Name: derivation, expansion, cryopreservation and characterization of brain microvascular endothelial cells from human induced pluripotent stem cells
Uploaded: 2020-11-19T14:00:00
Description: Watch this Scientific Journal Video about Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells at JoVE.com

Dette arbejde blev støttet af en National Institute of Mental Health Biobehavioral Research Awards for Innovative New Scientists (BRAINS) Award R01MH113858 (til R.K.), en National Institutes of Health Award KL2 TR002542 (PL). en National Institute of Mental Health Clinical Scientist Development Award K08MH086846 (til R.K.), en Sydney R Baer Jr Foundation Grant (til P.L.) Doris Duke Charitable Foundation Clinical Scientist Development Award (til R.K.), Ryan Licht Sang Bipolar Foundation (til R.K.), Phyllis & Amp; Jerome Lyle Rappaport Foundation (til R.K.), Harvard Stem Cell Institute (til R.K.) og af Steve Willis og Elissa Freud (til R.K.). Vi takker Dr. Annie Kathuria for hendes kritiske læsning og feedback på manuskriptet.

Human iPSCs blev omprogrammeret fra fibroblaster af raske donorer ved hjælp af en protokol godkendt af Institutional Review Boards of Massachusetts General Hospital og McLean Hospital, og karakteriseret som beskrevet i tidligere undersøgelser44,45,46.
BEMÆRK: Kort, fibroblaster blev omprogrammeret til iPSC via mRNA-baserede genetiske omprogrammering47. IPSC'erne blev opretho...

Ændringer og fejlfinding
I denne protokol har vi foretaget nogle ændringer i brugen af en almindeligt anvendt ekstracellulær matrix og cellekultur medier under iPSC dyrkning til afledning af BMECs (Figur 1). Disse ændringer påvirkede ikke evnen til at udlede BMECS fra menneskelige iPSC'er som beskrevet i Lippmann-protokol1. Der blev anvendt en iPSC-linje fra en anden sund donor til at påvise , at denne ændrede protok...

Blod-hjerne barriere funktion Blod-hjerne barrieren (BBB) danner en grænse, der begrænser bevægelse af stoffer fra blodet til hjernen. BBB består af hjerne mikrovaskulære endotelceller (BMECs), der danner et monolag foring vaskulaturen. BMECs, sammen med astrocytter, neuroner, pericytes, microglia, og ekstracellulær matrix, danner neurovaskulær enhed. BMECs har en stramt reguleret paracellulær struktur, der gør det muligt for BBB at opretholde høj trans-endotel elektrisk modstand (TEER), som begrænser passiv diffusion og fungerer som en indikator for barriereintegritet1,2. BMECs har også proteiner, der hjælper med transcellulær bevægelse såsom endokytose, transcytose og transmigration samt ekstravasation af leukocytter under et immunrespons3. BMECs er afhængige af tilstrømning og efflux transportører til næring og fjernelse af affaldsprodukter, for at opretholde en homeostatic balance i hjernen3. F.eks. solute carrier family 2 member 1 (SLC2A1) er en tilstrømning transportør ansvarlig for flytning af glukose på tværs af BBB4, mens efflux transportører såsom ATP bindende kassette subfamily B medlem 1 (ABCB1) og ATP bindende kassette subfamily C medlem 1 (ABCC1) er ansvarlige for at returnere substrater tilbage i blodet3,5,6,7. ABCB1 substrater omfatter morfin, verapamil4, og antipsykotika såsom olanzapine og risperidone8, mens ABCC1 transportør har en række substrater, herunder sulfat konjugater, vincristine, og glucuronide konjugater4.
Anvendelse af BBB-modeller i psykiatriske lidelser BBB dysfunktion har været impliceret i en række neurologiske og psykiatriske lidelser, herunder skizofreni og bipolar lidelse9,10. For nylig bruges iPSC-afledte ex vivo cellulære modeller til at afhøre det cellulære og molekylære fundament af psykiatriske lidelser, men disse modeller tager i øjeblikket ikke højde for den potentielle rolle, som neurovasculature11,12,13. Det er hypotese, at perifere inflammatoriske cytokiner, der cirkulerer iblodet,kan have en negativ indvirkning på BBB14,15,16,17, men der er også tegn på paracellulær18,19,20,21,122, transcellulær23,24,25,26,27,28,29og ekstracellulær matrix20,29,30,31,32 abnormiteter, der bidrager til BBB-dysfunktion. Forstyrrelse af BBB kan resultere i, at indholdet af blodet kommer ind i hjernen parenchyma og aktiverer astrocytter og / eller mikroglia for at frigive proinflammatoriske cytokiner, som igen indleder en inflammatoriskreaktion 33, der kan have skadelige virkninger på hjernen34. BMECs er den primære komponent i BBB og undersøge strukturen og funktionen af disse celler kan forbedre forståelsen af BBB dysfunktion i neurologiske og psykiatriske lidelser.
Alternative BMEC-modeller Forud for udviklingen af effektive protokoller for at udlede BMECs fra iPSCs1, 6,35,36, forskere havde ansat udødeliggjort BMECs37 at studere BBB funktion. Mange af disse modeller opnåede imidlertid ikke ønskelige BBB-fænotyper, et sådant fysiologisk interval af TEER-værdier38,39. Brug af iPSCs har den fordel at bevare den genetiske baggrund af den person, hvorfra cellerne er afledt. Forskere arbejder aktivt på at etablere iPSC-afledte ex vivo mikromiljømodeller, der opsummerer strukturen og funktionen af den menneskelige hjerne. Forskere har udviklet metoder til at udlede BMECs, der er strukturelt og fysiologisk ligner BMECs findes in vivo. Metoder til opnåelse af rensede populationer af iPSC-afledte BMEC'er kræver en række forskellige trin , hvor protokollerne er optimeret i de seneste år1,6,35,36. Generelt er iPSC-afledte BMECs dyrket i Essential 6 (E6) medium i 4 dage, efterfulgt af 2 dage i humant endotel serumfrit medium (hESFM) suppleret med grundlæggende fibroblast vækstfaktor (bFGF), retinosyre (RA) og B27 supplement. Cellerne er derefter dyrket på en kollagen IV (COL4) og fibronectin (FN) matrix for at opnå >90% homogenE BMECs1.
BMEC'ernes identitet bekræftes af immunfluorescens, der viser co-ekspressionen af BMEC-proteiner, herunder blodplade-endotelcelle vedhæftningsmolekyle-1 (PECAM1), SLC2A1 og stramme krydsproteiner som stramt krydsprotein 1 (TJP1), occludin (OCLN) og claudin-5 (CLDN5)6. Spirende assays er blevet brugt til at bekræfte det angiogene potentiale i iPSC-afledte BMECs. 6 BBB-integriteten af BMEC'er vurderes ved tilstedeværelsen af fysiologiske IN VITRO TEER-værdier (~2000Ω x cm2)37 og målelig aktivitet for effluxtransportører som ABCB1 og ABCC11,6,36. Lippmann-gruppens seneste metodologiske fremskridt har ført til iPSC-afledte BMEC-protokoller med reduceret eksperimentel variation og forbedret reproducerbarhed1. Det vides dog ikke, om de kan udvides og passeres ud over underkulturfasen. Vores ændrede protokol har til formål at løse dette problem ved at videregive iPSC-afledte BMECs efter dag 8 og vurdere, om de kan udvides yderligere til at bevare BBB-egenskaber efter kryopræservering. Mens ingen undersøgelser har beskrevet passaging af iPSC-afledte BMECs, findes der en protokol for BMEC cryopreservation, der bevarer fysiologiske BBB-egenskaber efter at have gennemgået en fryse-optøningscyklus40. Det er dog ikke kendt, at BMEC'er efter kryopræservering kan passeres og bevare BBB-egenskaber.
BMECs afledt af iPSC'er ved hjælp af Lippmann-protokollen er blevet brugt til at modellere BBB-forstyrrelser i neurologiske lidelser som Huntingtons Sygdom7. Sådanne iPSC-afledte BMEC'er er også blevet anvendt til at undersøge virkningerne af bakteriel infektion som Neisseria meningitidis eller Gruppe B Streptococcus på afbrydelse af blod-CSF-barrieren og BBB henholdsvis41,42. Også ved hjælp af iPSC-afledte BMECs fra 22q sletning syndrom patienter med skizofreni, forskere observeret en stigning i intercellulær vedhæftning molekyle-1 (ICAM-1), en stor vedhæftning molekyle i BMECs, der hjælper med rekruttering og ekstravasation af leukocytter i hjernen43. Tilsammen viser disse undersøgelser nytten af iPSC-afledte BMECs til at studere BBB-forstyrrelser i komplekse neuropsykiatriske lidelser.

<table>
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		<tr>
			<td>2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate</td>
			<td>Sigma Aldrich</td>
			<td>D6883-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma Aldrich</td>
			<td>A6964-100mL</td>
			<td></td>
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			<td>Alexa Fluor 488 Donkey anti-Mouse IgG</td>
			<td>Life Technologies</td>
			<td>A-21202</td>
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			<td>Alexa Fluor 555 Donkey anti-Rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A-31572</td>
			<td></td>
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			<td>B27 Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
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			<td>CD31 (PECAM-1) (89C2) Mouse mAb</td>
			<td>Cell Signaling</td>
			<td>3528S</td>
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		<tr>
			<td>CLDN5 (Claudin-5)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35-2500</td>
			<td></td>
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		<tr>
			<td>Collagen IV from human placenta</td>
			<td>Sigma Aldrich</td>
			<td>C5533-5mg</td>
			<td></td>
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		<tr>
			<td>Corning 2 mL Internal Threaded Polypropylene Cryogenic Vial&#160;</td>
			<td>Corning</td>
			<td>&#160;8670</td>
			<td></td>
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		<tr>
			<td>Corning&#160;Costar Flat Bottom Cell Culture Plates (6-wells)</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
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		<tr>
			<td>Corning&#160;Falcon Flat Bottom Cell Culture Plates (24-wells)</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
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		<tr>
			<td>Corning Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts (12-wells)</td>
			<td>Corning</td>
			<td>3460</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (without phenol red)</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;A1413202</td>
			<td></td>
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		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2438-50mL</td>
			<td></td>
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		<tr>
			<td>Donkey serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663-10ML</td>
			<td></td>
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		<tr>
			<td>DPBS (+/+)</td>
			<td>Gibco/Thermo Fisher Scientific</td>
			<td>14040-117</td>
			<td></td>
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		<tr>
			<td>Epithelial Volt/Ohm (TEER) Meter (EVOM2) STX2</td>
			<td>World Precision Instruments</td>
			<td>N/A</td>
			<td></td>
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			<td>Essential 6 Medium (Thermo Fisher)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1516401</td>
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		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td></td>
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			<td>Fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-2mg</td>
			<td></td>
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		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413202</td>
			<td></td>
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		<tr>
			<td>Hanks&#39; Balance Salt Solution with calcium and magnesium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human endothelial serum-free medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11111044</td>
			<td></td>
		</tr>
		<tr>
			<td>InCell Analyzer 6000</td>
			<td>General Electric</td>
			<td>N/A</td>
			<td></td>
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		<tr>
			<td>Invitrogen&#160;Countess Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MK-571</td>
			<td>Sigma Aldrich</td>
			<td>M7571-5MG</td>
			<td></td>
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		<tr>
			<td>NutriStem</td>
			<td>Stemgent</td>
			<td>01-0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Occludin</td>
			<td>Thermo Fisher Scientific</td>
			<td>33-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Perkin Elmer Envision 2103 multi-plate Reader</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human VEGF 165</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic (154 a.a.)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine 123</td>
			<td>Sigma Aldrich</td>
			<td>83702-10MG</td>
			<td></td>
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			<td>SLC2A1 (GLUT-1)</td>
			<td>ThermoFisher</td>
			<td>PA1-21041</td>
			<td></td>
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			<td>SOX17</td>
			<td>Cell Signaling</td>
			<td>81778S</td>
			<td></td>
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		<tr>
			<td>TJP-1 (ZO-1)</td>
			<td>ThermoFisher</td>
			<td>PA5-28869</td>
			<td></td>
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			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787-50ML</td>
			<td></td>
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		<tr>
			<td>Trypan Blue Stain (0.4%) for use with the Countess Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
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		<tr>
			<td>Valspodar (Sigma) (cyclosporin A)</td>
			<td>Sigma Aldrich</td>
			<td>SML0572-5MG</td>
			<td></td>
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		<tr>
			<td>Versene solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>15040066</td>
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		<tr>
			<td>Y-27632 dihydrochloride (ROCK inhibitor)</td>
			<td>Tocris/Thermo Fisher Scientific</td>
			<td>1254</td>
			<td></td>
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derivation, expansion, cryopreservation and characterization of brain microvascular endothelial cells from human induced pluripotent stem cells

Hjernemikrobielle endotelceller (BMECs) kan skelnes fra menneskeskabte pluripotente stamceller (iPSCs) for at udvikle ex vivo cellulære modeller til undersøgelse af blod-hjerne-barriere (BBB) funktion. Denne ændrede protokol indeholder detaljerede trin til at udlede, udvide og kryopræservere BMEC'er fra menneskelige iPSC'er ved hjælp af en anden donor og reagenser end dem, der er rapporteret i tidligere protokoller. IPSCs behandles med essentielle 6 medium i 4 dage, efterfulgt af 2 dage af menneskelige endotel serum-fri kultur medium suppleret med grundlæggende fibroblast vækstfaktor, retinosyre, og B27 supplement. På dag 6, er celler sub-dyrket på en kollagen / fibronectin matrix i 2 dage. Immunocytochemistry udføres på dag 8 for BMEC-markøranalyse ved hjælp af CLDN5, OCLN, TJP1, PECAM1 og SLC2A1. Vestlig blotting udføres for at bekræfte BMEC-markørudtryk og fravær af SOX17, en endodermal markør. Angiogent potentiale demonstreres med en spirende analyse. Trans-endotel elektrisk modstand (TEER) måles ved hjælp af spisepindeelektroder og voltohmmeter fra dag 7. Efflux transportøraktivitet for ATP-bindingskassetteunderfamilie B-medlem 1 og ATP-bindende kassetteunderfamilie C-medlem 1 måles ved hjælp af en flerpladelæser på dag 8. En vellykket afledning af BMECs bekræftes af tilstedeværelsen af relevante cellemarkører, lave niveauer af SOX17, angiogent potentiale, transportaktivitet og TEER-værdier ~2000 Ω x cm2. BMECs udvides indtil dag 10, før de passerer på friskbelagte kollagen / fibronectin plader eller cryopreserved. Denne protokol viser, at iPSC-afledte BMEC'er kan udvides og passeres mindst én gang. Der blev dog observeret lavere TEER-værdier og dårligere lokalisering af BMEC-markører efter kryopræservering. BMECs kan bruges i co-kultur eksperimenter med andre celletyper (neuroner, glia, pericytes), i tre-dimensionelle hjerne modeller (organ-chip og hydrogel), for vaskularisering af hjernen organoider, og for at studere BBB dysfunktion i neuropsykiatriske lidelser.

BMEC Differentiering Et par kritiske skridt i denne protokol bør følges præcist (figur 1). E6 medium brug på dag 1 er vigtig, da det ofte bruges til at udlede neuroectoderm afstamning fra iPSCs inden for en relativt kort periode giver reproducerbare resultater på tværs af flere cellelinjer36. Et andet vigtigt skridt er på dag 4 i differentiering, hvor E6-medium skal skiftes til hESFM med fortyndet (1:200) B27, 20 ng/mL bFGF og 10 μM RA for ...

Watch this Scientific Journal Video about Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells at JoVE.com

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Denne protokol beskriver en tilpasset metode til at udlede, udvide og kryopreserve hjernens mikrovaskulære endotelceller opnået ved at differentiere humane inducerede pluripotente stamceller og til at studere blodhjernebarriereegenskaber i en ex vivo-model.

Afledning, ekspansion, kryopræservering og karakterisering af hjernemikrobielle endotelceller fra menneskeskabte pluripotente stamceller

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

BMECs generated from iPSC provide new experimental models for studying blood brain barrier function and dysfunction. We have used this protocol to examine whether BMECs retain their barrier properties after expansion and cryopreservation. This technique enables the generation of disease-specific BMECs from patients with disorders in which blood-brain barrier disruption plays a role in the pathophysiology of disease.To induce BMEC differentiation from human iPSCs, wash the confluent iPSC culture with DPBS one time before incubating the cells with the appropriate volume of enzymatic EDTA for approximately five minutes at 37 degrees Celsius. When a single cell suspension has been obtained collect the cells by centrifugation and resuspend the pellet in an appropriate volume of stem cell medium supplemented with 10 micromolar ROCK inhibitor for counting. Dilute the cells to 1.56 times 10 to the fourth cells per cubic centimeter concentration and seed two milliliters of cells into individual wells of a six-well flat bottom.After 24 hours in the cell culture incubator replace the supernatant in each well with E6 medium and return the plate to the incubator for four more days. To purify the iPSC-derived BMECs, coat the subculturing plates with the appropriate volume of a freshly prepared collagen IV and fibronectin solution per well and incubate the plates for a minimum of two hours at 37 degrees Celsius. On day six of differentiation collect the cells by centrifugation and resuspend the pellets in the appropriate volume of fresh human endothelial serum-free medium supplemented with B27, basic fibroblasts growth factor, and retinoic acid.For TEER analysis, plate the differentiated cells into each well of a collagen and fibronectin coated 12-transwell filtered plate. For efflux transporter activity analysis, plate the cells into each well of a collagen and fibronectin coated 24-well flat bottom plate. After 24 hours of subculture, replace the supernatant in each well with fresh human endothelial serum-free medium supplemented only with B27 supplement.To measure the blood brain barrier properties of the BMECs using TEER, charge the TEER instrument overnight and lightly wipe the instrument and chopstick electrodes with 70%ethanol before placing them into the safety hood. Switch on the power and calibrate the ohm meter according to the manufacturer's specifications. Plug in the electrodes and rinse them one time with 70%ethanol and one time with DPBS.Place the shorter end of the electrode into the atypical chamber of the insert and the longer end into the basolateral chamber of a blank well. After measuring the negative control response, quickly rinse the electrodes as demonstrated and measure the first experimental well. After all of the measurements have been recorded rinse chopstick electrodes again, gently wipe the electrodes and let them air dry in the safety hood.To assess the efflux transporter activity of the cells, on day eight of culturing treat the appropriate number of wells with a 10 micromolar efflux transporter inhibitor solution and place in an incubator for one hour at 37 degrees Celsius. At the end of the incubation, treat the cells with a 10 micromolar efflux transporter substrate solution for one hour in the incubator. At the end of the incubation, wash each well twice with 500 microliters of DPBS per well before lysing the cells with 500 microliters DPBS supplemented with 5%Triton-X per well.After five minutes, use a microplate reader to measure the fluorescence of the lysed cells at a 485 nanometer excitation and a 530 nanometer emission. For wells not used in the transporter assay, wash the cells twice with DPBS before fixing with 4%paraformaldehyde per cell nuclei quantification. After one day of culture in E6 medium, the cell morphology is similar to that of iPSCs.By day four, the cells are visibly distinct from iPSCs and cover most of the well. Two days of culture in human endothelial serum-free medium induces an elongated and cobblestone appearance in the cells. At day eight, individual cells form a mostly large cobblestone pattern.A sprouting assay can be performed to demonstrate the angiogenic potential of iPSC-derived BMECs. This leads to the development of tube-like structures after three days of growth factor treatment. The iPSC-derived BMECs generated using this protocol co-expressed tight junction proteins expressed in endothelial cells in the brain, lung, liver, and kidney, as well as vascular endothelial markers expressed at the blood brain barrier.As observed, 48 hours after subculture and medium change iPSC-derived BMECs demonstrate TEER values within the range described for iPSC-derived BMECs that were co-cultured with rat primary astrocytes. Efflux transporter activity was also observed at this stage. After cryopreservation, TEER measurements are lower compared to freshly derived BMECs.Western blot analysis reveals reduced levels of tight junction markers, such as occludin, while immunocytochemistry shows freckled and or frayed patterns of tight junction proteins. A too high or too low iPSC density will affect the differentiation efficiency and impact the development of appropriate blood brain barrier properties. This method enables the investigation of barrier properties cellular signaling and transporter activity using individualized BMECs to model the physiology and pathophysiology of the blood-brain barrier function.

Research

JoVE Journal

Neuroscience

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Effektiv derivering af humane Neuronal progenitorer og neuroner fra Pluripotente humane embryonale stamceller med småmolekyle Induktion

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Specifikationen af ​​Telencephalic glutamaterge neuroner fra humane pluripotente stamceller

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Isolering og dyrkning af endotelceller fra embryonale forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

En cGMP-gældende Expansion Metode til aggregater af humane neurale Stem og stamceller arvet fra pluripotente stamceller eller fostrets hjerne Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Feeder-fri Udledning af neurale Crest stamceller fra menneskelige pluripotente stamceller

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Instrueret Dopaminerg Neuron Differentiering fra menneskelige pluripotente stamceller

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

Isolering af Primary Murine Brain mikrovaskulære endotelceller

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

Isolering af primære Murine skeletmuskulatur mikrovaskulære Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Migration, Chemo-Attraction, og Co-Kultur Assays for Human Stem Cell-Afledt endotelceller og GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

Generering af humane neuroner og oligodendrocytter fra pluripotente stamceller til modellering af neuron-oligodendrocytinteraktioner

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...