Directed Differentiation of iPSCs to Microglia

iPSC-microglia were generated as described (McQuade et al., 2018 (link)). Briefly, iPSCs were directed down a hematopoietic lineage using the STEMdiff Hematopoesis kit (StemCell Technologies). After 10–12 days in culture, CD43⁺ hematopoteic progenitor cells are transferred into a microglia differentiation medium containing DMEM/F12, 2× insulin-transferrin-selenite, 2× B27, 0.5× N2, 1× glutamax, 1× non-essential amino acids, 400 µM monothioglycerol, and 5 µg/mL human insulin. Media was added to cultures every other day and supplemented with 100 ng/mL IL-34, 50 ng/mL TGF-β1, and 25 ng/mL M-CSF (Peprotech) for 28 days. In the final 3 days of differentiation 100 ng/mL CD200 (Novoprotein) and 100 ng/mL CX3CL1 (Peprotech) were added to culture.

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Shabestari S.K., Morabito S., Danhash E.P., McQuade A., Sanchez J.R., Miyoshi E., Chadarevian J.P., Claes C., Coburn M.A., Hasselmann J., Hidalgo J., Tran K.N., Martini A.C., Rothermich W.C., Pascual J., Head E., Hume D.A., Pridans C., Davtyan H., Swarup V, & Blurton-Jones M. (2022). Absence of microglia promotes diverse pathologies and early lethality in Alzheimer’s disease mice. Cell reports, 39(11), 110961.

Publication 2022

STEMdiff Hematopoesis kit IL-34 TGF-β1 M-CSF CD200 CX3CL1

Cd200 Cd43 Cx3cl1 Essential amino acids Hematopoesis Hematopoietic Human Il 34 Insulin Ipscs M csf Microglia Monothioglycerol Progenitor cells Selenite Tgf β1 Transferrin

Corresponding Organization : University of California, Irvine

Other organizations : Mater Research, University of Queensland, University of Edinburgh, Centre for Inflammation Research

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Variable analysis

independent variables

Differentiation of iPSCs down a hematopoietic lineage using the STEMdiff Hematopoesis kit
Culture duration of 10-12 days for hematopoietic progenitor cells
Microglia differentiation medium composition (DMEM/F12, 2x insulin-transferrin-selenite, 2x B27, 0.5x N2, 1x glutamax, 1x non-essential amino acids, 400 µM monothioglycerol, 5 µg/mL human insulin)
Supplementation of microglia differentiation medium with 100 ng/mL IL-34, 50 ng/mL TGF-β1, and 25 ng/mL M-CSF for 28 days
Supplementation of microglia differentiation medium with 100 ng/mL CD200 and 100 ng/mL CX3CL1 in the final 3 days of differentiation

dependent variables

Generation of iPSC-derived microglia

control variables

Culture duration of 10-12 days for hematopoietic progenitor cells
Microglia differentiation medium composition (DMEM/F12, 2x insulin-transferrin-selenite, 2x B27, 0.5x N2, 1x glutamax, 1x non-essential amino acids, 400 µM monothioglycerol, 5 µg/mL human insulin)

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