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

Human neutrophilic granulocytes form the largest pool of innate immune cells for host defense against bacterial and fungal pathogens. The dynamic changes that accompany the metamorphosis from a proliferating myeloid progenitor cell in the bone marrow into a mature non-dividing polymorphonuclear blood cell have remained poorly defined. Using mass spectrometry-based quantitative proteomics combined with transcriptomic data,we report on the dynamic changes of five developmental stages in the bone marrow and blood. Integration of transcriptomes and proteome unveils highly dynamic and differential interactions between RNA and protein kinetics during human neutrophil development,which can be linked to functional maturation of typical end-stage blood neutrophil killing activities. View Publication

The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm,leading to death by respiratory failure. Human experimental models to study phMNs are lacking,hindering the understanding of the mechanisms of phMN degeneration in ALS. Here,we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development,phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels,under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine,followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines,offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research,it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest. View Publication

Klotho,a well-known aging suppressor protein,has been implicated in neuroprotection and the regulation of neuronal senescence. While previous studies have demonstrated its anti-aging properties in human brain organoids,its potential to mitigate neurodegenerative processes triggered by ?-amyloid remains underexplored. In this study,we utilised human induced pluripotent stem cells (iPSCs) engineered with a doxycycline-inducible system to overexpress KLOTHO and generated 2D cortical neuron cultures from these cells. These neurons were next exposed to pre-aggregated ?-amyloid 1–42 oligomers to model the neurotoxicity associated with Alzheimer’s disease. Our data reveal that upregulation of KLOTHO significantly reduced ?-amyloid-induced neuronal degeneration and apoptosis,as evidenced by decreased cleaved caspase-3 expression and preservation of axonal integrity. Additionally,KLOTHO overexpression prevented the loss of dendritic branching and mitigated reductions in axonal diameter,hallmark features of neurodegenerative pathology. These results highlight Klotho’s protective role against ?-amyloid-induced neurotoxicity in human cortical neurons and suggest that its age-related decline may contribute to neurodegenerative diseases such as Alzheimer’s disease. Our findings underscore the therapeutic potential of Klotho-based interventions in mitigating age-associated neurodegenerative processes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13041-025-01199-6. View Publication

Organoids,derived from human induced pluripotent stem cells (hiPSCs),are intricate three-dimensional in vitro structures that mimic many key aspects of the complex morphology and functions of in vivo organs such as the retina and heart. Traditional histological methods,while crucial,often fall short in analyzing these dynamic structures due to their inherently static and destructive nature. In this study,we leveraged the capabilities of optical coherence tomography (OCT) for rapid,non-invasive imaging of both retinal,cerebral,and cardiac organoids. Complementing this,we developed a sophisticated deep learning approach to automatically segment the organoid tissues and their internal structures,such as hollows and chambers. Utilizing this advanced imaging and analysis platform,we quantitatively assessed critical parameters,including size,area,volume,and cardiac beating,offering a comprehensive live characterization and classification of the organoids. These findings provide profound insights into the differentiation and developmental processes of organoids,positioning quantitative OCT imaging as a potentially transformative tool for future organoid research. View Publication

Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However,methodologies for isolating functional synaptic structures from these models remain limited. Here,we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from air‐liquid interface cerebral organoids (ALI‐COs) at distinct developmental stages (Day 90 and 150). Notably,the method avoids density gradients,requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g.,GAP43) and classical synaptosomal proteins (e.g.,PCLO,BSN,SYN1). Transmission electron microscopy (TEM) confirmed the presence of membrane‐enclosed GCPs with fibrous content and mitochondria in Day 90 isolates,and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KCl‐induced depolarization,which triggered phosphorylation changes in growth cone proteins (GAP43,MARCKS,MARCKSL1),cytoskeletal regulators (DCLK1,SHTN1,MARK4,MAP1B) and protein kinases (CAMK2G,PRKCE) in Day 90 GCPs,as well as classical synaptic vesicle cycle proteins (SYN1,DNM1,RPH3A) at Day 150. Overall,this study establishes a centrifugation‐based protocol for isolating growth cones and immature synapses from human organoids,capturing key stages of synaptic development and enabling scalable,patient‐compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders. Synapses are implicated in several neurological disorders and psychiatric diseases. The emergence and wide use of neural organoids provide a new opportunity to study human synapses in healthy and disease settings. Therefore,we developed a simple method for the enrichment of synaptosomes and growth cone particles from forebrain organoids. The method is based on differential centrifugation,works with small tissue amounts,and is highly reproducible. We validated the functionality of the isolated structures using KCl stimulation and phosphoproteomics. The method enables detailed mapping of protein composition and function during growth cone pathfinding,synaptogenesis,and establishment of neural circuits in organoids. View Publication

The clinical application of T cells engineered with chimeric antigen receptors (CARs) for solid tumors is challenging. A major reason for this involves tumor immune evasion mechanisms,including the high expression of immune checkpoint molecules,such as the programmed death 1 (PD-1) ligands PD-L1 and PD-L2. The inducible expression of PD-L1 in tumors has been observed after CAR-T-cell infusion,even in tumors natively not expressing PD-L1. Furthermore,numerous types of pediatric cancer do not have suitable targets for CAR-T-cell therapy. Therefore,the present study aimed to develop novel CAR-T cells that target PD-L1 and PD-L2,and to evaluate their efficacy against pediatric solid tumors. A novel CAR harboring the immunoglobulin V-set domain of the human PD-1 receptor as an antigen binding site (PD-1 CAR-T) was developed without using a single-chain variable fragment. PD-1 CAR-T cells were successfully manufactured by adding an anti-PD-1 antibody,nivolumab,to the ex vivo expansion culture to prevent fratricide during the manufacturing process due to the inducible expression of PD-L1 in activated human T cells. The expression of PD-L1 (and PD-L2 to a lesser extent) was revealed to be highly upregulated in various pediatric solid tumor cells,which displayed no or very low expression initially,on in vitro exposure to interferon-γ and/or tumor necrosis factor-α,which are cytokines secreted by tumor-infiltrating T cells. Furthermore,PD-1 CAR-T cells exhibited strong cytotoxic activity against pediatric solid tumor cells expressing PD-L1 and PD-L2. Conversely,the effect of PD-1 CAR-T cells was significantly attenuated against PD-L1-positive cells coexpressing CD80,suggesting that the toxicity of PD-1 CAR-T cells to normal immune cells,including antigen presenting cells,can be minimized. In conclusion,PD-1 ligands are promising therapeutic targets for pediatric solid tumors. PD-1 CAR-T cells,either alone or in combination with CAR-T cells with other targets,represent a potential treatment option for solid tumors. View Publication

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines,of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice,we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH(br) cells) from the OS99-1 xenografts were much less frequent,averaging 3% of the entire tumor population,compared to those isolated directly from the OS99-1 cell line. ALDH(br) cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH(lo) cells),generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH(br) cells illustrated the stem cell characteristics of self-renewal,the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A,Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma. View Publication

In an attempt to bring pluripotent stem cell biology closer to reaching its full potential,many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included. View Publication

BACKGROUND Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has become an increasingly vital tool for modifying gene expression in a variety of cell types. Lentiviral transduction and electroporation are the two main approaches used to deliver CRISPR/Cas9 into cells. However,the application of CRISPR/Cas9 in primary hematopoietic cells has been limited due to either low transduction efficiency in terms of viral-based delivery or difficult selection and enrichment of transfected and edited cells with respect to electroporation of CRISPR/Cas9 ribonucleoprotein (RNP). METHODS In this study in vitro transcription was used to synthesize the guide RNA (gRNA),and plasmid pL-CRISPR.EFS.GFP was used as its DNA template. Then the in vitro transcribed gRNA was labeled with pCp-Cy5 via T4 ligase before incubating with Cas9 protein. Furthermore,CRISPR/Cas9 RNP was electroporated into primary CD34+ cells isolated from cord blood,and cell survival rate and transfection efficiency were calculated and compared to that of lentiviral transduction. RESULTS Here,we show that electroporation of CRISPR/Cas9 RNP resulted in higher cell viability compared to electroporation of CRISPR/Cas9 all-in-one plasmid,providing important findings for further studies in hematology via CRISPR/Cas9 technology. Moreover,we established a method for labeling in vitro-transcribed gRNA with fluorophore and the sorted fluorescent cells displayed higher knockout efficiency than nonsorted transfected cells. CONCLUSIONS Electroporation of fluorescence labeled CRISPR/Cas9 RNP is a perspective approach of gene editing. Our study provides an efficient and time-saving approach for genome-editing in hematopoietic cells. View Publication

BACKGROUND: The discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). AIM: To evaluate and compare five iMSC differentiation protocols,assessing their efficiency,phenotypic characteristics,and functional properties relative to primary mesenchymal stem cells (MSCs). METHODS: Five iMSC differentiation protocols were assessed: SB431542-based differentiation (iMSC1,iMSC3),an iMatrix-free method (iMSC2),growth factor supplementation (iMSC4),and embryoid body formation with retinoic acid (EB-iMSC). iMSC identity was confirmed according to the International Society for Cell & Gene Therapy 2006 criteria,requiring expression of surface markers (CD105,CD73,CD90) and absence of pluripotency markers. Functional assays were conducted to evaluate differentiation potential (osteogenic and adipogenic),proliferation,mitochondrial function,reactive oxygen species,senescence,and migration. RESULTS: All iMSC types expressed MSC markers and lacked pluripotency markers. EB-iMSC and iMSC2 showed enhanced osteogenesis (runt-related transcription factor 2; P ≤ 0.01 and P ≤ 0.0001,respectively),while adipogenic potential was reduced in iMSC2 (Adipsin; P ≤ 0.01) and EB-iMSC (Adipsin and peroxisome proliferator-activated receptor gamma; P ≤ 0.0001 and P ≤ 0.01,respectively). Proliferation was comparable or superior to bone marrow MSCs,except in iMSC1,with iMSC4 showing the highest rate (MTT assay; P values ranged from 0.01 to 0.001). Despite reduced mitochondrial health in iMSC3 and iMSC4 (P ≤ 0.001),reactive oxygen species levels were lower in all iMSCs (P values ranged from 0.001 to 0.0001),and senescence was significantly reduced in all iMSCs with the exception of iMSC1 (P values ranged from 0.01 to 0.0001). Migration was most reduced in iMSC4 (P ≤ 0.001 at 24 hours and P ≤ 0.0001 at 48 hours). CONCLUSION: While all protocols generated functional iMSCs,variations in differentiation,proliferation,and function emphasize the impact of protocol selection. These findings contribute to optimizing iMSC generation for research and clinical applications. View Publication

BACKGROUND MicroRNAs (miRs or miRNAs) regulate several biological processes in the cell. However,evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. Recently,we have shown that apoptosis-associated factors,such as p53 and caspases participate in the differentiation process of mouse neural stem (NS) cells. To identify apoptosis-associated miRNAs that might play a role in neuronal development,we performed global miRNA expression profiling experiments in NS cells. Next,we characterized the expression of proapoptotic miRNAs,including miR-16,let-7a and miR-34a in distinct models of neural differentiation,including mouse embryonic stem cells,PC12 and NT2N cells. In addition,the expression of antiapoptotic miR-19a and 20a was also evaluated. RESULTS The expression of miR-16,let-7a and miR-34a was consistently upregulated in neural differentiation models. In contrast,expression of miR-19a and miR-20a was downregulated in mouse NS cell differentiation. Importantly,differential expression of specific apoptosis-related miRNAs was not associated with increased cell death. Overexpression of miR-34a increased the proportion of postmitotic neurons of mouse NS cells. CONCLUSIONS In conclusion,the identification of miR-16,let-7a and miR-34a,whose expression patterns are conserved in mouse,rat and human neural differentiation,implicates these specific miRNAs in mammalian neuronal development. The results provide new insights into the regulation of neuronal differentiation by apoptosis-associated miRNAs. View Publication

Natural killer (NK) cell differentiation from pluripotent CD34(+) human hematopoietic stem cells or oligopotent lymphoid progenitors has already been reported. In the present study,long-term cultures of the CD56(-)/CD34(-) myeloid-like adherent cell fraction (ACF) from umbilical cord blood (UCB),characterized by the expression of CD14(+) as well as other myeloid markers,were set up with flt3 ligand (FL) and interleukin-15 (IL-15). The UCB/ACF gradually expressed the CD56 marker,which reached fairly high levels (approximately 90% of the cells were CD56(+)) by day 15. FL plus IL-15-driven ACF/CD56(+) cells progressively expressed a mature NK functional program lysing both NK- and lymphokine-activate killer (LAK)-sensitive tumor targets and producing high levels of interferon-gamma (IFN-gamma),granulocyte-macrophage colony-stimulating factor,tumor necrosis factor alpha,and IL-10 upon stimulation with IL-12 and IL-18. Similar results were obtained when highly purified CD14(+) cells from UCB were cultured with FL and IL-15. In contrast,UCB/CD34(+) cells cultured under the same conditions showed a delayed expression of CD56 and behaved functionally differently in that they exhibited NK but not LAK cytotoxicity and produced significantly fewer cytokines. Kinetic studies on the phenotype of UCB/ACF or UCB/CD14(+) cells cultured in the presence of FL and IL-15 showed a rapid decrease in CD14 expression after day 5,which reached levels of zero by day 20. Approximately 60% of the CD56(+) derived from the UCB/ACF or the UCB/CD14(+) cells coexpressed CD14 by day 5. Taken together,our data support the role of CD14(+) myeloid-like cells within UCB as a novel progenitor for lymphoid NK cells. View Publication