NL2017078A - A method for culturing myogenic cells, cultures obtained therefrom, screening methods, and cell culture medium. - Google Patents
A method for culturing myogenic cells, cultures obtained therefrom, screening methods, and cell culture medium. Download PDFInfo
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- NL2017078A NL2017078A NL2017078A NL2017078A NL2017078A NL 2017078 A NL2017078 A NL 2017078A NL 2017078 A NL2017078 A NL 2017078A NL 2017078 A NL2017078 A NL 2017078A NL 2017078 A NL2017078 A NL 2017078A
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Claims (28)
1. Een in vitro celcultuur omvattende een populatie van geëxpandeerde myogene progenitorcellen die zijn geproduceerd door expansie in cultuur gedurende ten minste 1 passage van ten minste één geïsoleerde C-Met + en Hnkl- myogene progenitor startcel, waarbij ten minste 90% van de cellen in genoemde populatie positief is voor myogenese merker MyoD, waarbij ten minste 50% van de cellen in de populatie negatief is voor myogenese merker Pax7, waarbij genoemde celcultuur verder een synthetisch kweekmedium omvat.
2. Celcultuur volgens conclusie 1, waarbij ten minste 70%, bij voorkeur ten minste 90% van de cellen in de populatie negatief is voor myogenese merker Pax7.
3. Celcultuur volgens conclusie 1 of conclusie 2, waarbij de populatie wordt geproduceerd door de expansie in cultuur van genoemde ten minste één geïsoleerde C-Met + en Hnkl- myogene progenitor startcel gedurende ten minste 7 passages.
4. Celcultuur volgens één van de conclusies 1-3, waarbij genoemde ten minste één geïsoleerde C-Met + en Hnkl- myogene progenitor startcel is geïsoleerd uit een myogene celcultuur geproduceerd door het kweken van een pluripotente stamcel (PSC) in aanwezigheid van (i) een Wnt agonist en/of een glycogeen synthase kinase 3 beta (GSK3B) remmer, en (ii) een FGF route activator.
5. Celcultuur volgens conclusie 4, waarbij genoemde PSC is een geïnduceerde PSC (iPSC) is, bij voorkeur een iPSC verkregen uit een cel van een gezond menselijk subject of een menselijke patiënt die lijdt, of vermoedelijk lijdt, aan een neuromusculaire aandoening.
6. Celcultuur volgens één van de conclusies 1-5, waarbij de ten minste één geïsoleerde C-Met + en Hnkl- myogene progenitor startcel is geïsoleerd door FACS.
7. Celcultuur volgens één van de conclusies 1-6, waarbij genoemde celcultuur een celtelling heeft van ten minste 106, bij voorkeur ten minste 109, liever ten minste 1012 cellen.
8. Celcultuur volgens één van de conclusies 1-7, waarbij de populatie van geëxpandeerde myogene progenitorcellen homogeen is.
9. Celcultuur volgens één van de conclusies 1-8, waarbij het synthetische kweekmedium foetaal runderserum (FBS) omvat, bij voorkeur in een concentratie van ongeveer 10% en een hoeveelheid van 90-110 ng/ml, bij grotere voorkeur ongeveer 100 ng/ml, FGF2.
10. Celcultuur volgens één van de conclusies 1-9, waarbij geëxpandeerde myogene progenitorcellen in genoemd kweekmedium expandeerbaar zijn in vitro en induceerbaar tot terminale differentiatie in myotubes en/of myovezels.
11. Een myogene progenitorcel van een celcultuur volgens één der conclusies 1-10, waarbij de myogene progenitorcel positief is voor myogenese merker MyoD en negatief is voor myogenese merker Pax7, bij voorkeur waarbij de cel geïsoleerd is.
12. Celcultuur volgens één van de conclusies 1-10, of de cel volgens conclusie 11, waarbij de celcultuur bevroren of gecryopreserveerd is.
13. Een celcultuur omvattende een populatie van geëxpandeerde myogene progenitorcellen in een synthetisch kweekmedium, verkrijgbaar middels een werkwijze omvattende de stappen van: a) het verschaffen van een pluripotente stamcel (PSC), bij voorkeur een iPSC; b) het kweken van genoemde PSC in een synthetisch kweekmedium dat de differentiatie ondersteunt van genoemde PSC naar een myogene cellijn gedurende (i) een eerste periode van 3-8 dagen in de aanwezigheid van tussen 2-5 microM, bij voorkeur ongeveer 3,5 microM aan CHIR99021, (ii) een tweede periode van 5-20 dagen in aanwezigheid van 10-30 ng/ml FGF2; en, optioneel, (iii) een derde periode van 10-20 dagen in de aanwezigheid van insuline-transferrine-selenium-ethanolamine (ITS-X), ter verschaffing van een celcultuur van gepredifferentieerde PSCs omvattende myogene progenitorcellen; c) het isoleren van ten minste één C-Met-i- en Hnkl- myogene progenitor startcel uit genoemde celcultuur omvattende myogene progenitorcellen cellen, bij voorkeur door FACS, ter verschaffing van een gezuiverde myogene cellijn; d) het expanderen van genoemde ten minste één geïsoleerde C-Met-ι- en Hnkl- myogene progenitor startcel in een synthetisch kweekmedium omvattende foetaal runderserum (FBS), bij voorkeur in een concentratie van ongeveer 10% (w/w) en 90-110 ng/ml FGF2 gedurende ten minste 1 passage, bij voorkeur gedurende ten minste 7 passages, ter verschaffing van een celcultuur omvattende een populatie van geëxpandeerde C-met+ en Hnkl-myogene progenitorcellen, waarbij ten minste 50%, bij voorkeur ten minste 90% van genoemde populatie van geëxpandeerde C-Met+ en Hnkl- myogene progenitorcellen positief zijn voor myogenese merker MyoD en negatief zijn voor myogenese merker Pax7.
14. Celcultuur volgens conclusie 13, waarbij de stap van het expanderen van genoemde ten minste één C-Met+ en Hnkl- myogene progenitor startcel in stap d) wordt uitgevoerd in een kweekmedium omvattende een ROCK remmer gedurende ten minste de cultivering voorafgaand aan de eerste passage, bij voorkeur waarbij de kweekmediumbasis DMEM-HG is.
15. Een myotube of my o vezel gevormd uit de celcultuur volgens één van de conclusies 1-10 of 12-14, eventueel op een scaffold.
16. De myotube of myovezel volgens conclusie 15, (i) die een fusie-index heeft van ten minste 60%, bij voorkeur ten minste 70%, (ii) waarin de myotube of myovezel sarcomeren vormt, (iii) waarbij de myotube of myovezel neuromusculaire verbindingen tot uitdrukking brengt, en/of (iv) waarbij de myotube of myovezel spontane contractie in kweek vertoont.
17. Werkwijze voor de productie van een celcultuur omvattende een populatie van geëxpandeerde myogene progenitorcellen in een synthetisch kweekmedium, omvattende de volgende stappen: a) het verschaffen van een pluripotente stamcel (PSC), bij voorkeur een iPSC; b) het kweken van genoemde PSC in een synthetisch kweekmedium dat de differentiatie ondersteunt van genoemde PSC naar een myogene cellijn gedurende (i) een eerste periode van 3-8 dagen in de aanwezigheid van tussen 2-5 microM, bij voorkeur ongeveer 3,5 microM, CHIR99021, (ii) een tweede periode van 5-20 dagen in aanwezigheid van 10-30 ng/ml FGF2; en optioneel, (iii) een derde periode van 10-20 dagen in de aanwezigheid van insuline-transferrine-selenium-ethanolamine (ITS-X), ter verschaffing van een eetcultuur van gepredifferentieerde PSCs omvattende myogene progenitorcellen; c) het isoleren van ten minste één C-Met-ι- en Hnkl- myogene progenitor startcel uit genoemde celcultuur omvattende myogene progenitorcellen cellen, bij voorkeur door FACS, ter verschaffing van een gezuiverde myogene cellijn; d) het expanderen van genoemde ten minste één geïsoleerde C-Met-ι- en Hnkl- myogene progenitor startcel in een synthetisch kweekmedium omvattende foetaal runderserum (FBS), bij voorkeur in een concentratie van ongeveer 10% (w/w) en 90-110 ng/ml FGF2 gedurende ten minste 1 passage, bij voorkeur gedurende ten minste 7 passages, ter verschaffing van een celcultuur omvattende een populatie van geëxpandeerde C-met-ι- en Hnkl-myogene progenitorcellen, waarbij ten minste 50%, bij voorkeur ten minste 90% van genoemde populatie van geëxpandeerde C-Met-ι- en Hnkl- myogene progenitorcellen positief zijn voor myogenese merker MyoD en negatief zijn voor myogenese merker Pax7.
18. De werkwijze volgens conclusie 17, waarbij de stap van het expanderen van genoemde ten minste één C-Met-ι- en Hnkl- myogene progenitor startcel in stap d) wordt uitgevoerd in een kweekmedium omvattende een ROCK remmer gedurende ten minste de cultivering voorafgaand aan de eerste passage, bij voorkeur waarbij de kweekmediumbasis DMEM-HG is.
19. Een in vitro werkwijze voor het screenen van een testverbinding die de functie moduleert van myogene cellen in een kweek volgens conclusies 1-10 of 12-14, een myotube of myovezel volgens conclusie 15 of 16, een cel volgens conclusie 11 of een myogene cel of celstructuur tussenliggend aan genoemde cel en genoemde myotube of myovezel in termen van differentiatiestatus, omvattende de stap van a) het in contact brengen van een testverbinding met een kweek volgens conclusies 1-10 of 12-14, een myotube of myovezel volgens conclusie 15 of 16, een cel volgens conclusie 11 of een myogene cel of celstructuur intermediair aan genoemde cel en genoemde myotube of myovezel in termen van differentiatiestatus; b) het waarnemen van een verandering in de functie, het fenotype, het proteoom, transcriptoom of interactoom van genoemde cellen, cellen in genoemde kweek, myotube of myofiber of intermediair, in vergelijking met een controle verbinding, en optioneel c) het selecteren van genoemde testverbinding in het geval een verandering wordt waargenomen in stap b) ter verschaffing van een kandidaat-medicijn voor de behandeling van skeletspierafwijkingen.
20. Gebruik van de kweek volgens conclusies 1-10 of 12-14, de cel volgens conclusie 11, de myotube of myovezel volgens conclusie 15 of 16, een cel volgens conclusie 11 of een myogene cel of celstructuur intermediair aan genoemde cel en genoemde myotube of myovezel in termen van differentiatiestatus, voor de in vitro screening van een testverbinding of geneesmiddel.
21. De cel volgens conclusie 11, myotube of myovezel volgens conclusie 15 of 16, een cel volgens conclusie 11 of een myogene cel of celstructuur intermediair aan genoemde cel en genoemde myotube of myovezel in termen van differentiatiestatus, voor toepassing als medicijn of in de vervaardiging van een medicijn.
22. De cel volgens conclusie 11, myotube of my o vezel volgens conclusie 15 of 16, een cel volgens conclusie 11 of een myogene cel of celstructuur intermediair aan genoemde cel en genoemde myotube of myovezel in termen van differentiatiestatus, voor toepassing bij de behandeling van een spieraandoening.
23. De cel, myotube of myogene cel of celstructuur voor toepassing volgens conclusie 22, waarbij de wijze van toediening door transplantatie is.
24. Een synthetisch kweekmedium voor de expansie van myogene progenitorcellen, omvattende uit een synthetische basismedium, bij voorkeur DMEM HG; foetaal runderserum (FBS), bij voorkeur in een concentratie van ongeveer 10%; en 90-110 ng/ml, bij grotere voorkeur ongeveer 100 ng/ml, FGF2.
25. Medium volgens conclusie 24, waarbij het synthetisch kweekmedium voor de expansie van geïsoleerde myogene progenitor cellen is, bij voorkeur myogene progenitorcellen geïsoleerd met behulp van FACS, en waarbij kweekmedium verder een ROCK remmer omvat.
26. Een werkwijze voor het differentiëren van geëxpandeerde myogene progenitorcellen tot myotubes en/of myovezels, omvattende de stappen van: a) het uitplaten van cellen van een celcultuur volgens conclusies 1-10 of 12-13 op een houder of plaat voor het houden van cellen, waarbij de houder of plaat is bekleed met een extracellulair matrix (ECM) eiwit, bij voorkeur extracellulaire matrix (ECM) gel, bij grotere voorkeur ECM gel van Engelbreth-Holm-Swarm muizen sarcoma fibrillair collageen en/of een combinatie van (rattenstaart) collageen type I en MaxGel ECM (E0282 Sigma), en/of fibrillair collageen en/of een component van ECM gel, en/of een synthetische nabootser van een ECM component, en/of collageen type I, of een combinatie daarvan; b) het kweken van de cellen in een kweekmedium zoals gedefinieerd in conclusie 24 of 25, of in een synthetisch kweekmedium omvattende DMEM; c) het vervanging van het kweekmedium van stap b) met een differentiatiemedium omvattende, of bestaande uit, (i) DMEM hoog glucose, DMEM laag glucose, of DMEM/F12, (ii) ongeveer 1% ITS-X, en (iii) 0,5%-5%, bij voorkeur ongeveer 1%, knock-out serum vervanger en/of 0,1-10 mg/ml, bij voorkeur ongeveer 0,5 mg/ml BSA; c) het toestaan dat de cellen myotubes of myovezels vormen.
27. Werkwijze volgens conclusie 26, waarbij iedere tweede dag ten minste 20%, bij voorkeur 30-60%, van genoemd differentiatiemedium wordt vervangen door vers differentiatiemedium.
28. Een synthetisch kweekmedium voor differentiatie van geëxpandeerde myogene progenitorcellen tot myotubes en/of myofibers, omvattende, of bestaande uit, (i) DMEM hoog glucose, DMEM laag glucose, of DMEM/F12, (ii) ongeveer 1% van ITS-X, en (iii) 0,5%-5%, bij voorkeur ongeveer 1%, knock-out serum vervanger en/of 0,1-10 mg/ml, bij voorkeur ongeveer 0,5 mg/ml BSA.
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