DK2898063T3 - Pankreatisk differentiering af pluripotente pattedyrsceller in vitro - Google Patents

Pankreatisk differentiering af pluripotente pattedyrsceller in vitro Download PDF

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DK2898063T3
DK2898063T3 DK13765696.3T DK13765696T DK2898063T3 DK 2898063 T3 DK2898063 T3 DK 2898063T3 DK 13765696 T DK13765696 T DK 13765696T DK 2898063 T3 DK2898063 T3 DK 2898063T3
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cells
pancreatic
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population
induction medium
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Ludovic Vallier
Hsin-Hua Cho
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Cambridge Entpr Ltd
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Claims (13)

1. Fremgangsmåde til fremstilling af en population af pankreatiske progenitor-celler, som omfatter: (i) at tilvejebringe en population af inducerede pluripotente (iPS-) celler, eventuelt humane iPS-celler; ii) at dyrke populationen i et definitiv endoderm (DE) -induktionsmedium til fremstilling af en population af definitiv endoderm-celler, hvor definitiv endoderm (DE) -induktionsmediet er et kemisk defineret medium, som omfatter en TGFp-ligand, fibroblastvækstfaktor (FGF), knoglemorfogenetisk protein (BMP), en PI3K-inhibitor og eventuelt en GSK33-inhibitor; iii) at dyrke populationen af definitiv endoderm-celler i et første pankreatisk induktionsmedium til fremstilling af en population af dorsale fortarmsceller, hvor det første pankreatiske induktionsmedium er et kemisk defineret medium, som omfatter en activin-antagonist; FGF; retinsyre; og en BMP-inhibitor; iv) at dyrke de dorsale fortarmsceller i et andet pankreatisk induktionsmedium, hvor det andet pankreatiske induktionsmedium er et kemisk defineret medium, som omfatter FGF, en BMP-antagonist, retinsyre og en hedgehog-signale-ringsinhibitor; v) at dyrke endodermcellerne i et tredje pankreatisk induktionsmedium, hvor det tredje pankreatiske induktionsmedium er et kemisk defineret medium, som omfatter FGF; hvorved der fremstilles en population af pankreatiske progenitorceller.
2. Fremgangsmåde ifølge krav 1, hvor definitiv endoderm (DE) -induktionsmediet er et kemisk defineret medium, som består af et grundmedium suppleret med activin, fibroblastvækstfaktor (FGF), knoglemorfogenetisk protein (BMP) og LY294002.
3. Fremgangsmåde ifølge krav 1 eller krav 2, hvor trin (ii) omfatter: (a) at dyrke populationen af pluripotente celler i DE-induktionsmediet, hvor DE-induktionsmediet yderligere omfatter en GSK33-inhibitor, (b) yderligere at dyrke populationen i definitiv endoderm-induktionsmediet, der magier GSK33-inhibitor; og (c) yderligere at dyrke populationen i et anterior definitiv endoderm (ADE) -induktionsmedium, som omfatter en TGFp-ligand og fibroblastvækstfaktor, til fremstilling af populationen af definitive endoderm (DE)-celler.
4. Fremgangsmåde ifølge krav 3, hvor anterior definitiv endoderm (DE) -induktionsmediet er et kemisk defineret medium, som består af et grundmedium suppleret med activin og fibroblastvækstfaktor (FGF).
5. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor det første pankreatiske induktionsmedium er et kemisk defineret medium, som består af et grundmedium suppleret med SB-431542; FGF; retinsyre; og noggin.
6. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor det andet pankreatiske induktionsmedium er et kemisk defineret medium, som består af et grundmedium suppleret med FGF; retinsyre; noggin; og KAAD-cy-clopamin.
7. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor det tredje pankreatiske induktionsmedium er et kemisk defineret medium, som består af et grundmedium suppleret med FGF og eventuelt retinsyre.
8. Fremgangsmåde ifølge et hvilket som helst af de foregående krav omfattende at modne de pankreatiske progenitorceller til fremstilling af en population af endokrine pancreasceller.
9. Fremgangsmåde ifølge krav 8, hvor de pankreatiske progenitorceller modnes ved i) dyrkning i et første endokrint induktionsmedium og ii) dyrking i et andet endokrint induktionsmedium til fremstilling af populationen af pankreatiske endokrine celler, hvor det første endokrine induktionsmedium er et kemisk defineret medium omfattende en Notch-signaleringsinhibitor og eventuelt retinsyre; og det andet endokrine induktionsmedium er et kemisk defineret medium uden differentieringsfaktorer eller uden andre differentieringsfaktorer end retinsyre.
10. Fremgangsmåde ifølge krav 9, hvor det første endokrine induktionsmedium er et kemisk defineret medium bestående af et suppleret grundmedium og N-[N-(3,5-difluorphenacetyl)-1 -alanyl]-S-phenylglycin-t-butylester (DAPT) og eventuelt retinsyre; og det andet endokrine induktionsmedium er et kemisk defineret medium bestående af et suppleret grundmedium og eventuelt retinsyre.
11. Fremgangsmåde ifølge et hvilket som helst af de foregående krav omfattende ekspandering, opbevaring, dyrkning eller bevarelse af populationen af pankreatiske progenitorceller eller endokrine pancreasceller.
12. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, omfattende at blande populationen af pankreatiske progenitorceller, endokrine pancreasceller eller endokrine pancreasceller med en terapeutisk acceptabel ex-cipiens.
13. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor iPS-cellerne stammer fra et individ med en genetisk sygdom eller en genetisk baggrund, der er forbundet med en pancreassygdom.
DK13765696.3T 2012-09-20 2013-09-16 Pankreatisk differentiering af pluripotente pattedyrsceller in vitro DK2898063T3 (da)

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GBGB1216796.1A GB201216796D0 (en) 2012-09-20 2012-09-20 In vitro pancreatic differentiation
PCT/EP2013/069188 WO2014044646A1 (en) 2012-09-20 2013-09-16 In vitro pancreatic differentiation of pluripotent mammalian cells

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EP (1) EP2898063B1 (da)
JP (1) JP6463681B2 (da)
CN (1) CN104755607B (da)
AU (1) AU2013320408A1 (da)
CA (1) CA2924511C (da)
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GB (1) GB201216796D0 (da)
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