Single-cell sequencing has allowed us to perform multiple analysis of different cell types in a large number of specimens and in culture samples (14). Thus, by means of single-cell sequencing using the GBM samples, cells with high proliferation and differentiative capacity were defined as GSCs. different subtypes of cells and further analyze the evolutionary relationship between each subtype of tumor cells, as well as immune cells. First, we identified subtypes of GSCs in surgical specimens according to the high proliferation characteristics. Then, we constructed the coculture model of T cells and GSCs. We cross-validated the DNA expression patterns in the GCSs in the established coculture model and surgical specimens. An ideal similarity was detected. Further, we depicted an evolution routine for GSCs in surgical specimens. The astrocytes showed a strong evolutionary relation with GSCs. Since T cells showed various characteristics in those two data sources, we defined the coculture model as the initial stage of tumor progression and the specimens as the advanced stage of tumor. Finally, we simulated the fold change of the immune checkpoint in both T cells and GSCs in those two data sources. The inhibiting checkpoint resulted in an advanced tumor stage. Above all, the model is an ideal tool for unveiling the interaction between peripheral T cells and GSCs, simulating the early microenvironment during tumorigenesis. Materials and Methods Isolation and Culture of Primary Cells Tumor tissues obtained during surgery were immediately immersed in the medium and transported to the laboratory on ice for further processing. The tissue was cleaned and shredded mechanically. The tissue was then enzymatically digested into single cells using trypsin. The single cells were filtered using a 200-mesh filter and centrifuged (400 g) for 5 min. After treating the cells with red blood cell lysis, they were centrifuged again. The obtained cells were cultured in a serum-free medium containing DMEM/F12 (Gibco) supplemented with B27 (Gibco), basic fibroblast growth factor (bFGF, 20 ng/mL), epidermal growth factor (EGF, 20 ng/mL), and heparin (2.5 mg/mL). Growth factors (bFGF and EGF) were added twice a week. Primary GSCs were enzymatically dissociated into single cells using Accutase (Sigma Aldrich) and thereafter routinely cultured in the serum-free medium that was replaced every 4C6 days. The stemness of GSCs was verified by multidirectional differentiation immunofluorescence staining (Figure 2A). Normal peripheral blood lymphocytes were obtained from healthy adult male donors. Isolation of peripheral blood T cells was performed following the protocol as previously described (9). In brief, peripheral ZL0420 blood mononuclear cells (PBMCs) were separated by density gradient centrifugation with Lymphoprep (STEMCELL). The PBMCs were resuspended in EasySep? Buffer (STEMCELL), and T cells were isolated following the manufacturer’s instruction (EasySep? Human T Cell Isolation Kit, STEMCELL). T cells were identified by CD3 staining flow cytometry (Figure 2A). Open in a separate window Figure 2 Similarity of cell grouping in the coculture model and surgical specimens. (A) The GSCs and T cells were verified by immunofluorescence staining and flow ZL0420 cytometry. OSP: oligodendrocyte specific protein. (B) The subgroups of cells in the coculture model. (C) Markers of proliferation and immunology in different cell groups. (D) The similarity of glioma stem cells and lymphocytes in the coculture model and surgical specimens. Peripheral blood T cells were cocultured with GSCs for 24 h the day after isolation without CD3/CD28 stimulation. 2 106 T cells, together with 1 106 GSCs, were directly mixed and resuspended in ImmunoCult?-XF T Cell Expansion Medium (STEMCELL) and were cocultured in a 37C 5% CO2 incubator. Construction of a Single-Cell RNA-Sequencing Library Single-cell RNA sequencing library construction of the tissue specimens obtained from GBM patients has been described in detail in our previous research (10). The cell preparation for coculture cellular model was done strictly in accordance with the official documentation of 10 Genomics ( Single-cell RNA sequencing was performed using Illumina (HiSeq 2000) according to the manufacturer’s instructions by Novogene (Beijing, China). Cell Clustering Using Seurat The cell clustering in GBM patients and coculture model of primary normal peripheral blood lymphocytes and GSCs was performed by the R package Seurat (version 3.0, Batch effect was removed before the clustering in GBM patients. Subsequently, MTS2 the cell clustering process in GBM patients and the coculture model were done in the same way. Firstly, cells that have had unique feature counts over 7,500 or 200 and 15% mitochondrial count were removed. Subsequently, after normalizing the data, nonlinear dimensional reduction of cells was carried out using UMAP with the default parameters. Finally, ZL0420 the cluster biomarkers were also obtained. In addition,.