A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states

Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the ‘druggable genome’. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting.


Areas for methodological improvement
Improved inducible CRISPRi/a machinery with more potent gene repression and activation in fully differentiated iTF-Microglia would enable the induction of CRISPRi/a at later stages during differentiation to avoid false-positive hits that affect microglial differentiation, such as CDK8 and TGFBR2 (Fig. 4b, Extended Data Fig. 5b-f).
Another goal for future technology development is further acceleration and enhancement of the microglial maturation. One potential concern about sustained expression of transgenic transcription factors is that this could promote certain microglial states over others. A protocol in which transcription factor expression is discontinued after day 8 (Extended Data Fig. 1a) can mitigate this concern. As with all currently available in vitro culture systems, microglia are slightly activated in monoculture and lose their unique homeostatic brain signature 1 . Previous research has shown that iPSC-microglia become more homeostatic in co-culture with neurons 2 , which is compatible with our own observation of enhanced ramification of iTF-Microglia in neuronal co-culture (Fig. 2f). Alternatively, optimizing the set of transcription factors used to generate iTF-Microglia may result in improved abundance of homeostatic microglia. CRISPRa screens in our current platform are a scalable strategy to identify additional transcription factors to promote microglial maturation and homeostasis, leading to ever more faithful models of human microglia.

Phagocytosis phenotypes of disease-associated genes
Coding mutations in profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS) 3 . PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how mutations in PFN1 cause ALS is unclear. The actin cytoskeleton is known to be important for the physiological functions of microglia, including migration and phagocytosis. We observed that PFN1 overexpression disrupts the actin cytoskeleton in iTF-Microglia with higher levels of F-actin. Recently, a study has shown that PFN1 is also involved in microglia activation, since knockdown of PFN1 inhibited M1 proinflammatory microglial polarization and promoted anti-inflammatory M2 microglia polarization after oxygen and glucose deprivation 4 . Introducing the ALS-associated mutations in the PFN1 gene in iPSCs will shed light on the impact of these specific mutations on the function of different relevant cell types, such as iPSC-derived neurons and microglia.
Genetic variants in the INPP5D locus are associated with an increased susceptibility to AD 5 and cerebrovascular function as well as tau and Ab levels in the cerebrospinal fluid of AD patients 6 . INPP5D encodes the lipid phosphatase SHIP1, which is selectively expressed in brain microglia. SHIP1 inhibits signal transduction initiated by activation of immune cell surface receptors, such as TREM2 7 . Intriguingly, INPP5D expression increases with AD progression, predominantly in plaque-associated microglia, and correlates with plaque density 8 . Given the results from our phagocytosis screen, INPP5D overexpression might result in microglia with deficient phagocytic capacity, resulting in increased Aβ deposition and neurodegeneration. Concordant with the findings from our genetic screen, a recent study found that pharmacological SHIP1/2 inhibitors promote microglial phagocytosis in vitro and in vivo 9 .

Regulators of the SPP1 state
Knockdown or pharmacological inhibition of MAPK14 strongly promoted adoption of the disease-associated SPP1-positive state. Previous work suggested a functional connection between SPP1 and MAPK14 in cancer cells, where SPP1 can activate the p38 MAPK signaling pathway, which comprises MAPK14 10 . MAPK14 was also recently predicted to be a unique network regulator in DAM 11 . However, our identification of MAPK14 as a regulator of the SPP1+ state is novel and enhances our understanding of modulators of microglia cell states.
We found that the SPP1-positive microglia state can be selectively depleted by genetic and pharmacological inhibition of CSF1R. CSF1R inhibitors have beneficial effects in mouse models of diseases including AD 12, 13 , tauopathy 14 and MS 15 . Intriguingly, CSF1R inhibition reduced SPP1 expression in the MS model, while homeostatic genes such as TMEM119 and P2RY12 were increased 15 , paralleling our finding that the SPP1 microglia state is selectively vulnerable to CSF1R inhibition. Additionally, disruption of CSF1-CSF1R signaling downregulated SPP1 in the cerebellum 16 . Combining CSF1R depletion and single cell profiling has enabled us previously to elucidate the differential effects of CSF1R inhibitors on microglia subtypes 17 . Following CSF1R inhibition, we found an enrichment of microglia states with elevated markers of inflammatory chemokines and proliferation and interestingly, in concordance with our findings in iTF-Microglia here, an upregulation of cell surface receptor CD74 17 . Others have reported compensatory upregulation of TREM2/b-catenin and IL-34 in microglia following conditional CSF1R KO 18 ; however, we did not find consistent upregulation of these factors in our iTF-microglia (Supplementary Table 9). Based on our new finding that CSF1R inhibition at low doses that are nontoxic to most microglia selectively depletes the SPP1+ population in iTF-Microglia, low-dose CSF1R inhibition might also give us a tool to study the SPP1+ population in mouse disease models.