Efficient derivation of microglia-like cells from human pluripotent stem cells

Abstract

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell-derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment.

Figure 1. induction of primitive myelogenesis from human pluripotent stem cells

(a) top panel: example image depicting the typical formation of cystic EBs bound by a single cell layer. lower panel: example of neuralized spheroid EB structures. Scale bars: 200μm. (b) typical appearance of endothelial lawns emerging from plated cystic EBs (on PDL coated plastic). Phase panels display the island formations with raised edges (left), and the progressive merger of such edges into raised ropes. Subsequent panels depict staining for VE-Cadherin (green), and c-kit (magenta). Scale Bars: 80μm. (c) higher magnification of raised structures surrounding islands (phase), staining for CD41 (green), and CD235a (magenta). Scale bar: 25μm. (d) after 2 weeks in suspension culture, cystic EBs can be plated to PDL coated plastic (left, phase contrast. Scale bar: 200μm), and large domains stain positive for the nucleus-localized transcription factor PU.1 (right, green). (e) Delamination of grape-like structures towards the luminal side from YS-EBs (red arrowheads, top left and right, scale bars:40 μm and 25μm, respectively). Putative myeloid cells are seen delaminating outward into the suspension medium (red arrowhead, bottom left, scale bar: 25μm). Homogeneous population of round motile cells seen delaminating and spreading away from the source YS-EB (Bottom right, scale bar: 80μm).

Figure 2. characterization of phagocytes delaminating from cystic YS-EBs

(a) differentiation protocol schematic showing the suspension culture (top row), and the selective adherent conditions (bottom row). (b) low magnification view of delaminated lawn after plating stained for nuclear DAPI (grey scale), nuclear PU.1 (magenta) and membrane CD11b/ITGAM (green pseudocolor). Merged PU.1 and CD11b channels are depicted in the right panel. Scale bar: 25μm. (c) high magnification view of ramified cell in resting culture viewed under phase contrast (grey scale) and stained for nuclear PU.1 (magenta) and cytoplasmic IBA1/AIF1. Merged PU.1 and IBA1 channels are depicted in the right panel. Scale bar: 5μm (d, e) FACS scatter plots of harvested pMGLs for CD11b and IBA1 (left) or CD45 (right). (f) left panel, example image depicting the migratory path (white dashed arrow) of a single pMGL on a fluorescent bead lawn (yellow), as well as intracellular accumulation of phagocytosed fluorescent beads (arrow head) Scale bar: 10μm. Right panel, example image depicting a cotton fiber opsonized by pMGLs (scale bar: 25μm). (g) extracted frames from supplementary movie 2 depicting fluorescent beads (red arrowheads) taken up by a single pMGL Scale bar: 3μm. h: Quantification of pMGL EdU incorporation, measured at 2 weeks and 2 months (mean ± s.e.m. of 2 biological replicates, t-test, P<0.05).

Figure 3. pMGLs adopt ramified morphologies over time and express specific markers of microglia

(a) example phase contrast images of depicting morphologies of human fetal microglia (hFMG; left) and pMGLs derived from human ES cells (hES pMGLs, middle) or induced pluripotent stem cells (iPS pMGLs, right). Scale bars: 25μm. (b) example high magnification images depicting morphologies of mouse primary neonatal microglia (mNMG, left), hFMG (middle) and hiPS pMGLs (right). scale bars: 10μm. (c) Example of phenotypic output in freshly plated MECP2 mutant (right panel) and isogenic wild-type cells (left panel, scale bar: 25μm). Average cell spread (surface) and perimeter are significantly lower in MECP2 mutant microglia (mean ± s.e.m. for 2 biological replicates, TTEST, p<0.05). (e–f) example images of ramified pMGLs (phase) stained for DAPI (magenta) and either TMEM119 (e, green) or P2RY12 (f, green). Scale bars: 10μm (g) Example confocal image representing an optical slice of wider field-of-view, stained for IBA1 (red), TMEM119 (green) and CD45 (white). Scale bar: 20μm.

Figure 4. pMGLs cytokine profiles in response to endotoxin challenge

(a) Baseline cytokine profiler assay from 10⁵ pMGLs conditioning 2mL of NGD in 24 hours. Top panel: 10′ exposure, showing baseline secretion of detectable CCL2, MIP1α/β, CXCL1 and IL8. Lower Panel: profile after stimulation for 24hours with 100ng/mL LPS and 20ng/mL IFN-γ. IL6, TNFα, MIP1α/β and CXCL10 display a dramatic increase. (b) map, to scale, of the spots blotted in panel a. Green highlights cytokines showing a significant upregulation. (c, d) Quantitative qPCR of TNFα (c) and IL6 (d) transcription in pMGLs at baseline (−) and after LPS stimulation (+). Data are presented as mean ± s.e.m. from 2 biological replicates, t-test significance is reported.

Figure 5. pMGLs recapitulate the consensus signature distinguishing primary microglia from other macrophages

(a) Quantification of normalized expression (counts from RNAseq data) of the indicated genes for pMGLs (black bars, N=5), primary human fetal microglia (red bars, hFMG, N=2), and differentiated human neural progenitors (red bars, Diff. NPCs, N=4). Data are presented as mean ± s.e.m. (b) Dendrogram depicting the results of unbiased hierarchical clustering of fMGs (N=2), pMGLs (N=5) and NPCs (N=4) derived in this study, compared to a published dataset (GSE73721) for adult primary microglia (N=3), fetal astrocytes (N=6), mature astrocytes (N=12), neurons (N=1) and whole cortex (N=4), using genes in Suppl. Table 8.

Figure 6. neural co-cultures enhance the microglial signature of pMGLs

(a) top: schematic representation of transwell culture system exposing pMGLs to conditioned medium from differentiating neuro-glial cultures. Bottom: schematic representation of direct re-aggregation after GFP transduction, as free-floating spheroids or 3D stacks in transwells. (b) Principal component analysis comparing RNAseq profiles from differentiated neural cultures (full squares, NPC1 in red, NPC2 in blue, NPC3 in green) to those of primary fetal microglia (fMG1 and fMG2, open triangles. fMG1+ NCM, closed triangle), and pMGLs (circles, pMGL1 in red, pMGL2 in blue, and pMGL3 in green) in the absence (open circles) or presence (full circles) of neural progenitor conditioned medium (+NCM). N=1 for each sequencing data point. (c) phase contrast (left) and fluorescence (right) images depicting the relative tiling position of GFP-labeled pMGLs in 3d culture on transwell. Scale bar: 200 μm. (d) representative image of a GFP-positive pMGL (grey scale) after 4 weeks in 3D neural stacks (live observation). Scale bar: 10μm. (e): Optical section through a fixed 3D neuroglial culture (without embedded pMGLs), stained for DAPI (blue, top), neuronal MAP2 (green, middle) and astrocytic GFAP (magenta, bottom). Scale bar: 50μm. (f) Maximum projection of supplementary movie 5 (scale bar: 2μm) pointing to rapidly extending (yellow arrowhead) and retracting (red arrowhead) protrusions in the 3D neuroglial cultures. (g) 1/5 frames of supplementary movies S5 and S6, showing branch movements over 300s. (h) Example montage of time-lapse images depicting GFP-labeled pMGLs (grayscale) response to localized cellular damage in 3D culture. Yellow arrowhead indicates the site of two-photon laser ablation after 5 minutes of acquisition. Red arrowheads point to microglia-like cells further away from injury, not reacting to the damage. Bottom right panel represent the color-coded individual trajectories of pMGLs during acquisition, highlighting radial migration towards the injury. Scale bar = 100μm.