Name: Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein (Video) | JoVE Journal
Uploaded: 2025-06-06T14:35:00.000+00:00
Duration: 10 min 59 s
Description: Learn to generate human iPSC-derived forebrain astrocytes for studying Fragile X Syndrome. This protocol enables investigation of metabolic dysregulation in glial cells and supports therapeutic development for neurodevelopmental disorders.

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5 minutes

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.

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
<ol>
	<li>One day before plating the iPSCs, coat a 6-well dish with 1:60 diluted Matrigel (an Extracellular Matrix [ECM]) in Advanced Dulbecco&#39;s Modified Eagle Medium/Ham&#39;s F-12 (DMEM/F12) and store at 2-8 &#176;C. 
	NOTE: Avoid storing Matrigel-coated dishes for &#62;5 days at 2-8 &#176;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 &#176;C and 5% CO2 for 1 h.</li>
	<li>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 &#176;C and 5% CO2 incubator before adding the hiPSCs.</li>
	<li>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 &#176;C.</li>
	<li>Next day, replenish the complete E8 medium without the ROCK inhibitor until the cells are 80% confluent (approximately 4-5 days).</li>
	<li>When they reach 80% confluency, detach the colonies enzymatically.
	<ol>
		<li>To passage the iPSCs, remove the spent media from the dish and add 1 mL per well of prewarmed (37 &#176;C) mixture of collagenase (2 mg/mL) and dispase (1 mg/mL) in a ratio of 1:1 and incubate at 37 &#176;C for more than 20-30 min to allow the iPSC colonies to lift. 
		NOTE: Do not leave the dishes at 37 &#176;C for more than 20-30 min; beyond this period, the colonies will disintegrate and cells will die.</li>
	</ol>
	</li>
	<li>When the colonies start to lift, take the dishes out from the incubator and neutralize the enzyme activity by adding 2 mL of Dulbecco&#39;s Phosphate-Buffered Saline (DPBS).</li>
	<li>Scrape off the colonies with DPBS and collect the suspension in a 15 mL conical tube using a wide-bore 10 mL serological pipette.</li>
	<li>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.</li>
	<li>Once the colonies settle down (approximately after 2 min), aspirate the DPBS enzyme mixture leaving approximately 1 mL in the tube.</li>
	<li>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.</li>
	<li>Alternatively, cryopreserve a proportion of colonies using 10% dimethyl sulfoxide (DMSO) as a cryoprotectant solution for future expansion and use.
	<ol>
		<li>Prepare cryopreservation medium freshly by mixing 90% complete E8 medium and 10% DMSO as a cryoprotectant solution and place it at 2-8 &#176;C until use.</li>
	</ol>
	</li>
	<li>To cryopreserve, follow steps 1.5-1.10.</li>
	<li>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 can&#160;damage cells.</li>
	<li>Immediately move the cryovials to a cryobox and keep in a -80 &#176;C freezer overnight.</li>
	<li>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.
	<ol>
		<li>To characterize hiPSCs using immunocytochemistry, plate the colonies on 1:60 Matrigel-coated, autoclaved 13 mm glass coverslips.&#160;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.</li>
		<li>After blocking, incubate the cultures with primary antibodies for 1 h and then wash 3x with PBS-T&#160;for 5 min each, followed by corresponding secondary antibodies (Table of Materials) in the dark for 1 h.</li>
		<li>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 &#181;m with 1 airy unit of pinhole diameter.</li>
	</ol>
	</li>
</ol>
2. Generation and characterization of astrocytic progenitor cells (APCs)
<ol>
	<li>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 medium14&#160;containing 50% Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM), 50% Ham&#39;s F-12 Nutrient Mix (F12), 5 mg/mL BSA, 1% Chemically Defined Lipid Concentrate (CD-Lipid), 450 &#181;M Monothioglycerol, 7 &#181;g/mL Insulin, 15 &#181;g/mL Transferrin, 1% Penicillin-Streptomycin supplemented with forebrain patterning mitogens N-acetyl Cysteine (1 mM), LDN 193189 (0.1 &#181;M), and SB431542 (10 &#181;M) 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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
	<li>For maturation of early gliospheres, replace bFGF-H with 20 ng/mL leukemia inhibitory factor (LIF) and maintain the spheres for 4 weeks.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>To passage:
	<ol>
		<li>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.</li>
		<li>Collect the cell suspension and centrifuge at 800 x&#160;&#609; for 2 min.</li>
		<li>Aspirate the supernatant, resuspend the cells in glial enrichment medium, and plate approximately 1 x&#160;106 cells/well on to a 1:80 Matrigel-coated 6-well dish.</li>
	</ol>
	</li>
	<li>For cryopreservation:
	<ol>
		<li>Resuspend the cells in a cold mixture of 90% corticosphere proliferation medium (without bFGF) and 10% cryoprotectant. Transfer the resuspended cells into cryovials.</li>
		<li>Immediately move the cryovials to a cryobox and keep in -80 &#176;C freezer overnight.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>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.</li>
</ol>
3. Generation and characterization of a homogeneous population of forebrain-specific astrocytes
<ol>
	<li>Differentiation of astrocytes from APCs
	<ol>
		<li>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).</li>
		<li>Confirm astrocytic identity by using immunostaining (as mentioned in steps 1.15.1-1.15.3.) with Glial fibrillary acidic protein (GFAP) and S100&#946; expression markers (Figure 2A,B).</li>
	</ol>
	</li>
	<li>De novo protein synthesis in APCs and astrocytes
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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 &#176;C and 5% CO2.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Mount the coverslips onto glass slides with mounting medium and use them for further image analysis.
		<ol>
			<li>Acquire images by confocal laser scanning at 405 nm, 488 nm, 561 nm, and 633nm.</li>
			<li>Capture images at 512 x 512 pixels; set the Z step size at 0.5 &#181;m with 1 airy unit of pinhole diameter.</li>
			<li>Maintain the microscopy and imaging parameters at a constant setting across cell types. Capture images for each biological replicate in the same session.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Adenosine 5&#8242;-triphosphate (ATP)-induced oscillatory calcium waves
	<ol>
		<li>Plate astrocytes on a glass bottom dish (35 mm) at 5 &#215; 103 cells/dish to measure cellular response to ATP.</li>
		<li>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 &#181;M 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 &#176;C.</li>
		<li>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.</li>
		<li>Record ATP-induced calcium responses by bath application of ATP at the final concentration of 5 mM at 25th s (Figure 4A).</li>
		<li>Draw regions of interest around each cell using Fiji/ImageJ and calculate the F340/F380 ratio across all time points (Figure 4B).</li>
	</ol>
	</li>
	<li>Cell metabolic assays 
	NOTE: The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells were measured as per the manufacturer&#39;s protocol (see the Table of Materials).
	<ol>
		<li>One day prior to the assay, allow the astrocytes to adhere to the microplate (1.5 &#215; 104 seeding density) in CNTF medium and incubate at 37 &#176;C with 5% CO2.</li>
		<li>For the glycolysis stress assay:
		<ol>
			<li>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 &#176;C for 1 h. 
			NOTE: pH must be adjusted in a 37 &#176;C water bath.</li>
			<li>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 &#181;M oligomycin, and 50 mM 2-deoxy-glucose (2-DG) (Figure 5A).</li>
			<li>At the end of the test, lyse the cells and estimate total protein content.</li>
		</ol>
		</li>
		<li>For the mitochondrion stress assay:
		<ol>
			<li>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 &#176;C.</li>
			<li>Incubate the plate in a non-CO2 chamber at 37 &#176;C for 1 h. Meanwhile, insert the cartridge plate into the instrument to calibrate the sensor.</li>
			<li>Post calibration, replace the cartridge plate with the cell culture plate with a final concentration of the following kit components: 1.5 &#181;M oligomycin, 1 &#181;M carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 &#181;M rotenone/antimycin A (Figure 5C).</li>
			<li>At the end of the test, lyse the cells and estimate total protein content.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Alvarez-Mora, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+mitochondrial+function+and+dynamics+in+the+pathogenesis+of+FXTAS%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">Impaired mitochondrial function and dynamics in the pathogenesis of FXTAS.</a> Mol Neurobiol. 54 (9), 6896-6902 (2017).</li>
<li>Giulivi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dysfunction+in+autism%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Mitochondrial dysfunction in autism.</a> JAMA. 304 (21), 2389-2396 (2010).</li>
<li>Anitha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+region-specific+altered+expression+and+association+of+mitochondria-related+genes+in+autism%5BTitle%5D)+AND+%22Mol+Autism%22%5BJournal%5D)">Brain region-specific altered expression and association of mitochondria-related genes in autism.</a> Mol Autism. 3 (1), 12(2012).</li>
<li>Teves, J. M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parkinson%27s+disease+skin+fibroblasts+display+signature+alterations+in+growth%2C+redox+homeostasis%2C+mitochondrial+function%2C+and+autophagy%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Parkinson's disease skin fibroblasts display signature alterations in growth, redox homeostasis, mitochondrial function, and autophagy.</a> Front Neurosci. 11, 737(2018).</li>
<li>Yao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+bioenergetic+deficit+precedes+Alzheimer%27s+pathology+in+female+mouse+model+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Mitochondrial bioenergetic deficit precedes Alzheimer's pathology in female mouse model of Alzheimer's disease.</a> Proc Natl Acad Sci USA. 106 (34), 14670-14675 (2009).</li>
<li>Sonntag, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Late-onset+Alzheimer%27s+disease+is+associated+with+inherent+changes+in+bioenergetics+profiles%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Late-onset Alzheimer's disease is associated with inherent changes in bioenergetics profiles.</a> Sci Rep. 7 (1), 14038(2017).</li>
</ol>

The authors have no conflicts of interest to disclose.

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...

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,&#160;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 &#34;passive support&#34; 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.

<table><tbody><tr><td>1-Thioglycerol</td><td>Sigma-Aldrich</td><td>M6145</td><td></td></tr><tr><td>Accutase&amp;nbsp;solution</td><td>Sigma-Aldrich</td><td>A6964</td><td>Enzyme cell detachment medium&amp;nbsp;</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate magnesium salt</td><td>Sigma-Aldrich</td><td>A9187</td><td></td></tr><tr><td>Advanced DMEM/F-12</td><td>ThermoFisher Scientific</td><td>12634010</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>ThermoFisher Scientific</td><td>15240062</td><td></td></tr><tr><td>B-27 Supplement (50x), serum-free</td><td>ThermoFisher Scientific</td><td>17504044</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Chemically Defined Lipid Concentrate</td><td>ThermoFisher Scientific</td><td>11905031</td><td></td></tr><tr><td>Collagenase, Type IV, powder</td><td>ThermoFisher Scientific</td><td>17104019</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Worthington Biochemical Corporation</td><td>LK003170</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dispase II, powder</td><td>ThermoFisher Scientific</td><td>17105041</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>ThermoFisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Essential 8 Medium</td><td>ThermoFisher Scientific</td><td>A1517001</td><td></td></tr><tr><td>FXS1, FXS2&amp;nbsp;</td><td>Coriell Institute of Medical Research&amp;nbsp;</td><td>GM07072, GM05848</td><td>FXS patient cells</td></tr><tr><td>GlutaMAX Supplement</td><td>ThermoFisher Scientific</td><td>35050061</td><td>glutamine substitute</td></tr><tr><td>Ham&#039;s F-12 Nutrient Mix</td><td>ThermoFisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Healthy control cells&amp;nbsp;</td><td>Cedars-Sinai Medical Center&amp;nbsp;</td><td>ND30625</td><td>healthy control cells&amp;nbsp;</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma-Aldrich</td><td>H3149</td><td></td></tr><tr><td>IMDM</td><td>ThermoFisher Scientific</td><td>12440053</td><td></td></tr><tr><td>Insulin, human</td><td>Roche</td><td>11376497001</td><td></td></tr><tr><td>LDN 193189&amp;nbsp;</td><td>Stratech Scientific</td><td>S2618-SEL</td><td></td></tr><tr><td>Leukemia Inhibitory Factor human</td><td>Sigma-Aldrich</td><td>L5283</td><td></td></tr><tr><td>Matrigel&amp;nbsp;Growth Factor Reduced (GFR) Basement Membrane Matrix</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>ThermoFisher Scientific</td><td>11140050</td><td></td></tr><tr><td>Mouse FGF-basic (FGF-2/bFGF) Recombinant Protein</td><td>Peprotech</td><td>450-33</td><td></td></tr><tr><td>Mr. Frosty freezing container</td><td>ThermoFisher Scientific</td><td>5100-0001</td><td>cryobox</td></tr><tr><td>N-2 Supplement (100x)</td><td>ThermoFisher Scientific</td><td>17502048</td><td></td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>Neurobasal Medium</td><td>ThermoFisher Scientific</td><td>21103049</td><td></td></tr><tr><td>Nunc Biobanking and Cell Culture Cryogenic Tubes</td><td>ThermoFisher Scientific</td><td>377267</td><td></td></tr><tr><td>Nunc Cell-Culture Treated 6 well dish</td><td>ThermoFisher Scientific</td><td>140675</td><td></td></tr><tr><td>Papain Dissociation System</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Recombinant Human CNTF Protein, CF&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>257-NT-010</td><td></td></tr><tr><td>Recombinant Human EGF Protein, CF</td><td>R&amp;amp;D Systems</td><td>236-EG-01M</td><td></td></tr><tr><td>RevitaCell Supplement (100x)</td><td>ThermoFisher Scientific</td><td>A2644501</td><td></td></tr><tr><td>SB431542</td><td>Tocris</td><td>1614</td><td></td></tr><tr><td>Seahorse XFe24 Analyzer&amp;nbsp;</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>Seahorse XF Cell Mito Stress Test Kit</td><td>Agilent Technologies</td><td>103015-100</td><td></td></tr><tr><td>Seahorse XF Glycolysis Stress Test Kit</td><td>Agilent Technologies</td><td>103020-100</td><td></td></tr><tr><td>Tissue Culture Dishes-100 cm</td><td>Biostar Lifetech</td><td>TCD000100</td><td></td></tr><tr><td>Transferrin</td><td>Roche</td><td>10652202001</td><td></td></tr><tr><td>VWR Razor Blades</td><td>VWR International</td><td>55411-050</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primary antibody&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Dilution&lt;/strong&gt;</td></tr><tr><td>Oct4 (C-10)</td><td>Santa Cruz Biotechnology</td><td>sc-5279</td><td>Dilution: 1:250&lt;br/&gt; Secondary antibody: Goat anti-Mouse IgG, Alexa Fluor 568</td></tr><tr><td>Nanog</td><td>R &amp;amp; D Systems</td><td>AF1997</td><td>Dilution: 1:100&lt;br/&gt; Secondary antibody: Donkey anti-Goat IgG, Alexa Fluor 488</td></tr><tr><td>Vimentin</td><td>Abcam</td><td>Ab5733</td><td>Dilution: 1:500&lt;br/&gt; Secondary antibody: Goat anti-Chicken IgY, Alexa Fluor 488</td></tr><tr><td>NFIA</td><td>Abcam</td><td>Ab41851</td><td>Dilution: 1:500&lt;br/&gt; Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 568</td></tr><tr><td>GFAP-cy3</td><td>Sigma</td><td>C9205</td><td>Dilution: 1:500&lt;br/&gt; Secondary antibody: NA</td></tr><tr><td>GFAP</td><td>DAKO</td><td>Z0334</td><td>Dilution: 1:500&lt;br/&gt; Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 568</td></tr><tr><td>S100&amp;beta;</td><td>DAKO</td><td>IR504</td><td>Dilution: 1:500&lt;br/&gt; Secondary antibody: Goat anti-Rabbit IgG, Alexa Fluor 488</td></tr><tr><td>Anti-Nuclei Antibody, clone 235-1</td><td>Merck Millipore</td><td>MAB1281</td><td>Dilution: 1:1000&lt;br/&gt; Secondary antibody: Goat anti-Mouse IgG1, Alexa Fluor 555</td></tr><tr><td>&lt;strong&gt;Secondary antibodies&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Dilution&lt;/strong&gt;</td></tr><tr><td>Goat anti-Mouse IgG, Alexa Fluor 568</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A11004</td><td>1:1000</td></tr><tr><td>Donkey anti-Goat IgG, Alexa Fluor 488</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A11055</td><td>1:1000</td></tr><tr><td>Goat anti-Chicken IgY, Alexa Fluor 488</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A11039</td><td>1:1000</td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 568</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A11011</td><td>1:1000</td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 488</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A11034</td><td>1:1000</td></tr><tr><td>Goat anti-Mouse IgG1, Alexa Fluor 555</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>A21127</td><td>1:1000</td></tr></tbody></table>

generation and characterization of human induced pluripotent stem cell-derived astrocytes lacking fragile x messenger ribonucleoprotein

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

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 Mothers&#160;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.

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...

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.

Fragile X syndrome (FXS), a leading inherited cause of autism spectrum disorder and intellectual disability, has been studied extensively using rodent ...

generation-characterization-human-induced-pluripotent-stem-cell

Research

JoVE Journal

Neuroscience

The video demonstrates a method to generate astrospheres from progenitors from human pluripotent stem cells. It involves the growth and proliferation of stem cells with nutrient media containing growth factors and kinase inhibitors. These cells then aggregate and differentiate into astrocytes, forming astrospheres.

Generating and Maintaining Astrospheres Using Human Pluripotent Stem Cells

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human induced Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Learn to generate human iPSC-derived forebrain astrocytes for studying Fragile X Syndrome. This protocol enables investigation of metabolic dysregulation in glial cells and supports therapeutic development for neurodevelopmental disorders.

Generation and Characterization of Human Induced Pluripotent Stem Cell-derived Astrocytes Lacking Fragile X Messenger Ribonucleoprotein (Video) | JoVE Journal

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

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