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

A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified,but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety,providing sustained drug release,and simultaneously delivering multiple drugs to target tissues and cells. Here,we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4+ T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4+ T cells from virologically suppressed patients. Furthermore,our targeted nanocarriers selectively activated CD4+ T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes,and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure. View Publication

BACKGROUND It is clinically important to maintain high viability and potency of umbilical cord blood units (CBUs) for transplantation during thawing. In the absence of a standard thawing protocol,this study was designed to develop one based on the consensus practice of transplant centers and address the shortage of dextran 40 thawing solution. STUDY DESIGN AND METHODS Frozen CBU aliquots were thawed using dextran 40 thawing solution while manipulating temperature and volume of diluent and mode of dilution. The effects of these on CD45+ and CD34+ cell viability were measured through annexin V and SYTOX green staining. The developed protocol was then used to compare dextran 40 and PLASMA-LYTE A thawing solutions and finally tested on whole CBUs. RESULTS Step-by-step investigations resulted in the development of a protocol that thaws and dilutes CBUs with room temperature diluent to five times the original volume using two sequential dilutions separated by equilibration times. PLASMA-LYTE A diluent provided superior viability of CD45+ and CD34+ cells than dextran 40 and recovered more colony-forming units. However,both diluents were equally effective in maintaining stability of the thawed CBU for 4 hours. Moreover,the stem cell-enriched CD34+CD38- subpopulations appeared more resistant to cryoinjuries than their CD34+CD38+ counterpart. CONCLUSION The developed thawing protocol recovers viable CD45+ and CD34+ cells above the standard thresholds and maintains CBU potency. PLASMA-LYTE A for thawing solution proved to be an efficient alternative to dextran 40. Finally,greater dilution should be avoided to maintain the viability of CD45+ cells and maximize graft cell dose. View Publication

Myeloid Derived Suppressor Cells (MDSCs) play important roles in constituting the immune suppressive environment promoting cancer development and progression. They are consisted of a heterogeneous population of immature myeloid cells including polymorphonuclear MDSC (PMN-MDSC) and monocytes MDSC (M-MDSC) that are found in both the systemic circulation and in the tumor microenvironment (TME). While previous studies had shown that all-trans retinoic acid (ATRA) could induce MDSC differentiation and maturation,the very poor solubility and fast metabolism of the drug limited its applications as an immune-modulator for cancer immunotherapy. We aimed in this study to develop a drug encapsulated liposome formulation L-ATRA with sustained release properties and examined the immuno-modulation effects. We showed that the actively loaded L-ATRA achieved stable encapsulation and enabled controlled drug release and accumulation in the tumor tissues. In vivo administration of L-ATRA promoted the remodeling of the systemic immune homeostasis as well as the tumor microenvironment. They were found to promote MDSCs maturation into DCs and facilitate immune responses against cancer cells. When used as a single agent treatment,L-ATRA deterred tumor growth,but only in immune-competent mice. In mice with impaired immune functions,L-ATRA at the same dose was not effective. When combined with checkpoint inhibitory agents,L-ATRA resulted in greater anti-cancer activities. Thus,L-ATRA may present a new IO strategy targeting the MDSCs that needs be further explored for improving the immunotherapy efficacy in cancer. View Publication

Rationale: IgA can induce activation of neutrophils which are the most abundant cell type in blood,but the development of IgA as therapeutic has been confounded by its short half-life and a weak ability to recruit NK cells as effector cells. Therefore,we generated an X-shaped antibody (X-body) based on the principle of molecular self-assembly that combines the activities of both IgG and IgA,which can effectively recruit and activate NK cells,macrophages,and neutrophils to kill tumor cells. Methods: X-body was generated by using a self-assembly strategy. The affinity of the X-body with the antigen and Fc receptors was tested by surface plasmon resonance. The shape of X-body was examined using negative staining transmission electron microscopy. The tumor cell killing activity of X-body was assessed in vitro and in multiple syngeneic mouse models. To explore the mechanism of X-body,tumor-infiltrating immune cells were analyzed by single-cell RNA-seq and flow cytometry. The dependence of neutrophil,macrophage,and NK cells for the X-body efficacy was confirmed by in vivo depletion of immune cell subsets. Results: The X-body versions of rituximab and trastuzumab combined the full spectrum activity of IgG and IgA and recruited NK cells,macrophages,and neutrophils as effector cells for eradication of tumor cells. Treatment with anti-hCD20 and anti-hHER2 X-bodies leads to a greater reduction in tumor burden in tumor-bearing mice compared with the IgA or IgG counterpart,and no obvious adverse effect is observed upon X-body treatment. Moreover,the X-body has a serum half-life and drug stability comparable to IgG. Conclusions: The X-body,as a myeloid-cell-centered therapeutic strategy,holds promise for the development of more effective cancer-targeting therapies than the current state of the art. View Publication

Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells,followed by the migration,differentiation,and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens,such as FGF8. Disruption of this developmental balance can lead to brain malformations,which underlie a range of complex neurodevelopmental disorders,including epilepsy,autism,and intellectual disabilities. Studying the early stages of human brain development,whether under normal or pathological conditions,remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently,human brain organoids have emerged as a powerful in vitro alternative,allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures,where neural progenitors and neurons are grown on flat surfaces,3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However,3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore,few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning. • To address these limitations,we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs),our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation,NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors,where they self-organize into neural rosettes and neuroepithelial structures,surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids,leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity,enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas. Key features • This protocol builds on the method established by Chambers et al. [1] for generating 2D neural progenitors,followed by dissociation and reaggregation into 3D brain organoids. • For optimal growth and maturation,telencephalic organoids are cultured in spinning mini-bioreactors [2] or on orbital shakers. • The protocol enables the generation of telencephalic neural progenitors in 10 days and produces 3D telencephalic organoids containing neocortical neurons within one month of culture. • Addition of morphogens in the culture medium (example: FGF8) enhances cellular heterogeneity,promoting the emergence of distinct brain domains within a single organoid. View Publication

ABSTRACTCell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors,especially hematological diseases. Complete remission rates (CRR) > 80%–97% and 50%–90% overall response rates (ORR) have been achieved with a treatment based on CAR‐T cells in patients with malignant B‐cell tumors that have relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most patients treated with CAR‐T cells develop high‐grade cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity syndrome (ICANS). However,the unprecedentedly high CRR and ORR have led to the approval of six CAR‐T cell therapeutics by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA),prompting researchers to improve existing products and develop new ones. By now,around 1000 clinical trials based on CAR‐T cells are registered at ClinicalTrials.gov: 82% are for hematological diseases,while the remaining 16% are for solid tumors. As a result of this increased research,an enormous amount of conflicting information has been accumulated in the literature,and each group follows its manufacturing protocols and performs specific in vitro testing. This review aimed to combine and compare clinical and preclinical information,highlighting the most used protocols to provide a comprehensive overview of the in vitro world of CAR‐T cells,from manufacturing to their characterization. The focus is on all steps of the CAR‐T cell manufacturing process,from the collection of patient or donor blood to the enrichment of T cells,their activation with anti‐CD3/CD28 beads,interleukin‐2 (IL‐2) or IL‐7 and IL‐15 (induction of a more functional memory phenotype),and their transfection (viral or non‐viral methods). Automation is crucial for ensuring a standardized final product. View Publication

Seven female individuals with multiple congenital anomalies,developmental delay and/or intellectual disability have been found to have a genetic variant of uncertain significance in the mediator complex subunit 12 gene ( MED12 c.3412C>T,p.Arg1138Trp). The functional consequence of this genetic variant in disease is undetermined,and insight into disease mechanism is required. We identified a de novo MED12 p.Arg1138Trp variant in a female patient and compared disease phenotypes with six female individuals identified in the literature. To investigate affected biological pathways,we derived two induced pluripotent stem cell (iPSC) lines from the patient: one expressing wildtype MED12 and the other expressing the MED12 p.Arg1138Trp variant. We performed neural disease modelling,transcriptomics and protein analysis,comparing healthy and variant cells. When comparing the two cell lines,we identified altered gene expression in neural cells expressing the variant,including genes regulating RNA polymerase II activity,transcription,pre-mRNA processing,and neural development. We also noted a decrease in MED12L expression. Pathway analysis indicated temporal delays in axon development,forebrain differentiation,and neural cell specification with significant upregulation of pre-ribosome complex gene pathways. In a human neural model,expression of MED12 p.Arg1138Trp altered neural cell development and dysregulated the pre-ribosome complex providing functional evidence of disease aetiology and mechanism in MED12-related disorders. The online version contains supplementary material available at 10.1186/s10020-025-01365-5. View Publication

Here,we present a protocol for isolating and culturing mouse photoreceptors in a minimal,chemically defined medium free from serum. We describe steps for retina dissection,enzymatic dissociation,photoreceptor enrichment,cell culture,extracellular vesicles (EVs) enrichment,and EV ultrastructural analysis. This protocol,which has been verified for cultured cells derived from multiple murine strains,allows for the study of several aspects of photoreceptor biology,including EV isolation and nanotube formation. For complete details on the use and execution of this protocol,please refer to Kalargyrou et al. (2021). 1 Subject areas: Cell Biology,Molecular Biology,Neuroscience View Publication

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7,termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity,some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity,several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus,as well as sequence following the Sec7 domain,also had high activity. The mutant lacking 630 N-terminal amino acids was,however,only 1% as active,as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5,and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated,but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at textless/=50 microM. The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200. Thus,the Sec7 domain of p200,like that of Sec7 itself (Sata,M.,Donaldson,J. G.,Moss,J.,and Vaughan,M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,4204-4208),plays a role in BFA inhibition as well as in GEP activity,although the latter is markedly modified by other structural elements. View Publication

T-cell-mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis,allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL-2-inducible tyrosine kinase (Itk),an enzyme acting downstream of the T-cell receptor (TCR),in T-cell-dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor,Compound 44,created by chemical optimization of an initial high-throughput screening hit,inhibited Itk's activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition,our data reveal that human Itk,comparable to its murine homologue,is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally,silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR-induced lymphokine secretion. In conclusion,Itk represents an interesting new target for the therapy of T-cell-mediated inflammatory skin diseases. View Publication

It is widely accepted that canonical Wnt (cWnt) signaling is required for the differentiation of osteoprogenitors into osteoblasts. Furthermore,tumor-derived secretion of the cWnt-antagonist Dickkopf-1 (Dkk-1) is known to cause bone destruction,inhibition of repair and metastasis in many bone malignancies,but its role in osteosarcoma (OS) is still under debate. In this study,we examined the role of Dkk-1in OS by engineering its overexpression in the osteochondral sarcoma line MOS-J. Consistent with the known role of Dkk-1 in osteoblast differentiation,Dkk-1 inhibited osteogenesis by the MOSJ cells themselves and also in surrounding tissue when implanted in vivo. Surprisingly,Dkk-1 also had unexpected effects on MOSJ cells in that it increased proliferation and resistance to metabolic stress in vitro and caused the formation of larger and more destructive tumors than controls upon orthotopic implantation. These effects were attributed in part to upregulation of the stress response enzyme and cancer stem cell marker aldehyde-dehydrogenase-1 (ALDH1). Direct inhibition of ALDH1 reduced viability under stressful culture conditions,whereas pharmacological inhibition of cWnt or overexpression of ALDH1 had a protective effect. Furthermore,we observed that ALDH1 was transcriptionally activated in a c-Jun-dependent manner through a pathway consisting of RhoA,MAP-kinase-kinase-4 and Jun N-terminal Kinase (JNK),indicating that noncanonical planar cell polarity-like Wnt signaling was the mechanism responsible. Together,our results therefore demonstrate that Dkk-1 enhances resistance of OS cells to stress by tipping the balance of Wnt signaling in favor of the non-canonical Jun-mediated Wnt pathways. In turn,this results in transcriptional activation of ALDH1 through Jun-responsive promoter elements. This is the first report linking Dkk-1 to tumor stress resistance,further supporting the targeting of Dkk-1 not only to prevent and treat osteolytic bone lesions but also to reduce numbers of stress-resistant tumor cells. View Publication

Characterization and modeling of biological neural networks has emerged as a field driving significant advancements in our understanding of brain function and related pathologies. As of today,pharmacological treatments for neurological disorders remain limited,pushing the exploration of promising alternative approaches such as electroceutics. Recent research in bioelectronics and neuromorphic engineering have fostered the development of the new generation of neuroprostheses for brain repair. However,achieving their full potential necessitates a deeper understanding of biohybrid interaction. In this study,we present a novel real-time,biomimetic,cost-effective and user-friendly neural network capable of real-time emulation for biohybrid experiments. Our system facilitates the investigation and replication of biophysically detailed neural network dynamics while prioritizing cost-efficiency,flexibility and ease of use. We showcase the feasibility of conducting biohybrid experiments using standard biophysical interfaces and a variety of biological cells as well as real-time emulation of diverse network configurations. We envision our system as a crucial step towards the development of neuromorphic-based neuroprostheses for bioelectrical therapeutics,enabling seamless communication with biological networks on a comparable timescale. Its embedded real-time functionality enhances practicality and accessibility,amplifying its potential for real-world applications in biohybrid experiments. Beaubois et al. introduce a real-time biomimetic neural network for biohybrid experiments,providing a tool to study closed-loop applications for neuroscience and neuromorphic-based neuroprostheses. View Publication