HRP20191213T1 - Postupci za reprogramiranje stanica i njihove upotrebe - Google Patents
Postupci za reprogramiranje stanica i njihove upotrebe Download PDFInfo
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- HRP20191213T1 HRP20191213T1 HRP20191213TT HRP20191213T HRP20191213T1 HR P20191213 T1 HRP20191213 T1 HR P20191213T1 HR P20191213T T HRP20191213T T HR P20191213TT HR P20191213 T HRP20191213 T HR P20191213T HR P20191213 T1 HRP20191213 T1 HR P20191213T1
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Claims (8)
1. Postupak za dobivanje neuronskih stanica sličnih matičnim stanicama (NSLC), koji obuhvaća:
1) dovođenje u kontakt DNK i/ili kromatina osigurane stanice prvog tipa koja nije NSLC s histonskim acetilatorom, inhibitorom deacetilacije histona, demetilatorom DNK, i/ili inhibitorom DNK metilacije;
2) povećanje unutarstaničnih razina u stanici prvog tipa Musashi1 (Msi1) polipeptida i/ili Neurogenin 2 (Ngn2) polipeptida; i
3) postavljanje stanice iz koraka (2) u uvjetima uzgoja kulture koji podržavaju transformaciju stanice prvog tipa u NSLC u dovoljnom vremenskom periodu da se omogući stabilna ekspresija pluraliteta gena čija ekspresija je karakteristika fenotipskih i/ili funkcionalnih svojstava NSLC;
pri čemu se na kraju spomenutog vremenskog perioda dobiva NSLC i pri čemu je spomenuta dobivena NSLC dalje naznačena sa stabilnom represijom pluraliteta broja gena koji su izraženi u stanici prvog tipa.
2. Postupak prema zahtjevu 1, pri čemu povećane unutarstanične razine jednog ili više polipeptida obuhvaćaju prolaznu transfekciju stanice prvog tipa sa ekspresijskim vektorom koji omogućava ekspresiju Musashi1 (Msi1) i/ili Neurogenin 2 (Ngn2) polipeptida; Musashi 1 (Msi1) i polipeptida metil-CpG vezujućeg domena proteina 2 (MBD2); Neurogenin 2 (Ngn2) i metil-CpG vezujućeg domena proteina 2 (MBD2); i Musashi1 (Msi1), Neurogenin 2 (Ngn2) polipeptida, i polipeptida metil-CpG vezujućeg domena proteina 2 (MBD2).
3. Postupak prema zahtjevu 1 ili 2, pri čemu NSLC izražava jedan ili više od Sox2, Nestin, GFAP, Msi1 i Ngn2.
4. Postupak prema bilo kojim zahtjevima 1-3, pri čemu je stanica prvog tipa izabrana iz grupe koja obuhvaća: embrionske matične stanice, odrasle matične stanice, progenitorske stanice, stanice koje su izvedene iz mezoderma, endoderma ili ektoderma, matične stanice koje su dobivene iz adipoznog tkiva, mezenhimalne matične stanice, hematopoetske matične stanice, prekursorske stanice koje su izvedene iz kože, stanice folikula dlake, fibroblast, keratinocit, epidermalnu stanicu, endotelnu stanicu, epitelnu stanicu, granuloznu epitelnu stanicu, melanocit, adipocit, hondrocit, hepatocit, B limfocit, T limfocit, granulocit, makrofag, monocit, mononuklearnu stanicu, stanicu otočića gušteraće, sertolijevu stanicu, neuron, glijalnu stanicu, srčanu mišićnu stanicu, i drugu mišićnu stanicu.
5. Postupak prema bilo kojem prethodnom zahtjevu, pri čemu je inhibitor DNK metilacije izabran iz grupe koja obuhvaća 5-azacitidin, 5-aza-2-deoksicitidin, 1-β-D-arabinofuranozil-5-azacitozin, dihidro-5-azacitidin, zebularin, i RG108; inhibitor deacetilacije histona je izabran iz grupe koja obuhvaća valproinsku kiselinu, fenilbutirat, Trihostatin A, Na-butirat, 5 benzamid, i ciklične tetrapeptide; i DNK demetilator je metil-CpG vezujući domena protein 2 (MBD2).
6. Postupak prema bilo kojem prethodnom zahtjevu, pri čemu tako dobivena NSLC sadrži svaku od sljedećih karakteristika:
i) sposobnost samoobnavljanja ili proliferacije;
ii) nije kancerogena stanica;
iii) stabilna je i ne održava se umjetno prisilnom ekspresijom gena ili sličnim sredstvima i može se održavati u standardnom mediju neuronskih matičnih stanica;
iv) može se razlikovati od najmanje jedne progenitorske stanice, prekursorske stanice, somatske stanice i drugog više diferenciranog staničnog tipa iz iste linije, osim ako je stanica terminalno diferencirana somatska stanica;
v) ne pokazuje nekontrolirani rast, formaciju teratoma, i in vivo formaciju tumora.
7. Postupak prema zahtjevima 1, 2 ili 4 do 6, pri čemu tako dobivena NSLC sadrži jednu ili više od sljedećihrakteristika:
- ekspresiju jednog ili više markera matičnih stanica koji su izabrani iz grupe koja obuhvaća Sox2, Nestin, GFAP, Msi1, i Ngn2;
- smanjenu ekspresiju jednog ili više gena specifičnih za stanicu prvog tipa;
- formira neurosfere u testu formiranja kolonije neurosfera;
- sposobna je za kultivaciju u supenziji ili adherentnoj kulturi;
- sposobna je za proliferaciju bez prisustva egzogenog sredstva za reprogramiranje tijekom 1 mjeseca;
- sposobna je da se dijeli svakih 36 sati na niskom pasažu;
- pozitivna je na aktivnost telomeraze;
- sposobna je za diferencijaciju u stanicu koja je slična neuronu, stanicu sličnu astrocitu, stanicu sličnu oligodendrocitu i njihovim kombinacijama;
- smanjenu ekspresiju telomeraze i jednog ili više neuronskih markera matičnih stanica;
- da ima jedan ili više morfoloških neuritu-sličnih procesa (aksona i/ili dendrita) većih od promjera jedne stanice u dužini;
- ekspresiju najmanje jednog neuronski-specifičnog antigena koji je izabran iz grupe koja obuhvaća neuronski-specifični tubulin, protein 2 koji se veže za mikrotubule, NCAM, i marker za neurotransmiter;
- ekspresiju jednog ili više funkcionalnih neuronskih markera;
- sposobna za oslobađanje jednog ili više neurotrofnih faktora;
- negativna u testu formiranja kolonije tumora;
- negativna za tumorski rast kod SCID miševa;
- negativna za rast teratoma kod SCID miševa;
- sposobna da značajno unaprijedi jednu ili više funkcionalnih mjera poslije postavljanja adekvatnog broja NSLCs u prazninu u modelu ablacije mozga;
- sposobna za značajno unapređenje ili održavanje jedne ili više funkcionalnih mjera poslije ubrizgavanja adekvatnog broja NSLCs u EAE modelu; ili
- sposobna za poboljšanje jedne ili više funkcionalnih mjera značajnije nego hNPCs u CNS ozlijedi ili neurodegnerativnim modelima.
8. Postupak prema bilo kojim prethodnim zahtjevima, pri čemu je dobiven pluralitet NSLCs i pri čemu su spomenuti pluraliteti NSCLs organizirani u trodimenzionalnoj strukturi.
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JP6002581B2 (ja) * | 2010-02-05 | 2016-10-05 | オークランド ユニサービシズ リミテッドAuckland Uniservices Limited | 細胞プログラミング |
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