Development and Validation of a Simplified Method to Generate Human Microglia from Pluripotent Stem Cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE117829 | GEO | 2018/12/05
REPOSITORIES: GEO
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Project description:Development and Validation of a Simplified Method to Generate Human Microglia from Pluripotent Stem Cells
| PRJNA483247 | ENA
Project description:BACKGROUND:Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS:iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGF?-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGF? signaling (IDE1) can be used to replace recombinant TGF?1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered. CONCLUSION:We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.
| S-EPMC6303871 | biostudies-literature
Project description:We developed a simplified proteomics sample preparation method that increased the sensitivity and reproducibility of large-scale plasma samples. Peptide loss was reduced while sensitivity was increased by eliminating the peptide clean-up step and using disposable trap column tips. The simplified High-Throughput Plasma Proteomics (sHTPP) method was evaluated using a systemic approach. We identified and quantified more than 500 proteins in non-depleted plasma samples without missing values as the DIA approach improved in terms of identification and quantification. Furthermore, 96-well plate sample handling with a multi-channel pipet allows for high-throughput sample analysis. In a large-scale clinical trial with 300 plasma samples, we used the method to demonstrate robust and accurate quantifications.
2023-04-03 | PXD038669 | Pride
Project description:An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells
| PRJNA796518 | ENA
Project description:Generating primordial germ cells (PGCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but currently entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days, with higher efficiencies and improved consistency across multiple hPSC lines. We used our simplified differentiation platform and single-cell RNA-sequencing to uncover new insights into PGC specification. Transient WNT activation for 12 hours followed by WNT inhibition specified PGCs; by contrast, sustained WNT instead induced primitive streak. Thus, somatic (primitive streak) and PGCs are related—yet distinct—lineages segregated by temporally-dynamic signaling. Pluripotency factors are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor strong transcriptional similarities with fetal PGCs. We report single-cell RNA-sequencing data of in vitro differentiated hPSCs (H9) cells into human Primordial germ cell-like cells (PGCLCs), profiled at different time-points of differentiation - Day 0 (H9), D0.5, D3.5 (sorted) and D3.5 unsorted population and a negative control of hPSCs differentiated into definitive endoderm (DE) (using the protocol described in (Loh et al., 2014, Cell Stem Cell 14, 237-252)). The data comprises of 5 single-cell RNA-sequencing libraries (H9, D0.5, D3.5S, D3.5U and DE) generated using 10xChromium Single cell RNA-expression platform and 10xGenomics Chromium version 2 chemistry with a targeted capture of ~6000 cells each. Single cell libraries were sequenced on Illumina HiSeq4000 platform as paired-end 150bp reads.
2023-08-09 | GSE157475 | GEO
Project description:A systems-level understanding of a small but essential population of cells in development or adulthood (e.g., somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. Experiment Overall Design: To verify the ability of the developed method, we prepared total RNA from ES cells and diluted it to the single-cell level (10 pg). Eight array data from the cDNA samples amplified independently from the 10 pg RNA (amplified samples) were compared to each other, and to eight array data from the undiluted 5 ug RNA (nonamplified controls). Since all the materials were the same, all of the observed variations were attributable to the methodogoly.
2008-06-13 | E-GEOD-4308 | biostudies-arrayexpress
Project description:Generating primordial germ cells (PGCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but currently entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days, with higher efficiencies and improved consistency across multiple hPSC lines. We used our simplified differentiation platform, bulk RNA-seq and single-cell RNA-sequencing to uncover new insights into PGC specification. Transient WNT activation for 12 hours followed by WNT inhibition specified PGCs; by contrast, sustained WNT instead induced primitive streak. Thus, somatic (primitive streak) and PGCs are related—yet distinct—lineages segregated by temporally-dynamic signaling. Pluripotency factors are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor strong transcriptional similarities with fetal PGCs. We report single-cell RNA-sequencing data of in vitro differentiated hPSCs (H9) cells into human Primordial germ cell-like cells (PGCLCs), profiled at different time-points of differentiation - Day 0 (H9), D0.5, D3.5 (sorted) and D3.5 unsorted population and a negative control of hPSCs differentiated into definitive endoderm (DE) (using the protocol described in (Loh et al., 2014, Cell Stem Cell 14, 237-252)). The data comprises of 5 single-cell RNA-sequencing libraries (H9, D0.5, D3.5S, D3.5U and DE) generated using 10xChromium Single cell RNA-expression platform and 10xGenomics Chromium version 2 chemistry with a targeted capture of ~6000 cells each. Single cell libraries were sequenced on Illumina HiSeq4000 platform as paired-end 150bp reads. Bulk RNA seq libraries were sequnced as paired-end reads.
2023-08-09 | GSE210711 | GEO
Project description:Plant plasma membranes (PMs) play an important role in maintaining the stability of the intracellular environment and exchanging information with the external environment. Therefore, deciphering dynamics of PM proteome provides crucial information for elucidating cellular regulation in response to diverse stimuli. In the study, we developed a simplified method for enriching PM proteins in leaf and root tissues of a tropical forage Stylosanthes by combining differential centrifugation and Brij-58 treatment. Both immunoblot analysis and mass spectrometry demonstrated that the representation and abundance of PM proteins were increased in the enrichment fraction, and the contamination of other organellar proteins was decreased. A total of 425 and 387 proteins were predicted to be PM proteins in leaves and roots respectively. Functional analysis classified these PM proteins into six major categories (transporter, enzyme, receptor, membrane structure protein, vesicular trafficking and chaperone), and orthologs of many PM proteins regulating the responses to abiotic and biotic stresses have been detected. In addition, the PM localization of a newly identified receptor-like kinase, SgRKL1, has been verified. Together, these results show that the simplified PM enrichment method can be successfully applied to different plant tissue types and to study the dynamics of PM proteome of Stylosanthes in response to multiple stresses.
2022-12-14 | PXD037423 | Pride