Development and validation of a simplified method to generate human microglia from pluripotent stem cells

Abstract

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 TGFβ-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 TGFβ signaling (IDE1) can be used to replace recombinant TGFβ1, 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.

Keywords: Differentiation; GWAS; Hematopoietic precursor cells; IDE1; Microglia; Neurodegeneration; Phagocytosis; Stem cells; TGFB; iPSCs.

Fig. 1

A simplified microglial differentiation protocol can be used to produce large numbers of highly pure human microglia. Schematic showing the process of differentiation from iPSCs through the mesoderm lineage (days 0–3) and further promoting hematopoiesis (days 3–11). Primitive hematopoietic progenitor cells begin to appear on day 7 (black arrows) and by day 11 large numbers of round non-adherent HPCs are observed. Floating HPCs are then transferred into new medium to induce microglial differentiation for 27 days. The last 3 days of microglial differentiation include additional neuronal and astrocytic ligands to further educate microglia toward a brain-like, homeostatic environment. By day 38, large numbers of highly pure microglia that stain positively for both P2RY12 and TREM2 (> 94%) have been produced and are ready for experimentation

Fig. 2

iPS-microglia 2.0 are virtually identical to iPS-microglia generated using a more complex protocol. a Principle component analysis demonstrates that iPS-microglia 2.0 (dark blue) and iPS-microglia differentiated using our previously published protocol (blue) exhibit highly equivalent gene expression profiles that cluster closely with cultured human fetal and adult microglia (light blue and teal). Additionally, these cells are distinct from human CD14+ monocytes (purple) and CD16+ inflammatory monocytes (pink), and dendritic cells (maroon). b Principal component analysis using a gene list enriched for 882 microglial genes from (Gosselin et al., 2017), further demonstrates the equivalent gene expression between iPS-microglia and fetal and adult microglia. This analysis also highlights the trajectory of differentiation from iPSCs to Microglia and shows the separation between our protocol and monocytic and dendritic cell populations. c Volcano plot of differential expression analysis (p < 0.001, log2(FC) > 2) between iPS-HPC and iPS-HPC 2.0 samples (top) as well as iPS-microglia and iPS-microglia 2.0 (bottom). Significantly increased or decreased genes are shown in coral or blue respectively. d Heatmap using 882 microglial-enriched genes further demonstrates the highly similar gene expression profiles between iPS-microglia and iPS-microglia 2.0 and the close similarity of both cell populations to fetal and adult cultured microglia

Fig. 3

iPS-microglia 2.0 are distinct from CD14⁺ and CD16⁺ monocytes Microglia differentiated using our published protocol are distinct from CD14⁺ monocytes and CD16⁺ inflammatory monocytes. In order to ensure our iPS-microglia 2.0 are similarly distinct from monocytes, differential expression analysis was computed with DEseq2. a Volcano plots of differentially expressed genes comparing genes enriched in CD16⁺ monocytes (pink) with genes enriched in iPSmicroglia 2.0 (dark blue) or iPS-microglia (light blue) show many significant differences between monocytes and microglia. Venn diagrams and comparative fold change plots of differentially expressed genes show that the vast majority of differences are identical between iPS-microglia and iPS-microglia 2.0 when compared to CD16⁺ monocytes. Direct comparisons of the fold change expression level (TPM) of every gene are shown in comparative fold change plots which demonstrate the striking similarity of differential expression when iPS-microglia and iPS-microglia 2.0 are each compared to CD16⁺ monocytes. b The same is true for comparisons of iPS-microglia and iPS-microglia 2.0 with CD14⁺ monocytes (purple)

Fig. 4

iPS-microglia 2.0 exhibit equivalent substrate-dependent phagocytosis. iPS-microglia and iPS-microglia 2.0 were exposed to fluorescent beta-amyloid fibrils, pHrodo tagged S. aureus, or pHrodo tagged Zymosan A bioparticles from S. cerevisiae. Quantification of the percent of total cells with positive fluorescent signal and the mean fluorescence intensity of that signal is shown on the left. No significance differences were found between each differentiation type, demonstrating the equivalent functional activity of microglia generated by these two differentiation paradigms. Representative images of phase, CD45 staining, and the fluorescent signal of beta-amyloid (top), S. aureus (middle), and Zymosan A (bottom) are shown on the right. One representative image of 10,000 quantified images is shown for iPS-microglia 2.0 (top of each set) and iPS-microglia (bottom of each set)

Fig. 5

Transplanted iPS-microglia 2.0 display typical microglial markers and morphology comparable to our previously described iPS-microglia. Adult 2 month old MITRG mice were transplanted with (a-d & i-j, top rows) iPS-microglia 2.0 or (e-h & m-p, bottom rows) iPS-microglia. Brains were harvested 2 months post-transplant. Representative images of cortical (a-h) and hippocampal (i-p) transplanted cells demonstrate complex process ramification and typical tiling. Transplanted iPS-microglia and iPS-microglia 2.0 both express Iba-1 (Overlay images c, g, k, & o, red) and the microglia specific marker, P2RY12 (Overlay images d, h, l, & p, red) and demonstrate human nuclear staining (Ku80, green). Additionally, transplanted human microglia can be seen integrating and tiling with the endogenous mouse microglia population (Arrows indicate Iba1+/Ku80- cells)

Fig. 6

The small molecule compound IDE1 can be used in place of TGFβ-1 to produce iPS-microglia. IDE1 and IDE2 were added to microglia cultures in place of TGFβ-1 at the indicated concentrations. a Growth curves from the first 3.5 days of microglial differentiation show that IDE2 is insufficient to allow proliferation of these cells. In contrast, IDE1 (blue) at lower concentrations shows similar growth kinetics to TGFβ control cells (green). b Correlation matrix displaying all samples analyzed in this manuscript shows cells differentiated with IDE1 cluster closely with iPS-microglia 2.0 and are actually more similar to fetal and adult microglia than TGFβ control microglia. c Gene ontology analysis using the Reactome database displays differences between IDE1 treated cells and TGFβ (FDR < 0.001, FC > 2). Enrichment in IDE samples reflects an increased expression of genes within this GO family in IDE1 treated cells