Generation of Induced Microglial-like Cells

Human induced pluripotent stem cells (iPSCs) were maintained on Matrigel (BD Biosciences) in complete mTeSR Plus (STEMCELL Technologies, 100–0276). iPSCs were passaged every 5–6 days using ReLeSR dissociation reagent (STEMCELL Technologies, 05872) and used for hematopoietic stem cell differentiation with STEMdiff Hematopoietic kit (STEMCELL Technologies, 05310) followed by differentiation to induced microglial-like cells (iMGLs) using a previously published protocol [53 (link)]. iPSC lines were confirmed to have a normal karyotype (KaryoStat assay, Thermo Fisher Scientific). iMGLs were maintained and fed with a microglial medium supplemented with three factors (100ng/ml IL34, 50ng/ml TGFβ, 25ng/ml MCSF) for 25 days. On day 25, iMGLs were additionally supplemented with two factors (CX3CL1 and CD200, 100ng/ml each) for an additional three days. Mature iMGLs (day 28) were used for bulk RNA-seq analyses and functional assays.

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Podlesny-Drabiniok A., Novikova G., Liu Y., Dunst J., Temizer R., Giannarelli C., Marro S., Kreslavsky T., Marcora E, & Goate A.M. (2023). BHLHE40/41 regulate macrophage/microglia responses associated with Alzheimer’s disease and other disorders of lipid-rich tissues. bioRxiv.

Publication Preprint 2023

Matrigel complete mTeSR Plus ReLeSR dissociation reagent STEMdiff Hematopoietic kit KaryoStat assay

Assays Bulk Cd200 Cx3cl1 Hematopoietic Hematopoietic stem cell Human induced pluripotent stem cells Il34 Ipsc Karyotype Matrigel Mcsf Microglial cells Rna seq Stemcell Tgfβ

Corresponding Organization : Allen Institute for Brain Science

Other organizations : Karolinska Institutet, Karolinska University Hospital, New York University

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Variable analysis

independent variables

Duration of iMGL differentiation (28 days)
Supplementation of iMGL culture medium with IL34, TGFβ, MCSF, CX3CL1, and CD200

dependent variables

Phenotype and function of induced microglial-like cells (iMGLs)

control variables

Maintenance of human induced pluripotent stem cells (iPSCs) on Matrigel in mTeSR Plus medium
Passaging of iPSCs every 5-6 days using ReLeSR dissociation reagent
Differentiation of iPSCs to hematopoietic stem cells using STEMdiff Hematopoietic kit
Subsequent differentiation of hematopoietic stem cells to iMGLs using a published protocol
Confirmation of normal karyotype of iPSC lines

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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