DK2094833T3 - Differentiering af pluripotente celler til primære kimlagsprogenitorer - Google Patents

Differentiering af pluripotente celler til primære kimlagsprogenitorer Download PDF

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DK2094833T3
DK2094833T3 DK07824520.6T DK07824520T DK2094833T3 DK 2094833 T3 DK2094833 T3 DK 2094833T3 DK 07824520 T DK07824520 T DK 07824520T DK 2094833 T3 DK2094833 T3 DK 2094833T3
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Roger Pedersen
Ludovic Vallier
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Cambridge Entpr Ltd
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Claims (15)

1. Fremgangsmåde til fremstilling af en population af primære kimlags-progenitorceller, omfattende; at dyrke humane pluripotente celler i et humaniseret kemisk defineret medium omfattende polyvinylalkohol (CDM-PVA) og suppleret med en eller flere differentieringsfaktorer, hvor det humaniserede kemisk definerede medium er uden bestanddele fra ikke-humane dyr; og at lade de humane pluripotente celler differentiere til progenitorceller, hvor de primære kimlags-progenitorceller er ektoderme progenitorceller, og mediet er suppleret med FGF2 og en activin-antagonist, eller de primære kimlags-progenitorceller er mesoderme/endoderme progenitorceller, og mediet er supperet med activin, FGF2 og BMP4.
2. Fremgangsmåde ifølge krav 1, hvor de primære kimlags-progenitorceller er ektoderme progenitorceller, og mediet er suppleret med FGF2 og en activin-antagonist.
3. Fremgangsmåde ifølge krav 2, hvor det humaniserede CDM er suppleret med 1 til 100μΜ af activin-antagonisten SB431542 og/eller 1 til 100 ng/ml af FGF2.
4. Fremgangsmåde ifølge krav 1, hvor de primære kimlags-progenitorceller er mesoderme/endoderme progenitorceller, og mediet er suppleret med activin, FGF2 og BMP4.
5. Fremgangsmåde ifølge krav 4, hvor de humane pluripotente celler dyrkes ved en fremgangsmåde, der omfatter; (i) at dyrke de humane pluripotente celler i et humaniseret CDM omfattende polyvinylalkohol (CDM-PVA) og suppleret med activin og FGF2, (ii) endvidere at dyrke cellerne i humaniseret CDM-PVA suppleret med en FGF2-antagonist og activin og (iii) endvidere at dyrke cellerne i humaniseret CDM-PVA suppleret med FGF2, BMP4 og activin.
6. Fremgangsmåde ifølge krav 5, hvor koncentrationen af activin i trin (ii) er mindre end koncentrationen af activin i trin (i).
7. Fremgangsmåde ifølge krav 5 eller krav 6, hvor; den humaniserede CDM-PVA i trin (i) er suppleret med 5 til 20 ng/ml activin og 1 til 50 ng/ml FGF2; og/eller det humanisererede CDM i trin (ii) er suppleret med 1 til 50 μΜ af FGF2-antagonisten og 1 til 10 ng/ml activin; og/eller det humaniserede CDM i trin (iii) er suppleret med 1 til 100 ng/ml FGF2, 1 til 100 ng/ml BMP4 og 1 til 200 ng/ml activin.
8. Fremgangsmåde ifølge et hvilket som helst af kravene 5 til 7, hvor det humaniserede CDM i trin (iii) er suppleret med 1 to 100 ng/ml FGF2, 1 til 100 ng/ml BMP4 og 1 til 200 ng/ml activin, hvor CDM'et er suppleret med mere end 50 ng/ml activin, og de mesoder-me/endoderme progenitorceller er endoderme progenitorer.
9. Fremgangsmåde ifølge et hvilket som helst af kravene 5 til 7, hvor det humaniserede CDM er suppleret med 1 til 100 ng/ml FGF2, 1 til 100 ng/ml BMP4 og 1 til 200 ng/ml activin, hvor CDM'et er suppleret med mindre end 50 ng/ml activin, og de mesoder-me/endoderme progenitorceller er mesoderme progenitorer.
10. Fremgangsmåde ifølge krav 4, hvor den humanisererede CDM-PVA yderligere er suppleret med en phosphatidyl-3-inositolkinaseinhibitor.
11. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor det humaniserede CDM omfatter et basalt defineret dyrkningsmedium, insulin, transferrin, 1-thiolglycerol og polyvinylalkohol.
12. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, omfattende at isolere og/eller oprense progenitorcellerne.
13. Fremgangsmåde til at ekspandere en population af humane pluripotente celler, omfattende; at dyrke en eller flere humane pluripotente celler i et humaniseret CDM om fattende polyvinylalkohol (CDM-PVA) og suppleret med FGF2 og activin, fortrinsvis 1 til 100 ng/ml FGF2 og 1 til 200 ng/ml activin, hvor det humaniserede kemisk definerede medium er uden bestanddele fra ikke-humane dyr, og derved at ekspandere populationen af humane pluripotente celler.
14. Fremgangsmåde ifølge krav 13, hvor det humaniserede kemisk definerede medium omfatter et basalt defineret dyrkningsmedium suppleret med insulin, transferrin, 1-thiolglycerol og polyvinylalkohol.
15. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor de humane pluripotente celler ikke er humane embryonale stamceller (hESC'er).
DK07824520.6T 2006-11-09 2007-11-09 Differentiering af pluripotente celler til primære kimlagsprogenitorer DK2094833T3 (da)

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GBGB0622394.5A GB0622394D0 (en) 2006-11-09 2006-11-09 Differentiation of pluripotent cells
PCT/GB2007/004291 WO2008056166A2 (en) 2006-11-09 2007-11-09 Differentiation of pluripotent cells into primary germ layer progenitors

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WO2008056166A3 (en) 2008-07-03
EP2094833A2 (en) 2009-09-02
US8323971B2 (en) 2012-12-04
GB0622394D0 (en) 2006-12-20
WO2008056166A2 (en) 2008-05-15
US20100034785A1 (en) 2010-02-11

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