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

BACKGROUND In kidney transplantation,early allograft inflammation impairs long-term allograft function. However,precise mediators of early kidney allograft inflammation are unclear,making it challenging to design therapeutic interventions. METHODS We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. RESULTS Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period,they were also induced to express a high level of Ccl8,which,in turn,promoted recipient monocyte graft infiltration,their differentiation to resident macrophages,and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed,including CD4,CD8,and ?? T cells. Consequently,blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. CONCLUSIONS Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation. View Publication

Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

Progressive resistance exercise training (PRT) is the most effective known intervention for combating aging skeletal muscle atrophy. However,the hypertrophic response to PRT is variable,and this may be due to muscle inflammation susceptibility. Metformin reduces inflammation,so we hypothesized that metformin would augment the muscle response to PRT in healthy women and men aged 65 and older. In a randomized,double-blind trial,participants received 1,700 mg/day metformin (N = 46) or placebo (N = 48) throughout the study,and all subjects performed 14 weeks of supervised PRT. Although responses to PRT varied,placebo gained more lean body mass (p = .003) and thigh muscle mass (p {\textless} .001) than metformin. CT scan showed that increases in thigh muscle area (p = .005) and density (p = .020) were greater in placebo versus metformin. There was a trend for blunted strength gains in metformin that did not reach statistical significance. Analyses of vastus lateralis muscle biopsies showed that metformin did not affect fiber hypertrophy,or increases in satellite cell or macrophage abundance with PRT. However,placebo had decreased type I fiber percentage while metformin did not (p = .007). Metformin led to an increase in AMPK signaling,and a trend for blunted increases in mTORC1 signaling in response to PRT. These results underscore the benefits of PRT in older adults,but metformin negatively impacts the hypertrophic response to resistance training in healthy older individuals. ClinicalTrials.gov Identifier: NCT02308228. View Publication

The vascular wall is a source of progenitor cells that are able to induce skeletal repair,primarily by paracrine mechanisms. Here,the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First,purified human perivascular stem cells (PSCs) were observed to induce mitogenic,pro-migratory,and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic,pro-migratory,and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins,as demonstrated by EV trypsinization,or neutralizing antibodies for CD9 or CD81. Moreover,shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally,PSC-EVs stimulated bone repair,and did so via stimulation of skeletal cell proliferation,migration,and osteodifferentiation. In sum,PSC-EVs mediate the same tissue repair effects of perivascular stem cells,and represent an 'off-the-shelf' alternative for bone tissue regeneration. View Publication

Human cortical neural progenitor cell transplantation holds significant potential in cortical stroke treatment by replacing lost cortical neurons and repairing damaged brain circuits. However,commonly utilized human cortical neural progenitors are limited in yield a substantial proportion of diverse cortical neurons and require an extended period to achieve functional maturation and synaptic integration,thereby potentially diminishing the optimal therapeutic benefits of cell transplantation for cortical stroke. Here,we generated forkhead box G1 (FOXG1)-positive forebrain progenitors from human inducible pluripotent stem cells,which can differentiate into diverse and balanced cortical neurons including upper- and deep-layer excitatory and inhibitory neurons,achieving early functional maturation simultaneously in vitro. Furthermore,these FOXG1 forebrain progenitor cells demonstrate robust cortical neuronal differentiation,rapid functional maturation and efficient synaptic integration after transplantation into the sensory cortex of stroke-injured adult rats. Notably,we have successfully utilized the non-invasive 18F-SynVesT-1 PET imaging technique to assess alterations in synapse count before and after transplantation therapy of FOXG1 progenitors in vivo. Moreover,the transplanted FOXG1 progenitors improve sensory and motor function recovery following stroke. These findings provide systematic and compelling evidence for the suitability of these FOXG1 progenitors for neuronal replacement in ischemic cortical stroke. Human NPCs show promise for stroke treatment,but challenges remain in neuron diversity and maturation time. Here,the authors describe the generation of FOXG1 progenitors from iPSCs that quickly mature into functional cortical neurons,enhancing stroke recovery in rats. View Publication

IntroductionCRISPR/Cas9-edited induced pluripotent stem cells (iPSCs) are valuable research models for mechanistic studies. However,gene conversion between a gene-pseudogene pair that share high sequence identity and form direct repeats in proximity on the same chromosome can interfere with the precision of gene editing. Mutations in the human beta-glucocerebrosidase gene (GBA1) are associated with Gaucher disease,Parkinson’s disease,and Lewy body dementia. During the creation of a GBA1 KO iPSC line,we detected about 70% gene conversion from its pseudogene GBAP1. These events maintained the reading frame and resulted from GBA1-specific cleavage by CRISPR/Cas9,without disrupting the GBA1 gene.MethodTo increase the percentage of alleles with out-of-frame indels for triggering nonsense-mediated decay of the GBA1 mRNA,we supplied the cells with two single-stranded oligodeoxynucleotide (ssODN) donors as homology-directed repair (HDR) templates.ResultsWe demonstrate that HDR using the ssODN templates effectively competes with gene conversion and enabled biallelic KO clone isolation,whereas the nonallelic homologous recombination (NAHR)-based deletion rate remained the same.DiscussionHere,we report a generalizable method to direct cellular DNA repair of double strand breaks at a target gene towards the HDR pathway using exogenous ssODN templates,allowing specific editing of one gene in a gene-pseudogene pair without disturbing the other. View Publication

Genomic imprinting controls parental allele-specific gene expression via epigenetic mechanisms. Abnormal imprinting at the GNAS gene causes multiple phenotypes,including pseudohypoparathyroidism type-1B (PHP1B),a disorder of multihormone resistance. Microdeletions affecting the neighboring STX16 gene ablate an imprinting control region (STX16-ICR) of GNAS and lead to PHP1B upon maternal but not paternal inheritance. Mechanisms behind this imprinted inheritance mode remain unknown. Here,we show that the STX16-ICR forms different chromatin conformations with each GNAS parental allele and enhances two GNAS promoters in human embryonic stem cells. When these cells differentiate toward proximal renal tubule cells,STX16-ICR loses its effect,accompanied by a transition to a somatic cell-specific GNAS imprinting status. The activity of STX16-ICR depends on an OCT4 motif,whose disruption impacts transcript levels differentially on each allele. Therefore,a biallelically active embryonic enhancer dictates GNAS imprinting via different chromatin conformations,underlying the allele-specific pathogenicity of STX16-ICR microdeletions. STX16 microdeletions cause pseudohypoparathyroidism type-1B only on the maternal allele. Here,the authors show that the allele-specific pathogenicity reflects differential conformations of a biallelically active enhancer dictating GNAS imprinting. View Publication

Brain tumors are commonly treated with radiotherapy,but the efficacy of the treatment is limited by its toxicity to the normal tissue including post-irradiation contrast enhanced lesions often linked to necrosis. The poorly understood mechanisms behind such brain lesions were studied using cerebral organoids. Here we show that irradiation of such organoids leads to dose-dependent growth retardation and formation of liquid-filled cavities but is not correlated with necrosis. Instead,the radiation-induced changes comprise of an enhancement of cortical hem markers,altered neuroepithelial stem cell differentiation,and an increase of ZO1+/AQP1+/CLDN3+-choroid plexus (CP)-like structures accompanied by an upregulation of IGF2 mRNA,known to be expressed in CP and cerebrospinal fluid. The altered differentiation is attributed to changes in the WNT/BMP signaling pathways. We conclude that aberrant CP formation can be involved in radiation-induced brain lesions providing additional strategies for possible countermeasures. Human cerebral organoids provide insights into mechanisms behind the formation of choroid plexus (CP)-like structures that may contribute to radiation-induced brain image changes. View Publication

Huntington disease (HD) is a neurodegenerative disease caused by the abnormal expansion of a polyglutamine tract resulting from a mutation in the HTT gene. Oxidative stress has been identified as a significant contributing factor to the development of HD and other neurodegenerative diseases,and targeting anti-oxidative stress has emerged as a potential therapeutic approach. CHCHD2 is a mitochondria-related protein involved in regulating cell migration,anti-oxidative stress,and anti-apoptosis. Although CHCHD2 is highly expressed in HD cells,its specific role in the pathogenesis of HD remains uncertain. We postulate that the up-regulation of CHCHD2 in HD models represents a compensatory protective response against mitochondrial dysfunction and oxidative stress associated with HD. To investigate this hypothesis,we employed HD mouse striatal cells and human induced pluripotent stem cells (hiPSCs) as models to examine the effects of CHCHD2 overexpression (CHCHD2-OE) or knockdown (CHCHD2-KD) on the HD phenotype. Our findings demonstrate that CHCHD2 is crucial for maintaining cell survival in both HD mouse striatal cells and hiPSCs-derived neurons. Our study demonstrates that CHCHD2 up-regulation in HD serves as a compensatory protective response against oxidative stress,suggesting a potential anti-oxidative strategy for the treatment of HD. View Publication

The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs),whose role in antiviral immunity is elusive. Here,we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state,GLUT4 is docked at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9,TUG). Following RNA virus infection,GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs,preventing them from activating IFN-? responses. UBXN9 deletion prompts constitutive GLUT4 trafficking,sequestration of RLRs,and attenuation of antiviral immunity,whereas GLUT4 deletion heightens RLR signaling. Notably,reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall,our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs. View Publication