Differentiation of human induced pluripotent stem cells to authentic macrophages using a defined, serum-free, open-source medium

Abstract

The authors would like to make two corrections to the supplemental information for this manuscript. First, the formulation of XVIVO medium is stated to be the same as previously published by van Wilgenburg et al. (2013), and OXM is subsequently based on this formulation. In van Wilgenburg et al. (2013) the concentration of M-CSF used was 100 ng/mL; however, in the originally published version of our supplemental information, 50 ng/mL M-CSF was reportedly used. This value was incorrect as written, and the concentration was in fact 100 ng/mL, the same as in Wilgenburg et al. (2013). Therefore, Table S2 in the supplemental information has been corrected to reflect this, stating a final concentration of 100 ng/mL M-CSF in both OXM and XVIVO media. Secondly, in the originally published version of this paper, OXM Macrophage medium is described to be the same as OXM differentiation medium with only Tropolone and IL-3 omitted. However, in the originally published version of Table S2 in the supplemental information, the composition of OXM Macrophage medium is listed as 96.5% Advanced DMEM/F-12, 2mM GlutaMAX, 15mM HEPES, 10 ng/mL M-CSF, and 1% Penicillin-Streptomycin. The correct formulation is 96.5% Advanced DMEM/F-12, 2mM GlutaMAX, 15mM HEPES, 5 mg/mL human recombinant Insulin solution, 100 ng/mL M-CSF, and 1% Penicillin-Streptomycin. The supplemental information has now been corrected with the manuscript online and the authors sincerely apologize for any confusion these errors may have caused.

Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSCs to microglia

“…IL-34 is the main ligand for CSF1R in the brain, whereas M-CSF is the main ligand for CSF1R in the periphery, both mediating a similar intracellular response (46). Supplementation with M-CSF is known to promote a macrophage phenotype and has been used in multiple iPSC-macrophage protocols, and the scRNA-seq analysis here shows that M-CSF-alone induces a more macrophage-like phenotype (21,23,48). This supports a unique role of IL-34 in microglial development in the brain which cannot be replicated by peripheral M-CSF alone (49,50).…”

“…All differentiation reagents are listed in (Table S2) and all medium compositions are listed in (Table S3) . iPSC were cultured in ‘OxE8’ medium (21) (based on the published E8 medium formulation (22)) on Geltrex (Gibco A1413302) coated tissue culture dishes and passaged at 80% confluency with 0.5mM EDTA. Cells were incubated at 37°C, 5% CO 2 and fed daily with fresh OxE8 medium.…”

A systematic characterization of microglia-like cell occurrence during retinal organoid differentiation

“…Such cystic structures have also been mentioned in other microglia differentiation protocols ( Haenseler et al., 2017 ; Muffat et al., 2016 ; Vaughan-Jackson et al., 2021 ). One common factor that is frequently used to enhance for microglia is BMP4.…”

“…Recent hiPSC-derived microglia-like protocols have reported that bone morphogenetic protein 4 (BMP4) promotes microglia generation in vitro ( Abud et al., 2017 ; Douvaras et al., 2017 ; Guttikonda et al., 2021 ; Haenseler et al., 2017 ; Pandya et al., 2017 ; Takata et al., 2017 ) with some studies mentioning the development of cystic structures ( Haenseler et al., 2017 ; Muffat et al., 2016 ; Vaughan-Jackson et al., 2021 ). Because we sought insight into the tissue identity of the IBA1 + -cell enriched cystic compartments and we were confronted with the heterogeneity of our unguided protocol culture, we decided to enrich for the cystic compartments with a low-dosed BMP4 application one day after EB formation to the otherwise unchanged protocol ( Figure 3 A).…”

Zika virus-induces metabolic alterations in fetal neuronal progenitors that could influence in neurodevelopment during early pregnancy

“…Non-sendai reprogramming (Cytotune, Life Technologies) was used to reprogram fibroblast cells into hiPSCs. hiPSCs were cultured in defined, open-source medium termed OXE8 (47). Cells, resuspended as clumps by using 0.5 mM EDTA, were plated onto Geltrex TM precoated plates and cultured at 37 °C, 5% carbon dioxide.…”