Table 2.

Detailed summary of the molecular markers and functional tests used to characterize microglia-like cells

	Hematopoietic Progenitors	Microglia Precursors	Microglia-like Cells	Efficiency	Reproducibility
Protocol 1 – Muffat et al.	Morphology: cystic structure ICC: CD144 (CDH5), CD117 (KIT), CD41 (ITGA2B), CD235a (GYPA), PU.1 (SPI1)	Morphology: round, vacuolated cells, compact nucleus, filopodia, membrane ruffles ICC: CD11b (ITGAM), IBA1 (AIF1), PU.1 Flow: CD11b, IBA1, CD45 (PTPRC) Phagocytosis: latex bead assay Proliferation: EdU assay	Morphology: multiple thin first-order branches terminated by membrane ruffles ICC: TMEM119, P2Y12 (P2RY12), IBA1, CD45 Cytokines (CTRL, LPS + IFN-γ): Cytokine Array R&D Transcriptional Profile: RNA-Seq	Yield: 0.5-4x Purity: 97%	15 hiPSC-lines 3 hESC lines
Protocol 2 - Pandya et al.	Morphology: floating cells ICC: CD34, CD43 (SPN), CD45	not reported	Flow: CD11b, IBA1, CD45, CX3CR1, HLA-DR; Negative: CD80, CD86, CD206, CD200R Cytokines (CTRL, LPS): TNF-α, kit unspecified Phagocytosis: pHrodo-E.coli ROS: CellROX assay Transcriptional Profile: Microarray	Yield: 0.8-3x Purity: 9% (in mixed glial culture)	2 hiPSC lines
Protocol 3 – Abud et al.	Morphology: endothelial cells Flow: CD41 (ITGA2B), CD43, CD235a	ICC: PU.1, TREM2 Flow: CD11b (9%), CD45 (65%), CX3CR1 (22%); negative: CD117	Morphology: high nucleus to cytoplasm ratio ICC: PU.1, CX3CR1, TREM2, P2Y12, TGFBR1, PROS1, MERTK, ITGB5 Flow: CD11bint, CD45low-int Phagocytosis: pHrodo-E.coli, pHrodo-hS (CD11b-dependent, MERTK-independent) Cytokines (CTRL, LPS, IL-1β, IFN-γ): V-PLEX cytokine 30-plex Calcium Signaling: ADP-response (P2Y12-negated) Transcriptional Profile: RNA-Seq Transplantation: into MITRG mice	Yield: 30-40x Purity: 97%	10 hiPSC lines
Protocol 4 - Douvaras et al.	Morphology: adherent endothelial cells Flow: CD309 (KDR), CD235a (up to 6%)	Morphology: floating cells Flow: CD14, CD45, CX3CR1 (70%)	Morphology: ramified cells with motile processes ICC: CD11c, IBA1, P2Y12, TMEM119 Flow: CD11b, CD11c, CX3CR1, P2Y12 Phagocytosis: latex beads Cytokines (CTRL): Human XL Cytokine Array Kit Calcium Signaling: ADP-response Transcriptional Profile: RNA-Seq	Yield: 2x Purity: 68% (CD14+)	14 hiPSC lines 2 hESC lines
Protocol 5 - Haenseler et al.	Flow: CD34	Morphology: large, vesicle-rich, floating cells Flow: CD14, CD16, CD45, CD86, CD163, HLA-DR Phagocytosis: pHrodo-zymosan	Morphology: ramified (secondary branches) Flow: CD11b, CD11c, CD14, CD45, (CD33), (MERTK); Negative: HLA-DR Cytokines (CTRL, LPS/IFN-γ): Human XL Cytokine Array Kit (R&D), Luminex 100 Bio-Plex System (BioRad)	Yield: 10-43x Purity: ~ 100% (CD14+)	4 hiPSC lines
Protocol 6 - Takata et al.	not reported	Morphology: floating, round, vacuolated cells Flow: CD11b, CD14, CD45, CD163, CX3CR1 CyTOF: CD11b, CD14, CD45, CD48, CD62L, CD64, CD115, CD163, CX3CR1, HLA-DR, MARCO, MERTK Phagocytosis: latex beads, Aβ-TAMRA	Morphology: ramified cells ICC: IBA1 FLOW: CX3CR1 (increased expression compared to d25) Phagocytosis: latex beads, Aβ-TAMRA Cytokines (CTRL, LPS): Human Magnetic Luminex Assay (R&D) Transcriptional Profile: none	Yield: n.a. Purity: 94% (CD14+/CD45+)	1 hiPSC line