Name: generation of a human ipsc-based blood-brain barrier chip
Uploaded: 2020-03-02T14:30:00
Description: Watch this Scientific Journal Video about Generation of a Human iPSC-Based Blood-Brain Barrier Chip at JoVE.com

Vi vil gerne takke Dr. Soshana Svendsen for kritisk redigering. Dette arbejde blev støttet af Israel Science Foundation grant 1621/18, Ministeriet for Videnskab og Teknologi (MOST), Israel 3-15647, California Institute for Regenerativ Medicine grant ID DISC1-08800, Sherman Family Foundation, NIH-NINDS tilskud 1UG3NS105703, og ALS Association tilskud 18-SI-389. AH blev finansieret af Wallenberg Foundation (tilskudnummer 2015.0178).

1. Generation af iPSC-afledte neurale stamceller celler (iNpS)
<ol><li>Producere EZ-kugler fra iPSC kolonier som beskrevet nedenfor og som tidligere offentliggjort20,21,22.<ol>
<li>Kultur iPSC kolonier til sammenløb på kælderen membran matrix-belagt 6 brønd plader (0,5 mg / plade) i mTESR1 eller andre kommercielle medier (se Tabel over materialer).</li>
		<li>Fjern iPSC medium og erstatte med 2 ml EZ-sfær...

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Kombinationen af organ-on-chip-teknologi og iPSC-afledte celler i NVU lover nøjagtig modellering af den menneskelige BBB. Her giver vi en detaljeret protokol for enkel og robust anvendelse af den nyligt offentliggjorte iPSC-baserede BBB chip16. En oversigt over og timing af seeding paradigme er vist i figur 3. At opnå og vedligeholde barrierefunktioner, der er egnede til BBB-modellering, er det afgørende at generere et homogent iBMEC-monolag og bevare dets integrit...

BBB er en meget selektiv barriere, der adskiller CNS fra det cirkulerende blod. Det beskytter kritiske hjernefunktioner fra potentielt forstyrrende stoffer, faktorer, og xenobiotika samtidig tillader tilstrømning en næringsstoffer og andre metabolitter, der kræves for at opretholde hjernen homøostase1. BBB er en multicellulær NVU, hvor pericytter, astrocyt endfeet, og neuronale processer direkte kontakt hjernen mikrovaskulære endotelceller (BMECs). Disse interaktioner gør det muligt for BMECs at danne specialiserede barriereegenskaber , der understøttes af tætte og klæbende vejkryds2,3. Dannelsen af denne barriere begrænser paracellulær passage af molekyler, men det indeholder polariserede transportører til aktivt at transportere molekyler ind i CNS eller tilbage i blodet1. På grund af disse unikke barriere egenskaber, BBB udgør en væsentlig hindring for levering af biofarmaceutiske lægemidler i hjernen, og det anslås, at mindre end 5% af FDA-godkendte små molekyler kan nå CNS4.
Dyremodeller har været meget anvendt til at studere BBB penetrance og de molekylære mekanismer, der er involveret i BBB udvikling5. Mens dyremodeller trofast repræsenterer det komplekse flercellede in vivo-miljø, udelukker forskelle i bbb-transportørers udtryk og aktivitet samt substratspecificitet på tværs af arter ofte nøjagtig ekstrapolering af dyredata til mennesker6. Således er menneskebaserede modeller afgørende for at studere den menneskelige BBB og til brug i udviklingen af lægemidler, der er designet til at målrette CNS. Dette behov bliver endnu tydeligere med den stigende dominans af biologiske, menneskespecifikke lægemidler på lægemiddeludviklingsområdet. Akkumulere beviser tyder på, at en kompromitteret BBB er forbundet med en række alvorlige CNS lidelser, herunder hjernetumorer og neurologiske sygdomme7,8,9. Menneskelige modeller, der trofast afspejler disse sygdomme, har potentiale til både 1) at identificere nye veje, der kunne målrettes mod udvikling af lægemidler og 2) forudsige CNS penetrance, hvilket reducerer tid og ressourcer i prækliniske undersøgelser og muligvis faldende fejlrate i kliniske forsøg.
In vitro-modeller er blevet bredt implementeret for at undersøge samspillet mellem BMECs og andre celler i NVU og gennemføre skærme for potentielle BBB-permeable lægemidler10. For at genskabe centrale aspekter af den menneskelige BBB skal in vitro-modeller ne udvise fysiologisk relevante egenskaber (dvs. lav paracellulær permeabilitet og fysiologisk relevant transendotelelektrisk resistens [TEER] på tværs af det endotelmonolag). Desuden skal in vitro-systemets molekylære profil omfatte udtryk for repræsentative funktionelle transportsystemer. Typisk er in vitro-modeller sammensat af endotelceller, der er co-kulturfremstillet på en semipermeable membran med kombinationer af andre NVU celler til at forbedre BBB egenskaber11. Denne fremgangsmåde giver mulighed for enkel og relativt hurtig vurdering af barrierefunktionalitet og molekylepermeabilitet. Sådanne cellebaserede BBB-modeller kan etableres med animalske eller menneskelige cellekilder, herunder celler isoleret fra kirurgiske excisions eller udødeliggjorte BMEC linjer.
For nylig blev protokoller til differentiere menneskelige pluripotente celler i BMECs indført som en attraktiv kilde til in vitro menneskelige BBB modeller12,13. Inducerede pluripotente stamceller (iPSC)-afledte BMECs (iBMECs) er meget skalerbare, demonstrere afgørende morfologiske og funktionelle egenskaber af den menneskelige BBB, og bære genetik af patienten. I kultur danner iBMECs et monolag, der udtrykker stramme krydsmarkører og viser in vivo-lignende stramme krydskomplekser. Disse celler udtrykker også BBB-markører, herunder BBB-glukosetransportøren, glukosetransportør1 (GLUT1). Vigtigt, og i modsætning til andre alternative cellekilder for humane BMECs, iBMECs erhverve barriere egenskaber med værdier så højt som dem, der måles i vivo14,polarisere langs basolateralakse, og udtrykke funktionelle efflux pumper. Desuden glæder brugen af iPSC'er fra forskellige begge 1) sig over muligheden for at teste aspekter af BBB på en personlig medicinmåde, og 2) giver en fleksibel kilde til generering af yderligere celletyper af NVU. Generering af disse celler fra en isogen celle kilde til at skabe personlige BBB chips ville også støtte i forståelsen af inter individuelle forskelle i lægemiddelrespons, som er en væsentlig årsag til resistens eller kompromitteret respons på behandling observeret i kliniske undersøgelser.
Brug af iBMECs som monolag i en skål eller på en semi gennemtrængelig transwell indsats repræsenterer en kraftfuld tilgang til BBB modellering. Disse systemer har tendens til at være robuste, reproducerbare og omkostningseffektive. Desuden er funktionelle analyser som TEER og permeabilitet relativt enkle at udføre. Men, to-dimensionelle (2D) systemer undlader at opsummere 3D karakter in vivo væv, og de mangler de fysiologiske forskydning stress kræfter, der leveres af cirkulerende blod og blodlegemer. Dette begrænser det vaskulære endotheliums evne i disse modeller til at udvikle og vedligeholde iboende BBB-egenskaber og -funktioner.
Microengineered systemer foret med levende celler er blevet gennemført til at modellere forskellige organ funktionaliteter i et koncept kaldet organ-on-chips. Ved at genskabe in vivo-lignende flercellede arkitektur, vævsvæv grænseflader, fysisk-kemiske mikromiljøer, og vaskulær perfusion, disse mikromanipulerede platforme generere niveauer af væv og organ funktionalitet ikke muligt med konventionelle 2D-kultursystemer. De muliggør også høj opløsning, realtidsbilleddannelse og analyse af biokemiske, genetiske og metaboliske profiler svarende til levende celler i in vivovæv og organkontekst. Men en særlig udfordring af orgel-on-chip er, at design, fabrikation, og anvendelse af disse mikroengineered chips kræver specialiseret ingeniørekspertise, der normalt mangler i biologisk orienterede akademiske laboratorier.
Vi har for nylig kombineret iPSC og organ-on-chip teknologier til at generere en personlig BBB chip model15,16. For at overvinde de beskrevne teknologiske udfordringer bruges den kommercielt tilgængelige Chip-S1 sammen med kulturmodulet, et instrument, der er designet til at automatisere vedligeholdelsen af chipsene på en enkel og robust måde (Emulate Inc.). BBB-chippen genskaber interaktioner mellem neurale og endotelceller og opnår fysiologisk relevante TEER-værdier, som måles ved specialfremstillede organchips med integrerede guldelektroder17. Derudover viser BBB-chippen lav paracellulær permeabilitet, reagerer på inflammatoriske signaler på organniveau, udtrykker aktive efflux-pumper og udviser prædiktiv transport af opløselige biomarkører og biofarmaceutiske produkter. Især, BBB chips genereret fra flere personer indfanger de forventede funktionelle forskelle mellem raske personer og patienter med neurologiske sygdomme15.
Nedenstående protokol beskriver en pålidelig, effektiv og reproducerbar metode til generering af humane iPSC-baserede BBB-chips under dynamiske flowforhold. Der gives vejledning om typen af analyser og slutpunktsanalyser, der kan udføres direkte i BBB-chippen eller fra prøvetagningsspildevand. Protokollen viser således spektret af teknikker, der kan anvendes til evaluering af biologiske og funktionelle egenskaber og reaktioner i en menneskerelevant model.
En kort beskrivelse af den iPSC-baserede BBB-chip findes her. Menneskelige iPSCs er oprindeligt differentieret og formeret i vævkultur kolber som fritflydende aggregater af neurale sår, betegnes EZ-kugler. Den øverste kanal af Chip-S116,18,19 er seedet med dissociated EZ-kugler, der danner "hjernen side" af chippen, som celler differentiere over 7 dage i en blandet kultur af neurale stamsygdomme celler (iNPCs), iAstrocytter, og iNeurons. Menneskelige iPSCs er også differentieret i vævskulturplader i iBMECs. Den nederste kanal af chippen er seedet med iBMECs at danne "blod side", som de udvikler sig til at danne en endotelrør (Figur 1). Den porøse ekstracellulære matrix (ECM)-belagt membran, der adskiller de øverste og nederste kanaler 1) tillader dannelsen af celle-til-celle interaktioner mellem kanaler og 2) giver brugeren mulighed for at køre permeabilitet assays og billedceller i begge kanaler ved hjælp af en konventionel lys mikroskop.

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			<td>Sigma</td>
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			<td>Sigma</td>
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			<td>Dako</td>
			<td>Z0334</td>
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			<td>Invitrogen</td>
			<td>MA5-11315</td>
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			<td>Life Technologies</td>
			<td>35050038</td>
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			<td>Thermo Fisher Scientific</td>
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			<td>Platelet-poor plasma-derived bovine serum (PPP)</td>
			<td>Biomedical Technologies</td>
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			<td>Retinoic acid (RA)</td>
			<td>Sigma</td>
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			<td>S100&#946;</td>
			<td>Abcam</td>
			<td>ab6602</td>
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			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
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			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
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			<td>ZO-1 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>33-9100</td>
			<td></td>
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			<td>&#946;III-tubulin (Tuj1&#945;)</td>
			<td>Sigma</td>
			<td>T8660</td>
			<td></td>
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			<td>&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>31350010</td>
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generation of a human ipsc-based blood-brain barrier chip

Blodhjernebarrieren (BBB) er dannet af neurovaskulære enheder (NVM), der beskytter centralnervesystemet (CNS) fra en række faktorer, der findes i blodet, der kan forstyrre sarte hjernefunktion. BBB er derfor en væsentlig hindring for leveringen af terapeutiske lægemidler til CNS. Akkumulere nde beviser tyder på, at BBB spiller en central rolle i starten og progression af neurologiske sygdomme. Således er der et enormt behov for en BBB model, der kan forudsige penetration af CNS-målrettede lægemidler samt belyse BBB's rolle i sundhed og sygdom.
Vi har for nylig kombineret organ-on-chip og induceret pluripotente stamceller (iPSC) teknologier til at generere en BBB chip fuldt personlig til mennesker. Denne nye platform viser cellulære, molekylære og fysiologiske egenskaber, der er egnede til forudsigelse af narkotika og molekyle transport på tværs af den menneskelige BBB. Desuden har vi ved hjælp af patientspecifikke BBB-chips genereret modeller af neurologiske sygdomme og vist potentialet for personlig prædiktiv medicinapplikationer. Forudsat her er en detaljeret protokol, der viser, hvordan man genererer iPSC-afledte BBB-chips, begyndende med differentiering af iPSC-afledte hjernemikrovaskulære endotelceller (iBMECs) og resulterer i blandede neurale kulturer, der indeholder neurale stamlitter, differentierede neuroner og astrocytter. Også beskrevet er en procedure for såning celler i organchip og dyrkning af BBB chips under kontrolleret laminar flow. Endelig gives detaljerede beskrivelser af BBB-spånanalyser, herunder permecellulære permeabilitetsanalyser til vurdering af lægemiddel- og molekylepermeabilitet samt immuncytokemiske metoder til bestemmelse af sammensætningen af celletyper i chippen.

Figur 6A,B,C repræsenterer en BBB-chip, der er seedet med EZ-kugler på "hjernesiden" og iBMECs på "blodsiden"- bundlinjen. iBMECs var seedet først og fik lov til at vedhæfte natten over, hvorefter EZ-kugler var seedet. Chips blev derefter dyrket under statiske forhold med daglige medier udskiftning i syv dage. BBB-chippen blev derefter fastgjort ved hjælp af 4% PFA på RT i 10 min og vasket 3x med DPBS. Immuncytokemi blev udført på BBB-chippen ved hjælp af 1) nestin...

Watch this Scientific Journal Video about Generation of a Human iPSC-Based Blood-Brain Barrier Chip at JoVE.com

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

Blod - hjerne barrieren (BBB) er en multicellulær neurovaskulær enhed stramt regulere hjernen homøostase. Ved at kombinere menneskelige iPSCs og organ-on-chip teknologier, har vi genereret en personlig BBB chip, egnet til sygdom modellering og CNS narkotika penetrability forudsigelser. En detaljeret protokol er beskrevet for produktion og drift af BBB-chippen.

Generation af en menneskelig iPSC-baseret blod-hjerne barrierchip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Denne protokol vil vise, hvordan differentierede inducerede pluripotente stamceller kan sås på en organ-on-chip til at generere en fuldt menneskelig, personlig, mikrofluidisk blod - hjerne barriere, som kan bruges til at forudsige centralnervesystemet narkotika permeabilitet og til at studere neurologiske lidelser. Ved hjælp af et kommercielt tilgængeligt organ-on-chip, vil vi vise, hvordan enhver biologisk orienteret lab kan bruge denne teknologi til at generere en personlig, microfluidic blod - hjerne barrier-on-chip. Akkumulerende beviser tyder på, at BBB spiller en rolle i neurologiske sygdomme ved at generere personlige BBB chips stammer fra iPSCs af personer med en genetisk neurologisk sygdom.BBB's rolle inden for sundhed og sygdom vil kunne undersøges. Denne metode kan også være nyttig for pharma virksomheder, som kan bruge denne menneskelige BBB platform til at screene for penetrability af kandidat neurologiske lægemidler i den menneskelige hjerne. Anvendelsen af organ-on-chip teknologier kræver normalt specialiserede tekniske færdigheder.Brug kommercielt tilgængelige platforme gør det muligt at anvende denne teknologi i ethvert biologisk orienteret laboratorium. Til at begynde med skal du bringe skålen med de forberedte chips til biosikkerhedsskabet. Ved hjælp af en P200 pipette, forsigtigt vaske begge kanaler ved at tilføje 200 mikroliter af neurale differentiering medium i fjorden og trække væsken fra stikkontakten.Undgå kontakt med portene, omhyggeligt aspirere overskydende medier dråber fra overfladen af chippen. Ryst forsigtigt cellesuspensionen for at sikre en homogen cellesuspension. At frø iPSC-afledte neurale progenitor celler i den øverste kanal til at generere hjernen side, tilføje en P200 spids, der indeholder 30 til 100 mikroliter af celle suspension ved koncentrationen af en gange 10 til de seks celler pr milliliter til den øverste kanal indløb, og forsigtigt frigive spidsen fra pipetten.Tag en tom P200-pipette, tryk stemplet ned, sæt stemplet ind i den øverste kanaludtag, og træk forsigtigt enkeltcellesuspensionen gennem spidsen. Overfør chippen til mikroskopet for at kontrollere såningstætheden og den homogene fordeling af celler i den øverste kanal. Efter bekræftelse af celletætheden ved 20% dækning, straks placere chips i inkubatoren i to timer ved 37 grader Celsius.Derefter vaskes de celler væk, der ikke tillægger ved at tilføje frisk neural differentieringsmedium i indløbet og trække væsken via pipetten fra stikkontakten. Hold cellerne under statiske forhold ved 37 grader Celsius med en daglig neural differentiering medium udskiftning. Når iNPC'er har tilsluttet eller på en efterfølgende dag efter såning, bringes skålen med de forberedte spåner til biosikkerhedsskabet.Vask forsigtigt den nederste kanal med 200 mikroliter af endotelcellemedium. Undgå kontakt med portene, omhyggeligt aspirere dråber af overskydende endotel celle medium fra overfladen af chippen, og sørg for at forlade medium i begge kanaler. Ryst forsigtigt cellesuspensionen for at sikre en homogen cellesuspension.Ved hjælp af en P200-pipette udtøms 30 til 100 mikroliter af iPSC-afledt hjernemikrovaskulær endotelcellesuspension ved en koncentration af 14 til 20 gange 10 til de seks celler pr. milliliter, og spidsen sættes ind i det nederste kanalindtag. Det mest kritiske skridt for at opnå funktionelle barriere egenskaber er såning af hjernen mikrovaskulære endotelceller. Det er afgørende for frøceller ved korrekt tæthed for at undgå bobledannelse for at kontrollere homogen fordeling i organchippen.Tryk stemplet på en P200-pipette med en tom spids, indsæt det nederste kanaludtag, og slip langsomt pipettestemplen for forsigtigt at trække enkeltcellesuspensionen gennem den nederste kanal. Indsugning den overskydende celleaffjedring fra overfladen af spånen. Undgå direkte kontakt med indløbs- og udløbsportene for at sikre, at der ikke aspireeres en celleaffjedring ud af kanalerne.Overfør chippen til mikroskopet for at kontrollere såningstætheden. Den nederste kanal er fyldt med celler uden observerbare mellemrum imellem. Efter bekræftelse af den korrekte celletæthed, frø cellerne i de resterende chips.For at fastgøre cellerne på den porøse membran, som er placeret på toppen af den nederste kanal, invertere hver chip, og lad dem hvile i en chip vugge. Placer et lille reservoir, der indeholder steril DPBS inde i 150-millimeter skålen for at give fugtighed for cellerne. Inkuber chips ved 37 grader Celsius i cirka tre timer, eller indtil celler i den nederste kanal har tilsluttet.Når iPSC-afledte hjernen mikrovaskulære endotelceller har vedhæftet, vende chips tilbage til en oprejst position for at tillade celle fastgørelse til den nederste del af den nederste kanal. 48 timer efter såning af iPSC-afledte hjerne mikrovaskulære endotelceller, ækvilibrer temperaturen af endotelcellemediet ved opvarmning i et 37-graders Celsius-vandbad i en time. Derefter afgasse mediet ved inkubation under et vakuumdrevet filtreringssystem i 15 minutter.Dernæst desinfetgøre ydersiden af den bærbare modul emballage og bakker med 70% ethanol, tørre, og overføre dem til biosikkerhed kabinet. Åbn pakken, og placer modulerne i bakken Orient dem med beholderne mod bagsiden af bakken. Pipette tre milliliter af det forkvædrede, varme medier til hver indsugningsbeholder.Tilføj endotelcellekulturmedium til indløbsbeholderen på den nederste kanal og neuraldifferentieringsmedie til den øverste kanals indløbsreservoir på den øverste kanal. Derefter pipette 300 mikroliter af de præ-kvilibrerede, varme medier til hver stikkontakt reservoir, direkte over hver udløbsport. Placer op til seks bærbare moduler på hver bakke.Bring bakkerne til inkubatoren, og skub helt ind i kulturmodulet med bakkehåndtaget vendt udad. Vælg og kør den primære cyklus på kulturmodulet. Luk inkubatordøren, og lad kulturmodulet prime de bærbare moduler i ca. et minut.Priming-cyklussen er fuldført, når statuslinjen læser klar. Tag bakken ud af kulturmodulet, og bring den til biosikkerhedsskabet. Undersøg undersiden af hvert bærbart modul i biosikkerhedsskabet for at kontrollere, at de bærbare moduler med succes blev primet.Se efter tilstedeværelsen af små dråber i alle fire porte. Hvis et bærbart modul ikke viser dråber, skal du køre prime-cyklussen igen på disse moduler. Hvis der dryppede et medie på bakken, hvilket oftere forekommer af udløbsportene, skal bakken rengøres med 70 % ethanol.Dernæst skal du forsigtigt vaske begge kanaler af hver chip med varm, ligeudspecifik celle-specifikke kultur medium for at fjerne eventuelle bobler i kanalen, og placere små dråber af medier på toppen af hver indløb og udløb port. Sæt spånerne med luftfartsselskaber i de bærbare moduler, og placer op til seks på hver bakke. Sæt bakkerne i kulturmodulet.Programmer de relevante organchipkulturbetingelser, såsom strømningshastighed og stræk, på kulturmodulet. Så snart reguleringscyklussen er færdig, hvilket tager ca. to timer, starter de programmerede forhold. Derefter begynder kulturmodulet flow ved de forudindstillede organchipkulturforhold.Immunocytochemistry på iPSC-baserede blod - hjerne barriere chip syv dage efter såning viser EZ kugler differentieret i en blandet neuralcellepopulation i den øverste hjerne side kanal, herunder S100 beta-positive astrocytter, Nestin-positive neurale progenitor celler, samt GFAP-positive astrocytter, og beta III tubulin-positive neuroner. IPSC-afledte hjerne mikrovaskulære endotelceller seedet i bunden blod side kanal udtrykt GLUT-1 og PECAM-1. Evaluering af blod - hjerne barrieren chip permeabilitet viser, at organchippen seedet med iPSC-afledte hjerne mikrovaskulære endotelceller og EZ kugler havde en stram barriere i forhold til organ chips seedet med iPSC-afledte hjerne mikrovaskulære endotelceller alene.Organ chips seedet med EZ kugler alene viste ikke nogen barriere egenskaber. Fra den indledende overfladebehandling af kanalen til mediet skifter hver dag, undgå bobledannelse i kanalen. Under hele trinnet i den protokol, hvor overfladeaktiveringsgenage, ekstracellulære matrix eller celleholdigt medium skal indsættes gennem kanalerne, er det yderst vigtigt omhyggeligt at kontrollere, at der ikke findes nogen boble.Den permeabilitet assay kan udføres for at vurdere den menneskelige hjerne gennemtrængelighed af kandidat narkotika. Denne fremgangsmåde kan tjene til at teste potentielle terapeutiske. Anvendelsen af den iPSC-baserede BBB-chip gør det muligt at studere BBB's rolle i forskellige neurologiske sygdomme.I fremtiden kan en sådan platform være nyttig til prædiktive, personlige medicinapplikationer.

generation-of-a-human-ipsc-based-blood-brain-barrier-chip

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Generation af menneskelige fedt-stamceller gennem dedifferentiering af Ældre Adipocytter i Loft Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Generering af genetisk modificerede Organotypiske Skin Cultures Brug devitaliseret Menneskelig Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Generation af induceret pluripotente stamceller fra human melanoma Tumor-infiltrerende lymfocytter

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Defineret og skalerbar Generation af hepatocyt-lignende celler fra menneskelige pluripotente stamceller

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

Generation af iPSC-afledte menneskelige hjerne organoider til Model Tidlig forstyrrelser i nervesystemets

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

Generation og dyrkning af primære humane keratinocytter fra voksen hud

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Real-time måling af epitel barriere permeabilitet i menneskelige tarm Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

I Vitro Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Assay for blod-hjerne barriere integritet i Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

Generation af flercellede humane primære Endometriale Organoider

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...