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BRAIN Publication-derived

Generation and Characterization of Down Syndrome Microglia from hiPSC-Derived PMPs in Cerebral Organoids and Chimeric Mouse Models

Source Jin et al., 2022 · Rutgers University · 10.1016/j.stem.2022.06.007

👤 Mengmeng Jin, Ranjie Xu, Le Wang, Mahabub Maraj Alam, Ziyuan Ma, Sining Zhu, Alessandra C. Martini, Azadeh Jadali, Matteo Bernabucci, Ping Xie, Kelvin Y. Kwan, Zhiping P. Pang, Elizabeth Head, Ying Liu, Ronald P. Hart, Peng Jiang ⏱ 56 days 📋 17 phases 🧫 Human iPSC-derived primitive macrophage progenitors (PMPs) from Down syndrome patients and controls

Abstract

This protocol generates Down syndrome (DS) and control microglia from hiPSCs via primitive macrophage progenitor differentiation, and characterizes their development, synaptic pruning, and response to pathological tau in cerebral organoids and human-mouse chimeric brain models. DS microglia exhibit enhanced synaptic pruning and senescence in response to pathological tau, mediated by elevated type I interferon signaling.

Cell source
Human iPSC-derived primitive macrophage progenitors (PMPs) from Down syndrome patients and controls
Application
Disease modeling of Down syndrome microglial dysfunction and response to pathological tau; developmental and neurodegenerative disease research

Protocol overview

90 steps across 17 phases

hiPSC Culture and PMP Generation from Yolk Sac Embryoid Bodies Days 0–42 (6 weeks)
  1. 1 hiPSC Culture on Matrigel
  2. 2 Generate Yolk Sac Embryoid Bodies (YS-EBs)
  3. 3 Stimulate Myeloid Differentiation
  4. 4 Harvest Primitive Macrophage Progenitors (PMPs)
  5. 5 Verify Trisomy 21 Status via FISH and Gene Copy Number Assay
pNPC Generation and Characterization Days 0–42 (continuous with phase 1)
  1. 1 Differentiate hiPSCs to Primitive Neural Progenitor Cells (pNPCs)
  2. 2 Passage and Quality Control of pNPCs
Cerebral Organoid Generation Days 0–56 (8 weeks)
  1. 1 Prepare Organoid Cell Mixture
  2. 2 Initial Organoid Culture (Days 0–3)
  3. 3 Transfer to 6-Well Plates (Days 3–14)
  4. 4 Switch to Differentiation Medium (Day 15 onwards)
  5. 5 Harvest Organoids (Week 4–8)
In Vitro Differentiation of PMPs to Microglia Days 0–14 (2 weeks)
  1. 1 Culture PMPs in Microglial Differentiation Medium
  2. 2 Change Media at One Week
  3. 3 Collect Differentiated Microglia (Day 14)
Cell Transplantation into Neonatal Mouse Brain Day 0 (P0 injection), monitored for 3–4 months
  1. 1 Prepare PMPs for Transplantation
  2. 2 Anesthetize Neonatal Mice (P0)
  3. 3 Stereotaxic Cell Transplantation
  4. 4 Post-Injection Recovery
Preparation of Soluble S1 Fractions from Human Brain Tissue Day 0 (can be done on fresh or stored tissue)
  1. 1 Homogenize Human Brain Tissue
  2. 2 Ultracentrifuge Homogenate
  3. 3 Collect Supernatant (S1 Fraction)
  4. 4 Verify Tau Pathology by Western Blot
Intracerebral Tau Injection into Chimeric Mice Day 0 of tau injection (at 8–10 weeks post-PMP transplantation); effects assessed at 1–6 months post-injection
  1. 1 Anesthetize Adult Chimeric Mice
  2. 2 Stereotaxic Injection of Tau Extract
  3. 3 Post-Injection Recovery and Monitoring
  4. 4 Assess Tau Pathology at 2 Weeks Post-Injection
Flow Cytometry Analysis for IFNAR Expression Days 0–1 (analysis after tissue collection)
  1. 1 Prepare Single-Cell Suspensions from Chimeric Brains
  2. 2 Block Fc Receptors
  3. 3 Stain with IFNAR1 and IFNAR2 Antibodies
  4. 4 Add Secondary Antibodies (if applicable)
  5. 5 Fix Cells and Perform Flow Cytometry
RNA Isolation and qPCR for Gene Expression Analysis Days 0–1 (analysis after tissue collection)
  1. 1 Extract Total RNA from Tissue or Cells
  2. 2 Reverse Transcribe RNA to cDNA
  3. 3 Perform Real-Time qPCR
  4. 4 Data Analysis
Bulk RNA-seq on Primitive Macrophage Progenitors Days 0–7 (sample preparation and sequencing initiation)
  1. 1 Extract RNA and Assess Quality
  2. 2 Prepare cDNA Libraries
  3. 3 Perform 75 bp Paired-End Sequencing
  4. 4 Align Reads and Count Genes
  5. 5 Identify Differentially Expressed Genes (DEGs)
Single-Cell RNA Sequencing (scRNA-seq) of Microglial Chimeras Following Tau Injection Days 0–10 (tissue processing through data analysis)
  1. 1 Collect and Prepare Brain Tissue
  2. 2 Dissociate Brain Tissue
  3. 3 Remove Debris and Myelin
  4. 4 Magnetically Deplete Mouse Cells
  5. 5 Prepare Cells for scRNA-seq
  6. 6 Perform 10x Chromium Capture and Library Preparation
  7. 7 Sequence Libraries on Illumina NovaSeq 6000
  8. 8 Process Raw Sequencing Data
  9. 9 Perform Quality Control and Normalization
  10. 10 Differential Expression and Pathway Analysis
  11. 11 Trajectory Inference Analysis
shRNA Knockdown of IFNAR1/IFNAR2 in DS hiPSCs Days 0–42 (transduction through stable selection)
  1. 1 Transduce hiPSCs with IFNAR1/2 or Control shRNA Lentivirus
  2. 2 Replace Transduction Medium
  3. 3 Puromycin Selection (Days 3–14)
  4. 4 Confirm Knockdown by qPCR and Western Blot
  5. 5 Use Stable hiPSCs for Subsequent Differentiation
Electrophysiology: Whole Cell Patch Clamp Recording of mEPSCs Day 0 (at 3 months post-transplantation; ~1–2 hours per experiment)
  1. 1 Prepare ACSF and Cutting Solutions
  2. 2 Anesthetize and Perfuse Mouse Brain
  3. 3 Prepare Brain Slices
  4. 4 Prepare Internal Recording Solution
  5. 5 Record mEPSCs from CA1 Pyramidal Neurons
  6. 6 Analyze Electrophysiological Data
  7. 7 Verify Microglia-Neuron Proximity (Post-hoc Immunostaining)
Electrophysiology: Extracellular Hippocampal Recording (LTP and PPR) Day 0 (at 3 months post-transplantation; ~2–3 hours per animal)
  1. 1 Prepare Brain Slices
  2. 2 Recover Brain Slices
  3. 3 Record Field EPSPs
  4. 4 Measure Paired-Pulse Ratio (PPR)
  5. 5 Induce Long-Term Potentiation (LTP)
  6. 6 Analyze LTP Data
  7. 7 Post-Recording Immunostaining
Tissue Immunostaining and Confocal Microscopy Days 0–3 (fixation, staining, imaging)
  1. 1 Fix Brain Tissue or Organoids
  2. 2 Dehydrate Tissue in Sucrose
  3. 3 Freeze Tissue in OCT
  4. 4 Cryosection Tissue
  5. 5 Block and Permeabilize Tissue
  6. 6 Incubate with Primary Antibodies
  7. 7 Wash and Incubate with Secondary Antibodies
  8. 8 Mount Slides
  9. 9 Acquire Images with Confocal Microscopy
  10. 10 Process Images and Analyze
Behavioral Testing Days 0–7 (testing at 4–5 months post-transplantation; 5–10 minutes per test)
  1. 1 Blind Group Assignment
  2. 2 Open Field Test
  3. 3 Elevated Plus Maze Test
  4. 4 Novel Object Recognition Test
Golgi Staining and Dendritic Spine Density Analysis Days 0–5 (staining through analysis)
  1. 1 Anesthetize and Collect Brain
  2. 2 Perform Golgi Staining
  3. 3 Section Brain and Mount Slides
  4. 4 Acquire Microscopy Images
  5. 5 Quantify Dendritic Spine Density

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Jin et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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