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

Mature erythrocytes are known to lack major histocompatibility complex (MHC) proteins. However,the presence of MHC molecules on erythrocytes has been occasionally reported,though without a defined function. In this study,we designed erythrocyte conjugated solely with a fusion protein consisting of an antigenic peptide linked to MHC class I (MHC-I) protein,termed MHC-I‒Ery. The modified erythrocyte,decorated with the peptide derived from human papillomavirus (HPV) 16 oncoprotein E6/E7,effectively activated antigen-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from HPV16+ cervical cancer patients. Additionally,MHC-I‒Ery monotherapy was shown to inhibit antigen-positive tumor growth in mice. This treatment immediately activated CD8+ T cells and reduced suppressive myeloid cells in the spleen,leading to systemic anti-tumor activity. Safety and tolerability evaluations of MHC-I‒Ery in non-human primates further supported its clinical potential. Our results first demonstrated that erythrocytes equipped solely with antigen peptide‒MHC-I complexes can robustly stimulate the immune system,suggesting a novel and promising approach for advancing cancer immunotherapy. View Publication

Cystic Fibrosis (CF) is a common genetic disease in the United States,resulting from mutations in the Cystic Fibrosis transmembrane conductance regulator (cftr) gene. CFTR modulators,particularly Elexacaftor/Tezacaftor/Ivacaftor (ETI),have significantly improved clinical outcomes for patients with CF. However,many CFTR mutations are not eligible for CFTR modulator therapy due to their rarity. In this study,we report that a patient carrying rare complex CFTR mutations,c.1680-877G>T and c.3067_3072delATAGTG,showed positive clinical outcomes after ETI treatment. We demonstrate that ETI was able to increase the expression of CFTR harboring c.3067_3072delATAGTG in a heterologous system. Importantly,patient-derived nasal epithelial cells in an air–liquid interface (ALI) culture showed improved CFTR function following ETI treatment. These findings supported the initiation of ETI with the patient. Retrospective studies have suggested that the patient has shown small but steady improvement over the past two years in several clinical metrics,including lung function,body mass index (BMI),and sweat chloride levels. Our studies suggest that ETI could be beneficial for patients carrying c.3067_3072delATAGTG. View Publication

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes,with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations,leading to behavioural pathologies in humans,remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome,we narrowed this cellular phenotype to a single gene candidate,frizzled 9 (FZD9). At the neuronal stage,layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites,increased numbers of spines and synapses,aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain. View Publication

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor,cyclopamine,and the activators,Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes. View Publication

Animal models of the neuromuscular junction (NMJ) have been widely studied but exhibit critical differences from human biology limiting utility in drug and disease modelling. Challenges with scarcity,scalability,throughput,and ethical considerations further limit the suitability of animal models for preclinical screening. Engineered models have emerged as alternatives for studying NMJ functionality in response to genetic and/or pharmacological challenge. However,these models have faced challenges associated with their poorly scalable creation,sourcing suitable cells,and the extraction of reliable,quantifiable metrics. We present a turnkey iPSC-based model of the NMJ employing channelrhodopsin-2 expression within the motor neuron (MN) population driving muscle contraction in response to blue light. MNs co-cultured with engineered skeletal muscle tissues produced twitch forces of 34.7 ± 22.7 µN in response to blue light,with a response fidelity > 92 %. Histological analysis revealed characteristic punctate acetylcholine receptor staining co-localized with the presynaptic marker synaptic vesicle protein-2. Dose-response studies using botulinum neurotoxin showed loss of function in a dose- and time-dependent manner (EC50 - 0.11 ± 0.015 µg). Variability of the EC50 values between 2 different iPSC differentiations of both cell types and 2 users was less than 2 %. Further testing with the acute neurotoxins acetylcholine mustard and d-tubocurarine validated the biological relevance of the postsynaptic machinery of the model. This model marks a meaningful progression of 3D engineered models of the NMJ,providing engineered tissues at a throughput relevant to potency and screening applications with an abundant iPSC cell source and standardized hardware-software ecosystem allowing technology transfer across laboratories. View Publication

We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay,the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 x 10(5) cells. This was significantly higher than the frequency of 1 SRC in 3.0 x 10(6) adult BM cells or 1 in 6.0 x 10(6) mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus,the findings reported here will have important clinical as well as biologic implications. View Publication

Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However,a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here,we describe a non-genetic,label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes,neurons,smooth muscle cells),this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 $$V(rms). Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals,the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry,in which a cell's functionality is assessed rather than its expression profile or physical characteristics. View Publication