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Method Article

Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein

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DOI:

10.3791/68081

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June 6th, 2025

Ā ,Ā Ā ,Ā Ā ,Ā Ā ,Ā 
1Centre for High Impact Neuroscience and Translational Applications, TCG Centres for Research and Education in Science and Technology, 2Centre for Brain Development and Repair, 3National Centre for Biological Sciences, Tata Institute for Fundamental Research, 4Academy of Scientific and Innovative Research (AcSIR)

In This Article

Summary

Here we describe a protocol that facilitates the differentiation of human induced pluripotent stem cells into functional forebrain-specific astrocytes. This enables investigations into the role of glial cells in the pathogenesis of neurodevelopmental disorders, such as Fragile X Syndrome, and modeling of other brain disorders.

Abstract

Fragile X syndrome (FXS), a leading inherited cause of autism spectrum disorder and intellectual disability, has been studied extensively using rodent models. More recently, human stem cell-derived model systems have also been used to gain mechanistic insights into the pathophysiology of FXS. However, these studies have focused almost exclusively on neurons. Further, despite growing evidence for a key role of glia in neuronal function in health and disease, little is known about how human astrocytes are affected by FXS.

Therefore, in this study, we successfully developed a protocol that captures key spatiotemporal milestones of brain development and aligns with the process of gliogenesis as well. Together this offers a useful framework for studying neurodevelopmental disorders. First, we patterned the human induced pluripotent stem cells into the neuroectodermal lineage with dual Suppressor of MothersĀ against Decapentaplegic (SMAD) inhibition and small molecules. Subsequently, we utilized specific growth factors and cytokines to generate control (CTRL) and FXS patient-derived astrocytic progenitor cells (APCs). Treatment of APCs with ciliary neurotrophic factor, a differentiating cytokine, regulated and drove the progenitor cells towards astrocytic maturation, yielding forebrain-specific glial fibrillary acidic protein-expressing astrocytes.

We found that these astrocytes are functional, as evidenced by their calcium responses to ATP application, and they exhibit dysregulated glycolytic and mitochondrial metabolism in FXS. Taken together, these findings provide a useful experimental platform of human origin for the investigation of cell-autonomous and non-cell-autonomous consequences of alterations in astrocytic function caused by neurodevelopmental disorders.

Introduction

Fragile X Syndrome (FXS), a common inherited form of intellectual disability and autism spectrum disorder (ASD), is caused by the lack of fragile X messenger ribonucleoprotein (FMRP) produced by the fragile X messenger ribonucleoprotein 1 (FMR1) gene (OMIM: #300624,Ā https://www.omim.org/entry/300624). FMRP plays a role in the regulation of mRNA translation, mRNA granule formation and transport, and microRNA-mediated regulation of gene expression1. Thus, loss of FMRP impacts not just brain development but also adult brain function. Both mRNA transcript levels of FMR1 and immunostaining for FMRP in the brain have shown high neuronal expression, alongside significant expression in glial cells as well2. However, a vast majority of earlier studies in animal models of FXS focused primarily on neurons and aberrations in their function. Consequently, little is known about the role of glia in FXS3. Traditionally thought of as "passive support" cells4, there is accumulating evidence for astrocytes being critical in mediating a wide range of neuronal functions5,6, including promoting synaptogenesis7, refinement of developing neural circuits8, and neurotransmitter recycling9. In parallel, there is growing evidence for the role of astrocytes in disease pathogenesis and many neurological conditions have been associated with astrocytic dysfunction10.

While much of the earlier work using animal models of FXS focused on identifying and validating various molecular targets in neurons for treating FXS, these preclinical findings have not always led to successful clinical outcomes. Further, setbacks in recent clinical trials also underscore the need for human-based model systems. Models of neurological disorders based on human stem cell-derived brain cells offer a powerful strategy to bridge this gap between mechanistic insights from animal studies and limited success with clinical outcomes for patients. However, only a handful of these studies have focused on astrocytes and that too mostly on astrocytes that were spinal in origin. This, in turn, is relevant in light of studies showing that the structure and function of astrocytes vary between brain regions11,12. Thus, a better understanding of disease-induced changes in human astrocytes also needs to take into account these brain region-specific differences in astrocytes. However, models of neurodevelopmental disorders using human stem cell-derived astrocytes that are specific to the forebrain remain comparatively underexplored13. Hence, to begin to address these gaps, we describe protocols for generating forebrain-specific astrocytes from patient-derived induced pluripotent stem cells (iPSCs) carrying FXS mutations; further, we show that astrocytes are functional and display altered metabolism.

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Protocol

All experiments using human induced pluripotent stem cells (hiPSCs) (Table 1) were performed after obtaining appropriate institutional regulatory approvals. Figure 1A represents complete differentiation protocol of hiPSCs to mature forebrain-specific astrocytes.

1. Maintenance and expansion of hiPSCs

  1. One day before plating the iPSCs, coat a 6-well dish with 1:60 diluted Matrigel (an Extracellular Matrix [ECM]) in Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 (DMEM/F12) and store at 2-8 Ā°C.
    NOTE: Avoid storing Matrigel-coated dishes for >5 days at 2-8 Ā°C, as this may cause protein degradation in the ECM over time. To avoid drying of the dish, coat a minimum of 1 mL of 1:60 diluted Matrigel per one well of a 6-well dish and spread evenly across the dish. If using the same day, keep the plate in a humidified incubator at 37 Ā°C and 5% CO2 for 1 h.
  2. On the day of culturing the hiPSCs, remove the coating material and add 1 mL of complete Essential 8 medium (E8 basal medium with Essential 8 supplement) along with the ROCK inhibitorat 1x final concentration and keep the dish in a 37 Ā°C and 5% CO2 incubator before adding the hiPSCs.
  3. Resuspend the hiPSCs in complete E8 medium along with the ROCK inhibitor at 1x final concentration for better adherence of the colonies.
    NOTE: Do not prepare large quantities of culture medium. Prepare only for 3-4 days maximum and store at 2-8 Ā°C.
  4. Next day, replenish the complete E8 medium without the ROCK inhibitor until the cells are 80% confluent (approximately 4-5 days).
  5. When they reach 80% confluency, detach the colonies enzymatically.
    1. To passage the iPSCs, remove the spent media from the dish and add 1 mL per well of prewarmed (37 Ā°C) mixture of collagenase (2 mg/mL) and dispase (1 mg/mL) in a ratio of 1:1 and incubate at 37 Ā°C for more than 20-30 min to allow the iPSC colonies to lift.
      NOTE: Do not leave the dishes at 37 Ā°C for more than 20-30 min; beyond this period, the colonies will disintegrate and cells will die.
  6. When the colonies start to lift, take the dishes out from the incubator and neutralize the enzyme activity by adding 2 mL of Dulbecco's Phosphate-Buffered Saline (DPBS).
  7. Scrape off the colonies with DPBS and collect the suspension in a 15 mL conical tube using a wide-bore 10 mL serological pipette.
  8. Gently triturate the suspension 2-3x to break up the colonies using a 10 mL serological pipette, and allow the colonies to settle down.
    NOTE: Do not make the colonies into single cells; this may result in the cells not adhering to the dish and cause more cell death. Further, do not leave them as big colonies; this will lead to more differentiated colonies later.
  9. Once the colonies settle down (approximately after 2 min), aspirate the DPBS enzyme mixture leaving approximately 1 mL in the tube.
  10. Add 2 mL of DPBS to the tube, mix the colonies by tapping, and allow them to settle. Repeat this 2x to remove all the residual enzyme from the colonies. Remove as much supernatant as possible after the 2nd wash, resuspend the colonies in 1 mL of complete E8 Medium, and plate in a freshly prepared dish as mentioned in steps 1.2-1.3.
  11. Alternatively, cryopreserve a proportion of colonies using 10% dimethyl sulfoxide (DMSO) as a cryoprotectant solution for future expansion and use.
    1. Prepare cryopreservation medium freshly by mixing 90% complete E8 medium and 10% DMSO as a cryoprotectant solution and place it at 2-8 Ā°C until use.
  12. To cryopreserve, follow steps 1.5-1.10.
  13. During step 1.10, after the 2nd wash, allow the colonies to settle and remove the supernatant. To the colonies, add 1 mL of freshly prepared cold cryopreservation medium and transfer to cryovials.
    NOTE: After adding the cryopreservation medium to the colonies, quickly transfer the contents to cryovials. Delaying may result in a lower revival rate as DMSO is a cryoprotectant that canĀ damage cells.
  14. Immediately move the cryovials to a cryobox and keep in a -80 Ā°C freezer overnight.
  15. The next day, shift all the cryovials to a Liquid Nitrogen tank (LN2 tank) for future use.
    NOTE: hiPSCs should be routinely karyotyped using G-banding (Supplemental Figure S1) for any abnormalities, characterized using immunocytochemistry for pluripotency (Figure 1B), and tested for mycoplasma.
    1. To characterize hiPSCs using immunocytochemistry, plate the colonies on 1:60 Matrigel-coated, autoclaved 13 mm glass coverslips.Ā Once they are 40% confluent, wash the cells with PBS-T (PBS-0.1% Tween 20), fix with 4% paraformaldehyde for 10 min, permeabilize with 0.3% Triton X-100 in PBS for 10 min, and block with 3% Bovine Serum Albumin (BSA) in PBS for 1 h to prevent non-specific binding.
    2. After blocking, incubate the cultures with primary antibodies for 1 h and then wash 3x with PBS-TĀ for 5 min each, followed by corresponding secondary antibodies (Table of Materials) in the dark for 1 h.
    3. Mount the coverslips onto glass slides with mounting medium and acquire images by confocal laser scanning at 405 nm, 488 nm, 561 nm, and 633nm. Capture images at 512 x 512 pixels; set the Z step size at 0.5 Āµm with 1 airy unit of pinhole diameter.

2. Generation and characterization of astrocytic progenitor cells (APCs)

  1. Enzymatically lift the hiPSCs as mentioned in steps 1.5-1.10 and plate them onto a non-adherent suspension culture dish (100 mm) with chemically defined medium14Ā containing 50% Iscove's Modified Dulbecco's Medium (IMDM), 50% Ham's F-12 Nutrient Mix (F12), 5 mg/mL BSA, 1% Chemically Defined Lipid Concentrate (CD-Lipid), 450 ĀµM Monothioglycerol, 7 Āµg/mL Insulin, 15 Āµg/mL Transferrin, 1% Penicillin-Streptomycin supplemented with forebrain patterning mitogens N-acetyl Cysteine (1 mM), LDN 193189 (0.1 ĀµM), and SB431542 (10 ĀµM) for 7 days.
    NOTE: Small molecule inhibitors SB431542 and LDN-193189 (LDN) are inhibitors of bone morphogenetic protein and transforming growth factor-beta signaling pathways.
    From this point onwards, the medium was replenished once in 2 days or following the Monday/Wednesday/Friday protocol.
  2. Place the cell suspension on an orbital shaker at 40 rpm for 7 days under normoxic conditions to aid the development of corticospheres (Figure 1A).
  3. On day 8, transfer the corticospheres to a cell proliferation medium containing Advanced DMEM/F12 with 1% Antibiotic-Antimycotic, 1% N2 supplement, 1% glutamine substitute, 0.1% B27 supplement, and 2.5 ng/mL basic fibroblast growth factor (bFGF) for 7 days.
  4. Induce the spheres for glial specification by subjecting them to glial enrichment medium containing Advanced DMEM/F12 with 1% Antibiotic-Antimycotic, 1% N2, 1% glutamine substitute, 0.1% B27 supplement, 20 ng/mL epidermal growth factor (EGF), bFGF-H (20 ng/mL bFGF-5 mg/mL heparin) for 2 weeks to get early gliospheres (Figure 1A).
  5. For maturation of early gliospheres, replace bFGF-H with 20 ng/mL leukemia inhibitory factor (LIF) and maintain the spheres for 4 weeks.
  6. After 4 weeks in the maturation medium, maintain the spheres in glial enrichment medium for prolonged periods.To prevent aggregation and loss of viability, every 2 weeks, mechanically chop the gliospheres using a sterile industrial blade and replace the entire medium with DNase I to remove DNA fragments generated from chopping.
  7. Dissociate the gliospheres into monolayers of APCs using a Papain dissociation kit and plate onto a cell culture-treated adherent dish with 1:80 dilution of Matrigel coating.
  8. Propagate the APCs in glial enrichment medium until 80% confluent and enzymatically passage them using the enzyme cell detachment medium (see the Table of Materials).
  9. To passage:
    1. Remove the spent media and collect in a conical tube. To the cells, add the enzyme cell detachment medium and wait for 1-2 min. Once the cells begin to detach, add the spent media to neutralize the enzyme activity.
    2. Collect the cell suspension and centrifuge at 800 xĀ É” for 2 min.
    3. Aspirate the supernatant, resuspend the cells in glial enrichment medium, and plate approximately 1 xĀ 106 cells/well on to a 1:80 Matrigel-coated 6-well dish.
  10. For cryopreservation:
    1. Resuspend the cells in a cold mixture of 90% corticosphere proliferation medium (without bFGF) and 10% cryoprotectant. Transfer the resuspended cells into cryovials.
    2. Immediately move the cryovials to a cryobox and keep in -80 Ā°C freezer overnight.
    3. The next day, shift all the cryovials to a Liquid Nitrogen tank (LN2 tank) for future use.
      NOTE: APCs were cryopreserved using 10% DMSO until passage number 6.
  11. Characterize the dissociated APCs by immunostaining (as mentioned in steps 1.15.1-1.15.3) with vimentin and Nuclear Factor IA (NFIA) markers (Figure 1C,D) (See the Table of Materials for dilutions).
  12. To confirm forebrain regional specificity of hiPSC-derived APCs, test the cells for a forebrain marker such as human forkhead box G1 (hFOXG1) (positive) and hindbrain marker such as human Homeobox B4 (hHOXB4) (negative) using real-time qPCR (Figure 1E,F).
    NOTE: The primer sequences used for these experiments have been listed in Supplemental Table S1.

3. Generation and characterization of a homogeneous population of forebrain-specific astrocytes

  1. Differentiation of astrocytes from APCs
    1. Use Astrocytic differentiation medium (ADM) for 14 days to differentiate APCs into astrocytes. Astrocytic differentiation medium contains Neurobasal, 1% Antibiotic-Antimycotic, 1% glutamine substitute, 1% N2 supplement, 0.2% B27 supplement, 1% non-essential amino acid medium (NEAA), and 10 ng/mL ciliary neurotrophic factor (CNTF).
    2. Confirm astrocytic identity by using immunostaining (as mentioned in steps 1.15.1-1.15.3.) with Glial fibrillary acidic protein (GFAP) and S100Ī² expression markers (Figure 2A,B).
  2. De novo protein synthesis in APCs and astrocytes
    1. Maintain hiPSC-derived forebrain APCs and astrocytes on sterile autoclaved 13 mm glass coverslips and immunostain them as mentioned in steps 1.15.1-1.15.3.
    2. For de novo protein synthesis, use fluorescent non-canonical amino acid tagging (FUNCAT) method, explained briefly in steps 3.2.3-3.2.6 (Figure 3A).
    3. To ensure deficiency of methionine, remove the growth medium from the cultures and replace with methionine- and cysteine-free medium intermixed with 1 mM L-azidohomoalanine (AHA) for 30 min at 37 Ā°C and 5% CO2.
    4. Wash the cultures with PBS-T, fix with 4% paraformaldehyde for 10 min, permeabilize with 0.3% Triton X-100 in PBS for 10 min, and block with 3% BSA in PBS for 1 h to prevent non-specific binding.
    5. Incubate the cultures in the dark for 1 h at room temperature with Click cell chemistry reaction mix and Alkyne Alexa Fluor 647, followed by primary antibodies for 1 h and corresponding secondary antibodies (Table of Materials) for 1 h (Figure 3B).
    6. Mount the coverslips onto glass slides with mounting medium and use them for further image analysis.
      1. Acquire images by confocal laser scanning at 405 nm, 488 nm, 561 nm, and 633nm.
      2. Capture images at 512 x 512 pixels; set the Z step size at 0.5 Āµm with 1 airy unit of pinhole diameter.
      3. Maintain the microscopy and imaging parameters at a constant setting across cell types. Capture images for each biological replicate in the same session.
      4. Perform intensity measurements using any standard image analysis software (e.g., Fiji or Imaris). In Imaris, use the SURFACES module to yield volumetric measurements of cell bodies positive for fluorescent non-canonical amino acid tagging (FUNCAT) signal with Vimentin (APCs) and GFAP (astrocytes) limiting the voxel range (250-350) to remove debris.
        NOTE: Maintain the parameters across all biological replicates for both cell types.
  3. Adenosine 5ā€²-triphosphate (ATP)-induced oscillatory calcium waves
    1. Plate astrocytes on a glass bottom dish (35 mm) at 5 Ɨ 103 cells/dish to measure cellular response to ATP.
    2. Allow the cells to adhere to the glass bottom for 24 h, wash them 3x with HBSS (20 mM HEPES, 137 mM NaCl, 5 mM KCl, 10 mM Glucose, 1 mM MgCl2, pH = 7.3) without calcium, and incubate in culture medium with 5 ĀµM ratio metric dye Fura-2AM and 0.02% Pluronics F127 for 1 h at room temperature.
      NOTE: Fura-2AM is a light- and temperature-sensitive chemical; store at -20 Ā°C.
    3. Post incubation, wash 2x with culture medium and replace with 2 mM Ca2+ containing HBSS and image the cells at 2 FPS speed using a 60x oil objective (1.35 NA) in a focus drift-compensating inverted microscope.
    4. Record ATP-induced calcium responses by bath application of ATP at the final concentration of 5 mM at 25th s (Figure 4A).
    5. Draw regions of interest around each cell using Fiji/ImageJ and calculate the F340/F380 ratio across all time points (Figure 4B).
  4. Cell metabolic assays
    NOTE: The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells were measured as per the manufacturer's protocol (see the Table of Materials).
    1. One day prior to the assay, allow the astrocytes to adhere to the microplate (1.5 Ɨ 104 seeding density) in CNTF medium and incubate at 37 Ā°C with 5% CO2.
    2. For the glycolysis stress assay:
      1. Replace the cell culture medium with base medium supplemented with 2 mM glutamine (pH adjusted to 7.4) and incubate in a non-CO2 chamber at 37 Ā°C for 1 h.
        NOTE: pH must be adjusted in a 37 Ā°C water bath.
      2. Insert the cartridge plate into the instrument to calibrate the sensor. Post calibration, replace the cartridge plate with the cell culture plate at a final concentration of the following kit components: 10 mM glucose, 1 ĀµM oligomycin, and 50 mM 2-deoxy-glucose (2-DG) (Figure 5A).
      3. At the end of the test, lyse the cells and estimate total protein content.
    3. For the mitochondrion stress assay:
      1. Replace the CNTF medium with base medium supplemented with 2 mM glutamine, 1 mM pyruvate, 10 mM glucose (pH adjusted to 7.4) in a water bath at 37 Ā°C.
      2. Incubate the plate in a non-CO2 chamber at 37 Ā°C for 1 h. Meanwhile, insert the cartridge plate into the instrument to calibrate the sensor.
      3. Post calibration, replace the cartridge plate with the cell culture plate with a final concentration of the following kit components: 1.5 ĀµM oligomycin, 1 ĀµM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 ĀµM rotenone/antimycin A (Figure 5C).
      4. At the end of the test, lyse the cells and estimate total protein content.

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Results

Human induced pluripotent stem cell (hiPSC) colonies were maintained using commercially available defined medium and immunostained for pluripotency markers, Oct4 and Nanog (Figure 1B). FXS and CTRL APCs showed comparable highly enriched proportions of cells immunopositive for vimentin and NFIA (Figure 1C,D). We found that APCs derived from healthy and FXS hiPSCs showed substantial upregulation of FOXG1 c...

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Discussion

Here, we describe a method to generate human iPSC-derived astrocytes that serve as an assay platform for characterizing functional changes induced by FXS. These astrocytes are functionally viable in culture and exhibit various properties, as evidenced by a range of measurements carried out in the present study. A critical step in this protocol is the initial conversion of iPSCs to corticospheres using the enzymatic lifting method. At this stage, optimizing the incubation time for collagenase type IV and dispase is crucia...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

We thank Prof. Sumantra Chattarji for intramural Funds. We thank Prof. Gaiti Hasan for access to the calcium imaging setup, Central Imaging and Flow Facility-National Centre for Biological Sciences, Padmanabh Singh and Prangya Hota for proofreading and suggestions, and the Labmate Asia team for their assistance in performing Seahorse XF assays.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1-ThioglycerolSigma-AldrichM6145
AccutaseĀ solutionSigma-AldrichA6964Enzyme cell detachment mediumĀ 
Adenosine 5ā€²-triphosphate magnesium saltSigma-AldrichA9187
Advanced DMEM/F-12ThermoFisher Scientific12634010
Antibiotic-Antimycotic (100x)ThermoFisher Scientific15240062
B-27 Supplement (50x), serum-freeThermoFisher Scientific17504044
Bovine Serum AlbuminSigma-AldrichA9418
Chemically Defined Lipid ConcentrateThermoFisher Scientific11905031
Collagenase, Type IV, powderThermoFisher Scientific17104019
Deoxyribonuclease IWorthington Biochemical CorporationLK003170
Dimethyl sulfoxideSigma-AldrichD2650
Dispase II, powderThermoFisher Scientific17105041
DPBS, no calcium, no magnesiumThermoFisher Scientific14190144
Essential 8 MediumThermoFisher ScientificA1517001
FXS1, FXS2Ā Coriell Institute of Medical ResearchĀ GM07072, GM05848FXS patient cells
GlutaMAX SupplementThermoFisher Scientific35050061glutamine substitute
Ham's F-12 Nutrient MixThermoFisher Scientific11765054
Healthy control cellsĀ Cedars-Sinai Medical CenterĀ ND30625healthy control cellsĀ 
Heparin sodium salt from porcine intestinal mucosaSigma-AldrichH3149
IMDMThermoFisher Scientific12440053
Insulin, humanRoche11376497001
LDN 193189Ā Stratech ScientificS2618-SEL
Leukemia Inhibitory Factor humanSigma-AldrichL5283
MatrigelĀ Growth Factor Reduced (GFR) Basement Membrane MatrixCorning354230
MEM Non-Essential Amino Acids Solution (100x)ThermoFisher Scientific11140050
Mouse FGF-basic (FGF-2/bFGF) Recombinant ProteinPeprotech450-33
Mr. Frosty freezing containerThermoFisher Scientific5100-0001cryobox
N-2 Supplement (100x)ThermoFisher Scientific17502048
N-Acetyl-L-cysteineSigma-AldrichA9165
Neurobasal MediumThermoFisher Scientific21103049
Nunc Biobanking and Cell Culture Cryogenic TubesThermoFisher Scientific377267
Nunc Cell-Culture Treated 6 well dishThermoFisher Scientific140675
Papain Dissociation SystemWorthington Biochemical CorporationLK003150
Penicillin-StreptomycinThermoFisher Scientific15140122
Recombinant Human CNTF Protein, CFĀ R&D Systems257-NT-010
Recombinant Human EGF Protein, CFR&D Systems236-EG-01M
RevitaCell Supplement (100x)ThermoFisher ScientificA2644501
SB431542Tocris1614
Seahorse XFe24 AnalyzerĀ Agilent Technologies
Seahorse XF Cell Mito Stress Test KitAgilent Technologies103015-100
Seahorse XF Glycolysis Stress Test KitAgilent Technologies103020-100
Tissue Culture Dishes-100 cmBiostar LifetechTCD000100
TransferrinRoche10652202001
VWR Razor BladesVWR International55411-050
AntibodiesĀ 
Primary antibodyCompanyCatalog numberDilution
Oct4 (C-10)Santa Cruz Biotechnologysc-5279Dilution: 1:250
Secondary antibody: Goat anti-Mouse IgG, Alexa Fluor 568
NanogR & D SystemsAF1997Dilution: 1:100
Secondary antibody: Donkey anti-Goat IgG, Alexa Fluor 488
VimentinAbcamAb5733Dilution: 1:500
Secondary antibody: Goat anti-Chicken IgY, Alexa Fluor 488
NFIAAbcamAb41851Dilution: 1:500
Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 568
GFAP-cy3SigmaC9205Dilution: 1:500
Secondary antibody: NA
GFAPDAKOZ0334Dilution: 1:500
Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 568
S100Ī²DAKOIR504Dilution: 1:500
Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 488
Anti-Nuclei Antibody, clone 235-1Merck MilliporeMAB1281Dilution: 1:1000
Secondary antibody: Goat anti-Mouse IgG1, Alexa Fluor 555
Secondary antibodiesĀ Dilution
Goat anti-Mouse IgG, Alexa Fluor 568Thermo Fisher ScientificĀ A110041:1000
Donkey anti-Goat IgG, Alexa Fluor 488Thermo Fisher ScientificĀ A110551:1000
Goat anti-Chicken IgY, Alexa Fluor 488Thermo Fisher ScientificĀ A110391:1000
Goat anti-Rabbit IgG, Alexa Fluor 568Thermo Fisher ScientificĀ A110111:1000
Goat anti-Rabbit IgG, Alexa Fluor 488Thermo Fisher ScientificĀ A110341:1000
Goat anti-Mouse IgG1, Alexa Fluor 555Thermo Fisher ScientificĀ A211271:1000

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Fragile X SyndromeInduced Pluripotent Stem CellsAstrocyte Progenitor CellsAstrocyte DifferentiationGlial Enrichment MediumCalcium ImagingGlycolytic MetabolismMitochondrial RespirationImmunostaining MarkersNeurodevelopmental Disorders
Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein
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