title: hMG5
organism: Homo sapiens
description: Protocols: Animals were anesthetized using 5% isoflurane in oxygen and perfused transcardially with 0.1 M PBS. The human grafts were resected under a stereoscope (Zeiss SteREO Discovery.V8) and immersed in homogenization buffer: 1x HBSS (Life Technologies, 14175-095) containing 1% BSA, 1 mM EDTA. For mechanical dissociation, we used a 2-ml polytetrafluoroethylene pestle (Wheaton, 358026), as performed in Gosselin et al. (Gosselin et al., 2014, 2017). The homogenate was then pelleted, re-suspended in homogenization buffer and filtered through a 70-μm strainer. The filtered cells were first incubated 1:20 with a Fc receptor blocking antibody (Human TruStain FcX, BioLegend 422302) for 20 min, and then with the following cell surface marker antibody for 30 min (1:50 dilution): Anti-human CD45-APC/Cy7 (304014, clone HI30, BioLegend). Cells were washed twice, filtered again with a 70-μm strainer, and sorted on a BD Influx (100-μm nozzle, 22 PSI, 2-drop purity mode, sample chilling) as live/tdT+/hCD45+ cells. Zombie Violet (BioLegend; 1:500) was used for viability discrimination. Generation of forebrain organoids GFP-expressing hESC or subject-derived iPSC lines were used to generate forebrain organoids as described previously with minor modifications 50,67. Human iPSC colonies were detached before reaching confluency with collagenase Type IV (Gibco) and transferred to an Ultra-Low attachment 10-cm plate (Corning Costar) containing 10 ml hPSC medium consisting of DMEM:F12 (Invitrogen), 20% Knockout Serum Replacer (Gibco), 1× Non-essential Amino Acids (Invitrogen), 1× 2-mercaptoethanol (Gibco), 1× GlutaMAX (Invitrogen), 10 ng ml-1 FGF-2 (Peprotech) and ROCK inhibitor Y27632 (10 μM). Twenty-four hours later, the medium was replaced with induction medium containing hPSC media without FGF-2, 2 μM dorsomorphin (Cayman Chemical) and 2 μM A-083 (Stemgent). At day 5 the media were replaced with neural induction medium consisting of DMEM:F12 (Invitrogen), 1× N2 Supplement (Invitrogen), 1× Non-essential Amino Acids (Invitrogen), 1× GlutaMAX (Invitrogen), 10 μg ml-1 Heparin (Tocris), 1× Penicillin/Streptomycin (Gibco), 10 μM CHIR99021 (Tocris) and 1 μM SB-431542 (Tocris). Seven days after induction, organoids were embedded in 20-μl Matrigel (CultrexTM, Bio-Techne) droplets and continued to grow for an additional week in 6 cm Ultra-Low attachment plates (Corning Costar). From day 14 onwards, organoids were cultured in differentiation medium comprising DMEM:F12 (Invitrogen), 1× N2 and B27 Supplements (Invitrogen), 1× Non-essential Amino Acids (Invitrogen), 1× GlutaMAX (Invitrogen), 1× 2-Mercaptoethanol (Gibco), 1× Penicillin/Streptomycin (Gibco) and 2.5 μg ml-1 Insulin (Sigma), and transferred to an orbital shaker (65-75 rpm). At day 20, residual Matrigel was removed and media changes were performed every 2-3 days using the aforementioned differentiation medium. Colonization of cortical forebrain organoids through erythromyeloid progenitors (EMPs) EMPs were generated as previously described with minor modifications 18. Briefly, hESCs/iPSCs were dissociated using TrypLE (Invitrogen) and plated in a 6-cm tissue culture-treated plate (CytoOne USA Scientific) at a density of 400,000 cells per well with 10 μM ROCK inhibitor (Stem Cell Technologies). The next day, cells were changed to basal hematopoietic differentiation media supplemented with FGF2 (50 ng ml-1, Joint Protein Central), BMP4 (50 ng ml-1, Proteintech) Activin A (12.5 ng ml-1, R&D) ROCK inhibitor (1 μM, Stem Cell Technologies) and LiCl (2mM, Sigma) and grown under hypoxic conditions (5% CO2, 5% O2). Hematopoietic differentiation media consisted of IMDM (50%, Thermo Fisher Scientific), DMEM/F12 (50%), ITSG-X, (2% v/v, Thermo Fisher Scientific), L-ascorbic acid 2-Phosphate magnesium (64 mg ml-1; Sigma), monothioglycerol (400 mM), PVA (10 mg ml-1; Sigma), Glutamax (1X, Thermo Fisher Scientific), chemically defined lipid concentrate (1X, Thermo Fisher Scientific), non-essential amino acids (NEAA, Thermo Fisher Scientific 1X), Penicillin/Streptomycin (P/S Thermo Fisher Scientific, 1% V/V). On day 2, media was changed and supplemented with FGF2 (50 ng ml-1, Joint Protein Central) and VEGF (50 ng ml-1, R&D) and returned to hypoxic conditions. Following day 4, cells were placed into norm-oxic conditions (5% CO2, 21% O2) and kept in basal hematopoietic differentiation media supplemented with FGF2 (50 ng ml-1, Joint Protein Central), VEGF (50 ng ml-1, R&D) TPO (50 ng ml-1, Proteintech), SCF (10 ng ml-1, Proteintech), IL-6 (50 ng ml-1, R&D) and IL-3 (10 ng ml-1, R&D) until colonies released hematopoietic stem cells (usually between day 14 and 16). EMPs were then isolated using Fluorescence-activated cell sorting (FACS) by gating on tdT- and CD43-FITC (Biolegend, 315204)-positive cells. Co-culture of isolated EMPs with cortical organoids was performed using the indicated numbers (see manuscript and figure legends) in the above-indicated organoid differentiation media supplemented with 25 ng ml-1 M-CSF (R&D), 100 ng ml-1 IL34 (R&D) and 50 ng ml-1 TGFβ1 (R&D). The immunocompetent organoids that passed the quality control criteria (visible VZ-like regions and expansion of the CP) and showed sufficient EMP colonization were transplanted into the brain of NOD/SCID mice (JAX Stock: 001303) and kept for the indicated times (wpt - weeks post transplantation). TdT+/hCD45+ sorted cells were centrifuged and resuspended in PBS supplemented with 0.04% BSA to an adjusted concentration of 1,000 cells/μl. Single cells were then coupled to bar-coded beads in a droplet emulsion for the reverse transcription reaction using the 10X Genomics Chromium controller. Libraries were prepared following the standard protocol of 10X Single Cell 3' using v3.0 chemistry. TdT+/hCD45+ sorted cells were centrifuged and resuspended in PBS supplemented with 0.04% BSA to an adjusted concentration of 1,000 cells/μl. Single cells were then coupled to bar-coded beads in a droplet emulsion for the reverse transcription reaction using the 10X Genomics Chromium controller. Libraries were prepared following the standard protocol of 10X Single Cell 3' using v3.0 chemistry.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-05-11T00:15:04Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-05-11T00:15:04Z
attributes.tag: External Id
attributes.value: SAMEA112954507
attributes.tag: INSDC center name
attributes.value: Center for Organoid Systems Department of Psychiatry Technical University of Munich
attributes.tag: INSDC first public
attributes.value: 2023-05-11T00:15:04Z
attributes.tag: INSDC last update
attributes.value: 2023-05-11T00:15:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11522:hMG5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: iPSC derived cell line
attributes.tag: cell_type
attributes.value: microglial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo stage
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: forebrain organoid differentiation medium
attributes.tag: immunophenotype
attributes.value: tdT+/hCD45+
attributes.tag: organism part
attributes.value: forebrain
attributes.tag: progenitor cell type
attributes.value: embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-11522:hMG5
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: xenograft
attributes.value: organoid transplantation into NOD-SCID mice