Functional analysis and transcriptomic profiling of iPSC-derived macrophages and their application in modeling Mendelian disease

Abstract

Rationale: An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases.

Objective: To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDMs) and their isogenic human peripheral blood mononuclear cell-derived macrophage (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease.

Methods and results: We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (lipopolysaccharide+interferon-γ) activation. RNA-Seq revealed that nonpolarized (M0) as well as M1 or M2 (interleukin-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apolipoprotein A-I and high-density lipoprotein-3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease, an autosomal recessive disorder because of mutations in ATP-binding cassette transporter AI. Tangier disease-IPSDM also revealed novel defects of enhanced proinflammatory response to lipopolysaccharide stimulus.

Conclusions: Our protocol-derived IPSDM are comparable with HMDM at phenotypic, functional, and transcriptomic levels. Tangier disease-IPSDM recapitulated hallmark features observed in HMDM and revealed novel inflammatory phenotypes. IPSDMs provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.

Keywords: cholesterol; genomics; induced pluripotent stem cells; inflammation; macrophages.

Figure 1. Schematic figure of IPSDM differentiation

A stepwise protocol was used to differentiate iPSC to macrophages (IPSDM). Data shown are representative of 6 separate experiments.

Figure 2. IPSDMs are comparable to HMDMs at morphological and immunophenotypic levels

(A) and (C) IPSDM exhibited macrophage-like characteristics as well as expression of macrophage markers; (B) and (D) show morphologic and phenotypic similarity between HMDM and IPSDM. Data are representative of 6 separate experiments. In (C) and (D), isotype controls were shown as gray shaded and open histograms respectively.

Figure 3. Functional phenotypes in response to M1 (LPS+IFN-γ) and M2 (IL-4) polarization are comparable between IPSDM and HMDM

(A) Schematic protocol for HMDM and IPSDM polarization. (B) M1-HMDM and M1-IPSDM showed lower phagocytosis capacities (n=7 age/sex-matched subjects). HMDM and IPSDM derived from the same subject (Control-6) have comparable phagocytosis capacities (data represent mean of triplicate experiments). (C) ABCA1 and ABCG1 mRNA were upregulated in M1-HMDM and suppressed in M2-HMDM. M1-HMDM showed a clear pattern of increased efflux to apoA-I and HDL₃. IPSDM resembled HMDM in polarization-induced change in ABCA1/ABCG1 mRNA expression and cholesterol efflux. Each dot in the plots represents one subject. In addition, HMDM and IPSDM derived from Control-6 showed comparable efflux levels. (D) Representative cytokine array determining the relative level of 36 human cytokines and chemokines in culture media. HMDM and IPSDM derived from the same subject (Control-6) demonstrate similar cytokine profiles and response to polarization stimuli. (E) Cytokine array data of IPSDM and HMDM from 5 age/sex matched subjects are quantified by Image J and visualized.

Figure 4. RNA-Seq transcriptome analysis of iPSC, IPSDM and HMDM

(A) Schematic figure of RNA-Seq design and data analysis (n=3 subjects, Control-1, Control-3 and Control-4, with two iPSC clones per subject). (B) Multidimensional scaling (MDS) and (C) co-regulation analysis (CRA) confirmed the distinct transcriptome profile of iPSC from HMDM and IPSDM. M1 polarization profoundly affects the transcriptional profile while M2 polarization results in more subtle expression changes. (D) IPSDM showed expression of macrophage markers and the absence of markers of pluripotency and other hepatopoietic cells. (E) and (F) show the total number of genes (FPKM > 1% expression of all genes) and DE genes in iPSC vs. IPSDM, and IPSDM vs. HMDM. The top 10 gene ontology (GO) terms ranked by FDR-adjusted P values for DE genes are illustrated. The size of the circle is inversely correlated to FDR-adjusted P values of enrichment in the respective GO term. The thickness of the lines corresponds to similarity between two GO terms. Scatterplot (F) suggests the strong Pearson’s correlation (r=0.85) of transcriptome between HMDM and IPSDM. Green, DE genes with higher expression in M0-HMDM; Red, DE genes with higher expression in M0-IPSDM; Blue, non-DE genes.

Figure 5. IPSDM recapitulates transcriptome signature of M1 (LPS+IFN-γ) and M2 (IL-4) macrophage polarization

(A) and (B) Venn diagrams show the total number and the overlapped genes in M1 and M2 macrophages in HMDM and IPSDM. Scatterplots illustrate the correlation of Log₂ FC of DE genes in M0 vs. M1 (A) and M0 vs. M2 (B) polarization between HMDM and IPSDM. Only genes with FPKM >1% expression and also defined as DE genes in either HMDM or IPSDM were plotted. Genes with the same direction of change were highlighted in red for up-regulation and green for down-regulation. Genes with opposite direction of change were highlighted in purple. The DE genes of M1 vs. M2 are separated into those expressed at higher levels in M1 (C), or in M2 (D). Venn diagrams of the number of common DE genes in HMDM and IPSDM, and GO enrichment analysis of these common DE genes are performed and the top 10 GO terms ranked by FDR-adjusted P values are visualized. (E) The heat maps depict expression profiles of subsets of well-established macrophage polarization markers for selected M1-enriched (top) and M2-enriched (bottom) genes in HMDM and IPSDM.

Figure 6. TD (Tangier disease)-IPSDMs recapitulate hallmark phenotypes of impaired cholesterol efflux and reveal novel inflammatory phenotype in TD-IPSDM

(A) In HMDM, efflux of [³H]-labeled cholesterol to apoA-I was completely abolished, and efflux to HDL₃ was impaired in TD-1 and partially impaired in the heterozygote sibling. LXR agonists enhanced efflux in the Hetero-1 and age/sex/race-matched healthy control, but not in the TD patient. The TD-1 IPSDM faithfully reproduced the impaired cholesterol efflux function. (B) IPSDM of TD-2 and age/sex/race-matched Control-2 also showed abolished efflux to apoA-I and impaired efflux to HDL₃. The bar graph represents mean±SD of triplicate experiments for HMDM, and quadruplicate experiments of two clones per iPSC line of each subject for IPSDM. * P<0.05 vs. TD -9CisRA/22OH; # P<0.05 vs. TD +9CisRA/22OH; & P<0.05 for -9CisRA/22OH vs. +9CisRNA/22OH. (C) TD-HMDMs showed marginal increase in baseline expression of IL1B, IL8 and TNF, and are hypersensitive to LPS stimulus as evidenced by greater increases in inflammatory gene expression. IPSDM reproduce the enhanced inflammatory response in TD-HMDM. The ΔCts represent the mean cycle threshold for target genes relative to ACTB as the internal reference. n=4 replicates of Control-2 and TD-2.