Name: Generatie en karakterisering van door mensen geïnduceerde pluripotente stamcel-afgeleide astrocyten zonder fragiel X-boodschapperribonucleoproteïne (Video) | JoVE Journal
Uploaded: 2025-06-06T14:35:00.000+00:00
Duration: 10 min 59 s
Description: Leer hoe je functionele voorhersen-specifieke astrocyten genereert uit menselijke iPSCs voor onderzoek naar het fragiele X-syndroom. Deze methode onthult metabole dysregulaties en opent nieuwe therapeutische mogelijkheden.

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<table><tbody><tr><td>1-Thioglycerol</td><td>Sigma-Aldrich</td><td>M6145</td><td></td></tr><tr><td>Accutase&nbsp;oplossing</td><td>Sigma-Aldrich</td><td>A6964</td><td>Enzyme celdetacheringsmedium&nbsp;</td></tr><tr><td>Adenosine 5'-trifosfaat magnesiumzout</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), serumvrij</td><td>ThermoFisher Scientific</td><td>17504044</td><td></td></tr><tr><td>Bovine Serum Albumine</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Chemisch gedefinieerd lipidconcentraat</td><td>ThermoFisher Scientific</td><td>11905031</td><td></td></tr><tr><td>Collageenase, Type IV, poeder</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>Dimethylsulfoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dispase II, poeder</td><td>ThermoFisher Scientific</td><td>17105041</td><td></td></tr><tr><td>DPBS, geen calcium, geen 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&nbsp;</td><td>Coriell Institute of Medical Research&nbsp;</td><td>GM07072, GM05848</td><td>FXS-patiëntcellen</td></tr><tr><td>GlutaMAX-supplement</td><td>ThermoFisher Scientific</td><td>35050061</td><td>glutamine-substituut</td></tr><tr><td>Ham's F-12 voedingsmengsel</td><td>ThermoFisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Gezond controlecellen&nbsp;</td><td>Cedars-Sinai Medical Center&nbsp;</td><td>ND30625</td><td>gezonde controlecellen&nbsp;</td></tr><tr><td>Heparine natriumzout uit varkens darmmucosa</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, humaan</td><td>Roche</td><td>11376497001</td><td></td></tr><tr><td>LDN 193189&nbsp;</td><td>Stratech Scientific</td><td>S2618-SEL</td><td></td></tr><tr><td>Leukemieremmend factor humaan</td><td>Sigma-Aldrich</td><td>L5283</td><td></td></tr><tr><td>Matrigel&nbsp;Growth Factor Reduced (GFR) basaal membraanmatrix</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>Muis FGF-basic (FGF-2/bFGF) recombinant eiwit</td><td>Peprotech</td><td>450-33</td><td></td></tr><tr><td>Mr. Frosty vriescontainer</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-cysteïne</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 wells schaal</td><td>ThermoFisher Scientific</td><td>140675</td><td></td></tr><tr><td>Papaine dissociatie systeem</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td></td></tr><tr><td>Penicilline-Streptomycine</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Recombinant humaan CNTF-eiwit, CF&nbsp;</td><td>R&amp;D Systems</td><td>257-NT-010</td><td></td></tr><tr><td>Recombinant humaan EGF-eiwit, CF</td><td>R&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&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>Weefselkweek schotels-100 cm</td><td>Biostar Lifetech</td><td>TCD000100</td><td></td></tr><tr><td>Transferrine</td><td>Roche</td><td>10652202001</td><td></td></tr><tr><td>VWR Scherpe messen</td><td>VWR International</td><td>55411-050</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Antilichamen&nbsp;</td><td></td><td></td><td></td></tr><tr><td>Primair antilichaam</td><td>Bedrijf</td><td>Catalogusnummer</td><td>Verdunning</td></tr><tr><td>Oct4 (C-10)</td><td>Santa Cruz Biotechnology</td><td>sc-5279</td><td>Verdunning: 1:250 Secundair antilichaam: Goat anti-Mouse IgG, Alexa Fluor 568</td></tr><tr><td>Nanog</td><td>R &amp; D Systems</td><td>AF1997</td><td>Verdunning: 1:100 Secundair antilichaam: Donkey anti-Goat IgG, Alexa Fluor 488</td></tr><tr><td>Vimentin</td><td>Abcam</td><td>Ab5733</td><td>Verdunning: 1:500 Secundair antilichaam: Goat anti-Chicken IgY, Alexa Fluor 488</td></tr><tr><td>NFIA</td><td>Abcam</td><td>Ab41851</td><td>Verdunning: 1:500 Secundair antilichaam: Goat anti-Rabbit IgG, Alexa Fluor 568</td></tr><tr><td>GFAP-cy3</td><td>Sigma</td><td>C9205</td><td>Verdunning: 1:500 Secundair antilichaam: NA</td></tr><tr><td>GFAP</td><td>DAKO</td><td>Z0334</td><td>Verdunning: 1:500 Secundair antilichaam: Goat anti-Rabbit IgG, Alexa Fluor 568</td></tr><tr><td>S100&beta;</td><td>DAKO</td><td>IR504</td><td>Verdunning: 1:500 Secundair antilichaam: Goat anti-Rabbit IgG, Alexa Fluor 488</td></tr><tr><td>Anti-Nuclei Antilichaam, clone 235-1</td><td>Merck Millipore</td><td>MAB1281</td><td>Verdunning: 1:1000 Secundair antilichaam: Goat anti-Mouse IgG1, Alexa Fluor 555</td></tr><tr><td>Secundaire antilichamen&nbsp;</td><td></td><td></td><td>Verdunning</td></tr><tr><td>Goat anti-Mouse IgG, Alexa Fluor 568</td><td>Thermo Fisher Scientific&nbsp;</td><td>A11004</td><td>1:1000</td></tr><tr><td>Donkey anti-Goat IgG,

generatie en karakterisering van door mensen geïnduceerde pluripotente stamcel-afgeleide astrocyten zonder fragiel x-boodschapperribonucleoproteïne

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

Generatie en karakterisering van door mensen geïnduceerde pluripotente stamcel-afgeleide astrocyten zonder fragiel X-boodschapperribonucleoproteïne

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

Neurowetenschap

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

Astrocyten zijn erkend als veelzijdige cellen die deelnemen in de fundamentele biologische processen die essentieel zijn voor de normale ontwikkeling van de hersenen en functies binnen het centraal zenuwstelsel repareren. Hier presenteren we een snelle procedure voor pure muis astrocyten culturen te verkrijgen om de biologie van deze belangrijke klasse van het centrale zenuwstelsel cellen te bestuderen.

Isolatie en Cultuur van de Muis Corticale Astrocyten

Astrocyten een van de belangrijkste belangrijkste spelers in het centrale zenuwstelsel (CNS). Hier worden we melding van een praktische methode van gesekst hippocampus astrocyten cultuur protocol om de mechanismen die ten grondslag liggen aan de astrocyt functie in de mannelijke en vrouwelijke pasgeboren pups na in-vitro ischemie te bestuderen.

Sex Verschillen in Mouse hippocampus Astrocytes na<em&gt; In-vitro</em&gt; Ischemie

We wijzigen en implementeren van een eerder gepubliceerd protocol beschrijft de snelle, reproduceerbare en efficiënte differentiatie van humane geïnduceerde pluripotente stamcellen (hiPSCs) in prikkelende corticale neuronen 12. Specifiek onze modificatie maakt regeling van neuronale celdichtheid en gebruik op micro-electrode arrays elektrofysiologische eigenschappen op netwerkniveau meten.

Rapid neuronale differentiatie van geïnduceerde pluripotente stamcellen voor het meten van het netwerk activiteit op micro-electrode arrays

We beschrijven een protocol om menselijke huid-afgeleide fibroblasten te herprogrammeren in geïnduceerde Neuronal Progenitor Cells (iNPC's), en hun daaropvolgende differentiatie in geïnduceerde Astrocyten (iA's). Deze methode leidt tot een snelle en reproduceerbare generatie van iNPC's en iA's in grote hoeveelheden.

In vitro modellering voor neurologische ziekten met behulp van directe conversie van fibroblasten naar neuronale voorlopercellen en differentiatie in astrocyten

We hebben een methode ontwikkeld die menselijke astrocytenpopulaties verrijkt en isoleert van vers ingevroren weefsel voor gebruik in downstream moleculaire analyses.

Isolatie van volwassen menselijke astrocytenpopulaties uit versgevroren cortex met behulp van fluorescentie-geactiveerde kernensortering

We beschrijven een methode voor het differentiëren van door het ruggenmerg geïnduceerde pluripotent-afgeleide astrocyten en neuronen en hun co-cultuur voor elektrofysiologische registratie.

Oprichting van een elektrofysiologisch platform voor het modelleren van ALS met regionaal specifieke menselijke pluripotente stamcel-afgeleide astrocyten en neuronen

Twee gerelateerde methoden worden beschreven om subcellulaire gebeurtenissen te visualiseren die nodig zijn voor synaptische transmissie. Deze protocollen maken de real-time monitoring van de dynamiek van presynaptische calciuminstroom en synaptische vesikelmembraanfusie mogelijk met behulp van live-cell imaging van in vitro gekweekte neuronen.

Real-time fluorescerende meting van synaptische functies in modellen van amyotrofische laterale sclerose

Hier beschrijven we een gedetailleerd protocol om sterk verrijkte culturen van astrocyten te genereren die zijn afgeleid van verschillende regio's van het centrale zenuwstelsel van postnatale muizen en hun directe omzetting in functionele neuronen door de geforceerde expressie van transcriptiefactoren.

Isolatie en directe neuronale herprogrammering van muisastryoten

Wij hebben, op basis van kennis uit de ontwikkelingsbiologie en gepubliceerd onderzoek, een geoptimaliseerd protocol ontwikkeld om efficiënt A9 middenhersen-dopaminerge neuronen te genereren uit zowel menselijke embryonale stamcellen als menselijke geïnduceerde pluripotente stamcellen, wat nuttig zou zijn voor ziektemodellering en celvervangende therapie voor de ziekte van Parkinson.

Geregisseerd Dopaminerge Neuron Differentiatie van menselijke pluripotente stamcellen

Leer hoe je functionele voorhersen-specifieke astrocyten genereert uit menselijke iPSCs voor onderzoek naar het fragiele X-syndroom. Deze methode onthult metabole dysregulaties en opent nieuwe therapeutische mogelijkheden.

Generatie en karakterisering van door mensen geïnduceerde pluripotente stamcel-afgeleide astrocyten zonder fragiel X-boodschapperribonucleoproteïne (Video) | JoVE Journal

Generatie en karakterisering van door mensen geïnduceerde pluripotente stamcel-afgeleide astrocyten zonder fragiel X-boodschapperribonucleoproteïne

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