An adapted stem cell-derived microglia protocol for the study of microgliopathies and other neurological disorders

Background Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a primary microgliopathy caused by pathogenic variants in the colony-stimulating factor 1 receptor ( CSF1R ) gene. Since CSF1R signaling is crucial for microglia development, survival and function, induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial defects caused by ALSP patient-specific CSF1R variants. Methods Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an ALSP patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. Results The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to primary human microglia and with higher phagocytic and inflammatory competence at ∼3-fold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 ( P2RY12 ) expression. Finally, ALSP patient-derived cells showed surprisingly high phagocytic capacity, which was associated with higher lysosomal content. Conclusions We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory and phagocytic activities. ### Competing Interest Statement The authors have declared no competing interest. * ADP : adenosine diphosphate ALSP : adult-onset leukoencephalopathy with axonal spheroids and pigmented glia BSA : bovine serum albumin CCL3 : chemokine ligand 3 CD11b : cluster of differentiation 11b CD14 : cluster of differentiation 14 CD200 : cluster of differentiation 200 CD34 : cluster of differentiation 34 CD43 : cluster of differentiation 43 CD68 : cluster of differentiation 68 CSF1R : colony-stimulating factor 1 receptor CSF2R : colony-stimulating factor 2 receptor CX3CL1 : C-XC-3 motif chemokine ligand 1 CX3CR1 : C-XC-3 motif chemokine receptor 1 DMEM : Dulbecco’s Modified Eagle Medium EDTA : Ethylenediaminetetraacetic acid FBS : fetal bovine serum FITC : fluorescein isothiocyanate GAPDH : glyceraldehyde 3-phosphate dehydrogenase GAS6 : growth arrest specific 6 GM-CSF : granulocyte-macrophage colony-stimulating factor GPR34 : G-protein coupled receptor 34 IBA1 : Ionized calcium binding adaptor molecule 1 IFNψ : interferon gamma iHPC : induced pluripotent stem cell-derived hematopoietic progenitor cell IL-10 : interleukin 10 IL-1Ο : interleukin 1 beta IL-34 : interleukin 34 IL-6 : interleukin 6 iMGL : induced pluripotent stem cell-derived microglia iPSC : induced pluripotent stem cell LAMP1 : lysosomal-associated membrane protein 1 LPS : lipopolysaccharide LY96 : lymphocyte antigen 96 M-CSF : macrophage colony-stimulating factor MEM : Minimum Essential Medium MERTK : Mer tyrosine kinase MMP8 : metalloproteinase 8 mRNA : messenger ribonucleic acid NF-κB : nuclear factor kappa B NLRP3 : NLR family pyrin domain containing 3 OCT-3/4 : octamer binding transcription factor 3/4 P2RY12 : purinergic receptor P2Y12 PBMC : peripheral blood mononuclear cell PBMC-Mχπ : peripheral blood mononuclear cell-derived macrophages PCA : principal component analysis PROS1 : protein S P/S : penicillin/streptomycin qPCR : quantitative polymerase chain reaction qRT-PCR : real-time quantitative reverse transcription polymerase chain reaction RNA : ribonucleic acid SDS : sodium dodecyl sulfate SEM : standard error of the mean SIGLEC10 : sialic acid binding Ig-like lectin 10 SSEA-4 : stage-specific embryonic antigen-4 TGF-Ο1 : transforming growth factor beta 1 TLR4 : toll-like receptor 4 TMEM119 : transmembrane protein 119 TNF : tumor necrosis factor TREM2 : triggering receptor expressed on myeloid cell 2 YWHAZ : tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta