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 제품안내  세포배양   Stem Cell

hiPSC-Derived Microglia

Microglia (미세아교세포)는 뇌 발달, 신경 발생, 시냅스 가소성 및 항상성 유지에 핵심 역할을 하는 뇌의 면역 세포입니다. 특히 뇌세포에서 생성된 찌꺼기 및 죽은신경세포를 제거하는 등 중추신경계 면역계에서 중요한 역할을 담당합니다. 식세포 작용을 통해 beta-amyloid를 제거하여 알츠하이머를 지연시키지만, 이에의해 지나치게 활성화된 microglia는 오히려 알츠하이머나 파킨슨 등 뇌질환을 유발하기도 합니다. iPSC유래 Microglia를 이용하여 in vivo 실험을 대체할 수 있습니다.

AXOL
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특징
  • Microglia는 뇌 발달, 신경 발생, 시냅스 가소성 및 항상성 유지에 핵심 역할을하는 뇌의 면역 세포입니다.
  • serum-free Microglia Maintenance Medium은 Microglia의 유지를 효과적으로 도모합니다.
  • 병원균에 반응하여 싸이토카인을 생성하고 대식작용을 하는 microglia의 생리학적 특징을 그대로 재현합니다.
  • microglia-specific marker인 TMEM119를 발현합니다.
  • iPSC 유래 Microglia는 알츠하이머 병, 다발성 경화증 및 파킨슨 병에서 신경 염증을 조사하는데 이용됩니다.
  • Progenitor 형태 및 바로 사용이 가능한 96-well plate 형태로 제공합니다.(30,000cells/well)





 iPSC-derived microglia
 Cat. No. Product Name Starting Material
 ax1666 Human iPSC-derived Microglial Precursors   Cord blood CD34+ cells derived iPSCs 
 ax0666  Human iPSC-derived Microglia (in 96-well plate format Cord blood CD34+ cells derived iPSCs 
 
 Microglia culture media and reagents
 Cat. No. Product Name Quantity
 ax0660 Microglia Maintenance Medium  100 mL




  • Morphology Characterization


    Human iPSC-derived microglial precursors (monocytes) were seeded using Microglia Maintenance Medium
    into 96-well plates at a density of 100,000 cells/cm2. During maturation, spherical monocytes adhere strongly
    to the culture surface, displaying increasingly ramified morphology as they differentiate to microglia. Axol’s
    iPSC-Derived Microglia show dynamic surveillance behaviour, which is representative of their phenotype in vivo.

  • Immunocytochemistry



   Immunocytochemistry of Axol’s iPSC-derived microglia at Day 18 of in culture.

    Immunocytochemistry of Human iPSC-derived Microglia, after 18 days in culture, revealed the expression of
    IBA-1 and CX3CR1 (a chemokine receptor) as well as the microglial-specific markers TMEM119
    (a transmembrane protein), and P2RY12 (purinergic receptor). All 3 markers co-localized strongly with
    the myeloid marker IBA-1, confirming identity of brain-resident-like microglia.


  • Microglia comparison with macrophages


     A. Microglia and macrophages from the same donor were stained in parallel for the macrophage/microglia 
     markers IBA-1 (voltage-gated Ca2+ sensor) and CX3CR1 (chemokine receptor) as well as microglial-specific
     markers P2RY12 (purinergic receptor) and TMEM119 (transmembrane protein). Expression of P2RY12 and
     TMEM119 is either absent or significantly less in macrophages compared to microglia, confirming microglial
     identity. x10 Magnification; Scale bar=400um

     B. mRNA expression of 6 key microglial signature genes in iPSCs, iPSC-derived monocytes, iPSC-derived
     macrophages and iPSC-derived microglia were determined by qRT-PCR. Cells were lysed from starting iPSCs,
     Day0 (monocytes), Day14 (macrophages) or Day18 (microglia) prior to RNA isolation followed by RT. CQ1A,
     MERTK, P2RY12, GPR34, and TMEM119 are all differentially upregulated in microglia. The disease-associated
     marker TREM2 is expressed at roughly constant levels across the myeloid cell lineage. Fold change was
     calculated using the ΔΔCT method, with PDGB as an endogenous control and normalisation to iPSC.
     N=3 (iPSC), N=2 (macrophage), N=1 (monocyte, microglia), technical replicates were performed in triplicate
     (monocyte, macrophage, microglia) or duplicate (iPSC). Error bars are +- S.E.M.


  • Phagocytosis Assay

     Axol’s iPSC-derived microglia efficiently phagocytose pHrodo green Zymosan particles

     A) pHrodo is a fluorogenic dye that dramatically increases in fluorescence as the pH of the surroundings
      becomes more acidic, such is the case in the lysosome. pHrodo® Green Zymosan BioParticles®
      (Life Technologies) were added at 0.5 mg/mL to day 19-microglia plated at 1x105sup>/cm2, and incubated
      for 3hrs at 37oC, 5% CO2. Nuclei were counterstained with a live Hoechst 33342 stain and live cell imaging
      was carried out using an on-stage incubator connected to an EVOS Fl Auto imaging system (Life Technologies).
      Two activated microglial cells can be seen consuming increasing numbers of BioParticles® over time leading
      greater and more intense fluorescence emission. B) and C) Microglia seeded at 1x105/cm2 (B) or 3x105cm2
      (C) were pre-treated with 100 ng/mL lipopolysaccharide (LPS) for 1hr (B), 5, 10μM or 30μM Cytochalasin D
      (CytoD, phagocytosis inhibitor) or 1μM or 10μM Latrunculin A (Lat-A) for 30mins (B) or 1hr (C) before
      adding pHrodo® Green Zymosan BioParticles® at 0.5 mg/mL and incubating for 3hrs at 37oC, 5% CO2.
      Lat-A inhibited phagocytosis more strongly than CytoD, in line with Lat-A being a more potent inhibitor
      of actin polymerization. Fluorescence was quantified using a SpectraMax iD3 microplate reader (Molecular
      Devices), and values were blanked against a no-cell control well. N=4 for both experiments, One-way
      ANOVA *P<0.05, **P<0.01, ***P


  • Cytokine Response


     A and B) Stimulation with TLR agonists LPS (TLR1/2) and R848 (TLR7/8), as well as the cytokine IL1β and
     interferon gamma (IFNγ) induces changes in TNFα and IL-6 release.

     Microglia plated at 120,000/cm2 were stimulated with increasing concentrations of LPS, R848, IL1β, and IFNγ
     for 20hrs. The concentration of TNFα (A) and IL-6 (B) in the harvested supernatant were measured by alphaLISA.
     Except for LPS, there is a clear dose-dependent, positive correlation between simulant concentration and amount
     of cytokine released. N=1. Data kindly provided by Grünenthal GmbH, Germany.

  • Co-culture of microglia and cortical neurons



    A. Immunocytochemistry of Human iPSC-derived Microglia co-cultured with Human iPSC-derived cortical
    neurons reveals microglia specific maker expression TMEM119 and neuronal marker β-3 Tubulin.





  Protocol

  Application Note

  • Characterization of human iPSC-derived Microglia using live-cell imaging reveals hallmarks of morphology and function 

    This study characterized human iPSC-derived microglia using IncuCyte® Live Cell assays (Essen BioScience, part of the Sartorius Group) to validate their morphological and functional features, offering insights into their suitability for immune and neurodegenerative disease research. Live-cell functional assays quantified the ability of human iPSC-derived microglia to phagocytose apoptotic neuronal cells (efferocytosis) and neurodegenerative disease associated peptides.
주문정보(Ordering Information)
CATNUM PRODUCT SIZE
ax0666Human iPSC-derived Microglia (in 96-well plate format)1 vial
ax1666Human iPSC-derived Microglial Precursors1 vial
ax0660Microglia Maintenance Medium 100 mL