T-cell help by presenting internalized antigens recognized by their surface antigen receptor. Here,we demonstrate a highly efficient means whereby human B cells present lipid antigens to NKT cells,capturing the antigen using apolipoprotein E (apoE) and the low-density lipoprotein receptor (LDL-R). ApoE dramatically enhances B-cell presentation of alpha-galactosylceramide (alphaGalCer),an exogenous CD1d presented antigen,inducing activation of NKT cells and the subsequent activation of B cells. B cells express the LDL-R on activation,and the activation of NKT cells by B cells is completely LDL-R dependent,as shown by blocking experiments and the complete lack of presentation when using apoE2,an isoform of apoE incapable of LDL-R binding. The dependence on apoE and the LDL-R is much more pronounced in B cells than we had previously seen in dendritic cells,which can apparently use alternate pathways of lipid antigen uptake. Thus,B cells use an apolipoprotein-mediated pathway of lipid antigen presentation,which constitutes a form of innate help for B cells by NKT cells. View Publication