DK2951290T3 - Fremgangsmåde til fremsilling retinale pigmentepitel (rpe) -celler ud fra inducerede pluripotente stamceller (ipscs) - Google Patents

Fremgangsmåde til fremsilling retinale pigmentepitel (rpe) -celler ud fra inducerede pluripotente stamceller (ipscs) Download PDF

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DK2951290T3
DK2951290T3 DK14705645.1T DK14705645T DK2951290T3 DK 2951290 T3 DK2951290 T3 DK 2951290T3 DK 14705645 T DK14705645 T DK 14705645T DK 2951290 T3 DK2951290 T3 DK 2951290T3
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Kapil Bharti
Janine Davis
Marc Ferrer-Alegre
Sheldon S Miller
Arvydas Maminishkis
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Claims (31)

1. Fremgangsmåde til fremstilling af humane retinale pigmentepitel (RPE)-celler, hvilken fremgangsmåde omfatter: fremstilling af embryoide legemer ud fra inducerede humane pluripotente stamceller; dyrkning af de embryoide legemer i et første medium, som omfatter to Wnt-pathway-hæmmere og en Nodal-pathway-hæmmer; udpladning af de embryoide legemer på et matrigelovertrukket vævsdyrkningssubstrat i et andet medium, som (a) ikke omfatter basisk fibroblastvækstfaktor (bFGF); (b) omfatter en basisk fibro-blastvækstfaktor (bFGF)-hæmmer, de to Wnt-pathway-hæmmere og Nodal-pathway-hæmmeren; (c) omfatter 20 til 90 ng Noggin; og (d) omfatter 1 til 3% knockout-serumerstatning, til dannelse af differentierende RPE-celler; dyrkning af de differentierende RPE-celler i et tredje medium, som omfatter ACTIVIN A og Wnt3a; derpå dyrkning af cellerne i et fjerde, RPE-celle-, medium, som omfatter 5% føtalt serum, en kanonisk Wnt-hæmmer, en ikke-kanonisk Wnt-inducer samt Sonic- og FGF-pathway-hæmmere, idet der derved fremstilles humane RPE-celler.
2. Fremgangsmåde ifølge krav 1, hvor en af Wnt-pathway-hæmmerne er N-(2- aminoethyl)-5-chlorisoquinolin-8-sulfonamid (CK1-7), 3,5,7,8-tetrahydro-2-[4- (trifluormethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-on (XAV939), SFRP-1 ("secreted frizzled-related protein 1"), SFRP-2, SFRP-3, SFRP-4 eller SFRP-5.
3. Fremgangsmåde ifølge krav 1 eller krav 2, hvor det første medium omfatter 3,5 til 9 mM CK1-7.
4. Fremgangsmåde ifølge krav 1, hvor Nodal-pathway-hæmmeren er 4-(5- benzol[l,3]dioxol-5-yl-4-pyridin-2-yl-lH-imidazol-2-yl)-benzamid-hydrat, 4-[4-(1,3- benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]-benzamid-hydrat, 4-[4-(3,4- methylendioxyphenyl)-5-(2-pyridyl)-lH-imidazol-2-yl]-benzamid-hydrat (SB-431542), Lefty ("left-right determination factor") eller 2-(5-benzo[l,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridin-hydrochlorid-hydrat (SB-505124).
5. Fremgangsmåde ifølge krav 1 eller krav 4, hvor det første medium omfatter 3,5 til 9 mM SB-431542.
6. Fremgangsmåde ifølge et hvilket som helst af kravene 1-5, hvor det første medium er et retinacelleinducerende medium, som omfatter "Dulbecco's Modified Eagle's Medi- um" og F12 i et forhold på 1:1 i fravær af serum.
7. Fremgangsmåde ifølge et hvilket som helst af kravene 1-5, hvor bFGF-hæmmeren omfatter N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluor-2-[(2-fluor-4-iodphenyl)amino]-benzamid (PD0325901), N-[2-[[4-(diethylamino)butyl]amino-6-(3,5- dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(l,l-dimethylethyl)urea (PD173074), 2-(2-amino-3-methoxyphenyl)-4H-l-benzopyran-4-on (PD 98059), l-tert-butyl-3-[6-(2,6-dichlorphenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea (PD161570) eller 6-(2,6-dichlorphenyl)-2-[[4-[2-(diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8FI)-on-dihydrochlorid-hydrat (PD166285).
8. Fremgangsmåde ifølge et hvilket som helst af kravene 1-7, hvor det andet medium omfatter 0,5 til 2 mM PD0325901.
9. Fremgangsmåde ifølge et hvilket som helst af kravene 1-7, hvor det andet medium omfatter 3,5 til 9 mM CK1-7 og 3,5 til 9 mM SB-431542.
10. Fremgangsmåde ifølge krav 1, hvor det første medium omfatter 50 ng/ml DKK1 ("Dickkopf-related protein 1").
11. Fremgangsmåde ifølge et hvilket som helst af kravene 1-8, hvor det tredje medium omfatter 100 til 200 ng/ml ACTIVIN A.
12. Fremgangsmåde ifølge et hvilket som helst af kravene 1-11, hvor det tredje medium omfatter 75 til 150 ng/ml Wnt3a.
13. Fremgangsmåde ifølge et hvilket som helst af kravene 1-12, hvor den kanoniske Wnt-hæmmer er DKK1.
14. Fremgangsmåde ifølge krav 13, hvor det tredje medium omfatter 75 til 150 ng/ml DKK1.
15. Fremgangsmåde ifølge et hvilket som helst af kravene 1-14, hvor den ikke-kanoniske Wnt-induktor er Wnt5a.
16. Fremgangsmåde ifølge et hvilket som helst af kravene 1-15, hvor det fjerde medium omfatter 75 til 150 ng/ml Wnt5a, 75 til 150 ng/ml DKK1, 5 μΜ cyclopamin og/eller 10 μΜ SU5402.
17. Fremgangsmåde ifølge et hvilket som helst af kravene 1-15, hvor trin (a) omfat ter dyrkning af cellerne i nærvær af 1,5% knockout-serumerstatning.
18. Fremgangsmåde ifølge et hvilket som helst af kravene 1-17, hvor substratet er en transwell.
19. Fremgangsmåde ifølge et hvilket som helst af kravene 1-18, som omfatter ekspression af OCT4, SOX2, LIN28 og Nanog i en human føtal RPE-celle til fremstilling af de inducerede humane pluripotente stamceller.
20. Fremgangsmåde ifølge et hvilket som helst af kravene 1-19, hvor de inducerede humane pluripotente stamceller omfatter en nukleinsyre, som koder for en markør, der er operativt forbundet til en RPE-cellespecifik promotor.
21. Fremgangsmåde ifølge krav 20, hvor den retinaspecifikke promotor er en tyrosi-nasepromotor.
22. Fremgangsmåde ifølge et hvilket som helst af kravene 1-21, hvor de embryoide legemer dyrkes i det første medium i 48 timer.
23. Fremgangsmåde ifølge et hvilket som helst af kravene 1-22, hvor de embryoide legemer dyrkes i det andet medium i 18 til 24 døgn.
24. Fremgangsmåde ifølge krav 23, hvor de embryoide legemer dyrkes i det andet medium i tre uger.
25. Fremgangsmåde ifølge et hvilket som helst af kravene 1-24, hvor de differentierende RPE-celler dyrkes i det tredje medium i 18 til 24 døgn.
26. Fremgangsmåde ifølge krav 25, hvor de differentierende RPE-celler dyrkes i det tredje medium i tre uger.
27. Fremgangsmåde ifølge et hvilket som helst af kravene 1-26, hvor cellerne dyrkes i RPE-mediet i 12 til 16 døgn.
28. Fremgangsmåde ifølge krav 27, hvor cellerne dyrkes i RPE-mediet i to uger.
29. Fremgangsmåde ifølge et hvilket som helst af kravene 1-28, som yderligere omfatter bibeholdelse af RPE-cellerne i RPE-medium, der omfatter 5% føtalt serum.
30. Fremgangsmåde ifølge krav 29, som omfatter bibeholdelse af RPE-cellerne i RPE-medium, der omfatter 5% føtalt serum, i seks til otte uger.
31. Fremgangsmåde til bestemmelse af, om et middel af interesse øger differentieringen af RPE-celler, hvilken fremgangsmåde omfatter dyrkning af de embryoide legemer, der er fremstillet ud fra inducerede humane pluripotente stamceller omfattende en nukleinsyre, som koder for en første markør, der er operativt forbundet til en RPE-cellespecifik promotor, og omfattende en anden markør, der er operativt forbundet til en konstitutiv promotor, i et første medium, som omfatter to Wnt-pathway-hæmmere og en Nodal-pathway-hæmmer; udpladning af de embryoide legemer på et vævsdyrkningssubstrat i et andet medium, som (a) ikke omfatter basisk fibroblastvækstfaktor (bFGF); (b) omfatter en basisk fibro-blastvækstfaktor (bFGF)-hæmmer, de to Wnt-pathway-hæmmere og Nodal-pathway-hæmmeren; og (c) omfatter 20 til 90 ng Noggin, til dannelse af differentierende RPE-celler; dyrkning af de differentierende RPE-celler i et tredje medium, som omfatter ACTI- VIN A og Wnt3a; dyrkning af cellerne i et fjerde RPE-medium, som omfatter 5% føtalt serum, en kanonisk Wnt-hæmmer, en ikke-kanonisk Wnt-induktor samt Sonic- og FGF-pathway-hæmmere; idet der derved fremstilles humane RPE-celler, og i nærværelse af midlet af interesse; og evaluering af ekspressionen af den første markør i RPE-cellerne i sammenligning med ekspressionen af den anden markør, hvor en stigning i ekspressionen af den første markør i sammenligning med den anden markør indikerer, at midlet øger differentieringen af RPE-celler.
DK14705645.1T 2013-02-01 2014-01-31 Fremgangsmåde til fremsilling retinale pigmentepitel (rpe) -celler ud fra inducerede pluripotente stamceller (ipscs) DK2951290T3 (da)

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