HRP20220482T1 - Postupci in vitro diferencijacije neurona dopamina srednjeg mozga (mda) - Google Patents

Postupci in vitro diferencijacije neurona dopamina srednjeg mozga (mda) Download PDF

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HRP20220482T1
HRP20220482T1 HRP20220482TT HRP20220482T HRP20220482T1 HR P20220482 T1 HRP20220482 T1 HR P20220482T1 HR P20220482T T HRP20220482T T HR P20220482TT HR P20220482 T HRP20220482 T HR P20220482T HR P20220482 T1 HRP20220482 T1 HR P20220482T1
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stem cells
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Lorenz Studer
Stefan Irion
Mark Tomishima
Sonja KRIKS
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Memorial Sloan Kettering Cancer Center
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Claims (14)

1. Postupak in vitro diferencijacije pluripotentnih matičnih stanica obuhvaća: (a) dovođenje u kontakt više pluripotentnih matičnih stanica sa najmanje jednim inhibitorom TGFβ/aktivin-nodalnog signaliziranja; i (b) dovođenje u kontakt stanica sa najmanje jednim aktivatorom signaliziranja zvučnog ježa (SHH) i najmanje jednim aktivatorom signaliziranja wnt-signalnog puta (Wnt), pri čemu je koncentracija barem jednog aktivatora signaliziranja Wnt-signalnog puta povećana (i) između 2 dana i 6 dana od njegovog početnog kontakta sa stanicama i (ii) za između 250% i 1800% njegove početne koncentracije dovedene u kontakt sa stanicama, da bi se dobila populacija diferenciranih stanica koje izražavaju forkhead box protein A2 (FOXA2) i LIM homeobox transkripcioni faktor 1 alfa (LMX1A).
2. Postupak iz patentnog zahtjeva 1, još obuhvaća dovođenje u kontakt stanica sa najmanje jednim inhibitorom signaliziranja koštano morfogenetskog proteina (BMP) i Small Mothers Against Decapentaplegic (SMAD) signaliziranja.
3. Postupak iz patentnog zahtjeva 2, pri čemu se stanice dovode u kontakt sa najmanje jednim inhibitorom TGFβ/aktivin-nodalnog signaliziranja, najmanje jednim inhibitorom BMP/SMAD signaliziranja, i najmanje jednim aktivatorom signaliziranja zvučnog ježa (SHH) tokom između 4 dana i 10 dana, ili i do 7 dana, ili tokom najmanje 7 dana.
4. Postupak iz patentnog zahtjeva 2, pri čemu se stanice dovode u kontakt sa najmanje jednim aktivatorom signaliziranja Wnt signalnog puta tokom između 8 i 15 dana, ili tokom ili do 12 dana, ili tokom najmanje 12 dana.
5. Postupak iz bilo kojeg od patentnih zahtjeva 1-4, pri čemu koncentracija najmanje jednog aktivatora signaliziranja Wnt signalnog puta jeste povećana 4 dana od njegovog početnog kontakta sa stanicama.
6. Postupak iz bilo kojeg od patentnih zahtjeva 1-5, pri čemu povećanje koncentracije najmanje jednog aktivatora signaliziranja Wnt signalnog puta je 400% do 1450% od prvobitne koncentracije najmanje jednog aktivatora signaliziranja wnt signalnog puta dovedene u kontakt sa stanicama, ili 700% do 1050% prvobitne koncentracije najmanje jednog aktivatora signaliziranja wnt signalnog puta dovedenog u kontakt s tim stanicama.
7. Postupak iz bilo kojeg od patentnih zahtjeva 1-6, pri čemu povećanje koncentracije najmanje jednog aktivatora signaliziranja Wnt signalnog puta je povećanje do koncentracije od između 3 µM i 10 µM, ili 3 µM, ili 7,5 µM.
8. Postupak iz bilo kojeg od patentnih zahtjeva 1-7, pri čemu te diferencirane stanice izražavaju jednu ili više tirozinskih hidroksilaza (TH), utisnuti engrailed-1 (EN-1), i sa nuklearnim receptorom srodan-1 protein (NURR1), opcionalno pri čemu te diferencirane stanice ne izražavaju detektibilne nivoe familije uparenog boks proteina (PAX6) i/ili Ki67.
9. Postupak iz bilo kojeg od patentnih zahtjeva 1-8, pri čemu taj postupak još obuhvaća izlaganje populacije diferenciranih stanica uvjetima koji pogoduju sazrijevanju stanica u neuronima dopamina, opcionalno pri čemu ti uvjeti obuhvaćaju dovođenje u kontakt stanica sa neurotrofnim moždanim faktorom (BDNF), neurotrofnim glijalnim faktorom (GDNF), cikličnim adenozin monofosfatom (cAMP), transformišućim faktorom rasta beta 3 (TGFβ3), askorbinskom kiselinom (AA), i/ili DAPT.
10. Postupak iz bilo kojeg od patentnih zahtjeva 1-9, pri čemu se te pluripotentne stanice biraju iz grupe koja obuhvaća humane ne-embrione matične stanice, ne-embrione matične stanice primata, ne-embrione matične stanice glodavaca, humane embrione matične stanice, embrione matične stanice primata, embrione matične stanice glodara, pluripotentne matične stanice inducirane iz čovjeka, pluripotentne matične stanice inducirane iz primata, pluripotentne matične stanice inducirane iz glodavca, rekombianantne pluripotentne stanice čovjeka, rekombinantne pluripotentne stanice primata, i rekombinantne pluripotentne stanice glodavca.
11. Postupak iz bilo kojeg od patentnih zahtjeva 2-10, pri čemu (a) najmanje jedan inhibitor TGFβ/aktivin-nodalnog signaliziranja obuhvaća inhibitor TGFβ receptora, opcionalno pri čemu taj inhibitor TGFβ receptora obuhvaća 4-[4-(1,3-benzodioksol-5-il)-5-(2-piridinil)-1H-imidazol-2-il] benzamid (SB431542); i/ili (b) najmanje jedan inhibitor BMP i SMAD signaliziranja obuhvaća 4-(6-(4-(piperazin-1- il)fenil)pirazolo[1,5-a]pirimidin-3-il)hinolin (LDN193189), nogin, kombinaciju gore navedenih; i/ili (c) najmanje jedan aktivator SHH signaliziranja obuhvaća SHH protein, zaglađeni (SMO) agonist, ili kombinaciju gore navedenih, opcionalno (i) pri čemu taj SHH protein obuhvaća rekombinantni SHH, prečišćeni SHH, ili kombinaciju gore navedenih, i/ili (ii) pri čemu taj rekombinantni SHH obuhvaća SHH C25II, i/ili (iii) pri čemu zaglađeni (SMO) agonist obuhvaća purmorfamin; i/ili (d) najmanje jedan aktivator signaliziranja Wnt signalnog puta obuhvaća CHIR99021, Wnt3A, Wnt1, ili kombinaciju gore navedenih.
12. Postupak iz bilo kojeg od patentnih zahtjeva 1-11, pri čemu su diferencirane stanice koje izražavaju FOXA2 i LMX1A neuroni dopamina srednjeg mozga ili njihovi prekursori.
13. Postupak iz bilo kojeg od patentnih zahtjeva 1-12, pri čemu su pluripotentne matične stanice diferencirane u diferencirane stanice koje izražavaju FOXA2 i LMX1A ne kasnije od između 22 dana i 27 dana od njihovog početnog kontakta sa najmanje jednim inhibitorom TGFβ/aktivin-nodalnog signaliziranja.
14. Postupak iz bilo kojeg od patentnih zahtjeva 1-13, pri čemu te diferencirane stanice izražavaju detektibilni nivo CD142, opcionalno pri čemu taj postupak još obuhvaća odabir populacije stanica koje izražavaju CD142.
HRP20220482TT 2015-06-01 2016-06-01 Postupci in vitro diferencijacije neurona dopamina srednjeg mozga (mda) HRP20220482T1 (hr)

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US201562169379P 2015-06-01 2015-06-01
US201562169444P 2015-06-01 2015-06-01
EP16804350.3A EP3303564B1 (en) 2015-06-01 2016-06-01 Methods of in vitro differentiation of midbrain dopamine (mda) neurons
PCT/US2016/035312 WO2016196661A1 (en) 2015-06-01 2016-06-01 Methods of in vitro differentiation of midbrain dopamine (mda) neurons

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IL291532A (en) 2022-05-01
AU2022228216A1 (en) 2022-10-06
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