Fig 1.
Generation of iPSC-derived monocyte, macrophages and dendritic cells and their comparison to blood derived counterparts.
(A) Schematic illustrating the protocol and the samples generated including and used for downstream analysis. Also shown are representative images of the iPSC-derived cells both during the differentiation process and at terminal differentiation stages. (B) FACS analysis of the 2 fractions from the CD14 sorting step. Top row; CD14+ and bottom row; CD14- fractions respectively. Antibodies used are shown on the plots. Blue; CD16+ (DP; double positive) and red; CD16- fractions (CD14+).
Fig 2.
Phenotypic characterization of macrophages and dendritic cells.
(A) Phagocytotic capacity measuring S. aureus internalization after 2h and (B) LPS-induced cytokine release both comparing MDM and iPSdM. (C) Gating strategy and surface marker expression and (D) antigen uptake and processing of iPSdDC before and after LPS maturation. (E) Comparison of the antigen presentation capability of both MoDC and iPSdDC measured by T-cell proliferation (Mixed leukocyte reaction; MLR) and (F) cytokine release from iPSdDC before and after LPS activation. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Differentiation of iPSC into monocytes and derivatives results in loss of pluripotent markers and concomitant up regulation of lineage markers associated with differentiated cell types.
(A) Expression of lineage specific marker genes across all cell types., HSC; haematopoietic stem cell, NK cell; natural killer cell. Each column represents an individual sample and are grouped into cell type as shown. For the blood derived samples the sample has a donor designation (D1-3) for the IPSC-derived samples the sample has a designation that refers to the harvest day (Fig 1A). (B) Principal component analysis of all cells types generated in this study. PC1 v PC2 left panel, PC3 v PC2, right panel. Plots based on all genes.
Fig 4.
iPSC-derived monocytes show many similarities to PBMC with respect to transcriptional profile and differentiation capacity.
(A) Correlation scatter plot of the average rlog expression values for PBMo versus iPSdMo. Only genes with TPM>2 in either group are shown. Genes with fold change >4 in green and genes with fold change < -4 in blue. (B) Summary of functional groups enriched for the 1964 DE genes. Bar length is the–log2 of the enrichment p-value and the colour represents the z-score with green indicating functions activated in iPSdMo relative to PBMo and blue bars those activated in PBMo relative to iPSdMo. (C) PCA plot of the various iPSdMo, iPSdM, iPSdDC, PBMo, MDM and MoDC samples. The arrows illustrate the progression of either PBMo or iPSdMo to either MDM/MoDC or iPSdM/iPSdDc respectively.
Fig 5.
iPSC-derived macrophages and MDM show similar transcriptional and activation profiles.
(A) Correlation scatter plot of the average rlog expression values for MDM versus iPSdM. Only genes with TPM>2 in either group are shown. Genes with fold change >4 in green and genes with fold change < -4 in blue. (B) Summary of functional groups enriched for the 1964 DE genes. Bar length is the -log2 of the enrichment p-value and the colour represents the z-score with green indicating functions activated in iPSdM relative to MDM and blue bars those activated in MDM relative to iPSdM. (C) Heatmap illustrating changes in expression of 10 LPS inducible genes (left) for both MDM and iPSdM following LPS stimulation. (D) PCA plot iPSdM and MDM samples with and without LPS stimulation. Grey arrows show the progression from no LPS to 1h LPS to 6h LPS.
Fig 6.
iPSC-derived DC while similar transcriptionally to monocyte derived DC are more similar to blood derived DC.
(A) Correlation scatter plot of the average rlog expression values for MoDC versus iPSdDC. Only genes with TPM>2 in either group are shown. Genes with a fold change >4 are in green and genes with a fold change < -4 in blue. (B) Summary of functional groups enriched for the 3450 DE genes. Bar length is the -log2 of the enrichment p-value and the colour represents the z-score with green indicating functions activated in iPSdDC relative to MoDC and blue bars those activated in MoDC relative to iPSdDC. (C) Heatmap showing expression levels of genes identified in Villani as classifiers of the six DC subtypes across both types of monocytes and DC from this study.