Fig. 1

Single cell RNAseq reveals model-specific transcriptomic signatures in MDMi and resemblance to post-mortem and hiPSC-derived microglia. A Schematic illustrations of the differentiation of monocytes into MDMi as mono-cultures in 2D and 3D, and as 3D co-cultures with ReNcell VM. Immunofluorescence of MDMi in the different culture models with immunostaining against Tmem119 and P2ry12 in 2D; Trem2 and P2ry12 in 3D; and Iba1 (arrows) in 3D co-culture. ReNcell VM were stained for the neuron marker β3-tubulin (Tubb3) and the astrocyte marker Gfap. Scale bars, 100 μm. B Schematic of the methodology employed for single cells transcriptome analysis in monocytes and MDMi (2D, 3D, and 3D co-culture). Monocytes and MDMi from a single healthy donor were cultured, FACs-sorted for CD11b, fixed according to Chromium fixed RNA kit (10X Genomics), sequenced using Illumina NextSeq-2000 and analyzed (detailed protocol can be found in Methods). C UMAP plot showing the clustering of monocyte and MDMi (2D, 3D and 3D co-culture) single cell transcriptomic signatures. The total number of cells analyzed was: 9521 monocytes; 5608 MDMi in 2D; 5667 MDMi in 3D and 4543 MDMi in 3D co-culture. D Top 45 variable genes, with the most variable genes listed at the bottom and the most constant genes at the top of the list. Each row represents the level of expression of a selected key gene. The color of the dot represents the average expression z-score of the cells within the given cluster, while the size of the dot represents the % of cells within that cluster. E–G Combined UMAP plots of monocytes and MDMi (2D, 3D and 3D co-culture) show the expression of selected key myeloid, microglia and neurodegenerative disease-related genes. Each dot represents a cell and the normalized gene expression levels of the selected genes for each cell. The color gradient bar represents log-transformed expression values, with red and blue indicating maximum and minimum expression, respectively. H Distribution of clusters in monocytes and MDMi (2D, 3D and 3D co-culture) to find shared or unique pathways across the models. Pathways of these 10 clusters and can be found in Additional file 1: Fig. S7. I Venn diagram illustrating shared and/or unique clusters among all model systems. J, K Pseudo-bulk gene expression in monocytes and MDMi (2D, 3D, 3D co-culture) compared to human microglia. Human microglial genes were selected from post-mortem brain datasets (Olah et al.: Clusters 1–9) and Day 60 hiPSC-derived microglia (Svoboda et al.). Correlation tests were based on the following number of genes 13,798 (monocytes); 14,480 (2D); 14,220 (3D); 14,216 (3D co-culture). Spearman correlation coefficient (R) was used to examine the degree of correlation