Stemdiff hematopoietic kit

iPSC-microglia were generated as described in McQuade et al., 2018 (link) and McQuade and Blurton-Jones, 2021 (link). Briefly, iPSCs were directed down a hematopoietic lineage using the STEMdiff Hematopoietic kit (STEMCELL Technologies). After 10–12 days in culture, CD43+ hematopoietic progenitor cells were transferred into a microglia differentiation medium containing DMEM/F12, 2× insulin-transferrin-selenite, 2× B27, 0.5× N2, 1× GlutaMAX, 1× nonessential 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.

Differentiation of BiPSCs into HPC was performed using STEMdiff Hematopoietic Kit (STEMCELL Technologies, Vancouver, Canada). BiPSCs were cultured on Matrigel hESC-Qualified Matrix (CORNING, Corning, NY, USA), incubated with Gentle Cell Dissociation Reagent (STEMCELL Technologies) at room temperature for 4–6 min, removed using a scraper, and then, 50 μm-sized pieces were transferred to a matrigel-coated 12-well plate in Complete StemFit AK02N (100 pieces/well). Differentiation experiments were performed according to the instructions of the kit. BiPSCs with AID were cultured in the presence of 10 ng/mL doxycycline (Takara Bio, Kusatsu, Japan) to prevent AID expression. Concurrently, CD34⁺ cells were purified using MACS CD34 MicroBeads (Miltenyi Biotec Inc., Auburn, CA, USA) according to the manufacturer’s instructions, and the collected cells were used for phenotype analysis and a colony-forming assay.

iPSC colonies bearing desired mutation were differentiated using STEMdiff Hematopoietic kit obtained from STEMCELL Technologies (Catalog No. 05310) following the manufacturer’s protocol (outlined in Figure 2A). Thirty to 80 uniform sized colonies were plated in a six-well plate, and the next day media were exchanged with hematopoietic differentiation medium A, and on day 2 half media change was carried out with medium A. On day 3, medium A was removed, medium B added, and half media change was carried out on days 5, 7, and 10.

A detailed step-by-step protocol for generation of iMG from iPSCs is provided in the supplementary files. In brief, iPSCs were plated in Geltrex coated plates in mTeSR^TM1 media. In the first step of differentiation, STEMdiff Hematopoietic Kit (StemCell Technologies, Cat. No. 05310) was used for 12 days. The first three days were supplement A was used followed by nine days with supplement B for differentiation. The media was changed every other day. Cells were transferred to Poly-L-lysine (PLL) (Sigma, Cat. No. P4707-50ML) coated plates containing media C⁺⁺⁺ supplemented with astrocyte-conditioned media (ACM) in a 1:1 ratio. The complete composition of C⁺⁺⁺ and astrocytic conditioned media is provided in the supplementary step-by-step protocol. Growth factors were added freshly on the day of use. To check the differentiation efficiency, flow cytometry, immunocytochemistry and qPCR were performed to check for marker gene expression after every step. After 8 days in C⁺⁺⁺-ACM medium, floating cells were collected as microglia-like cells and used for functional or biochemical assays. Cells were collected every 2 to 3 days based on the confluency and plated on PLL coated plates in near homeostatic medium with freshly added growth factors, for functional assays. The composition of near homeostatic medium is provided in the supplementary step-by-step protocol.

Hematopoietic differentiation of iPSCs has been performed using a STEMDiff hematopoietic kit (STEMCELL Technologies, Grenoble, France, 05310) according to the manufacturer’s recommendations. Briefly, iPSCs (cultured on Geltrex with E8-flex medium) have been dissociated in aggregates of 50–100 μm by using EDTA (0.5 mM). A total of 50 aggregates have been seeded per well in a 12-well cell culture plate (Corning, Hazebrouck, France, 3513) coated with Geltrex. The medium was changed according to the manufacturer’s instruction, and cells were harvested on day 12 of hematopoietic differentiation using TrypLE select (ThermoFisher Scientific, Illkirch, France, 12604021). For single-cell transcriptomic assays, cells were harvested on day +5, day +9, and day +13 of hematopoietic differentiation.