Avledning av Cardiac stamceller fra embryonale stamceller
Summary
In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.
Abstract
Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.
Introduction
Hjertesykdom er fortsatt den ledende årsak i verden i dag, og dødsrater har vært tilnærmet uendret de siste to tiårene (American Heart Association). Det er et kritisk behov for å utvikle nye terapeutiske strategier for effektivt å forebygge eller reversere hjertesvikt. En lovende strategi er cellebasert behandling etter den raske utviklingen av stamcelle biologi 1. I denne forbindelse, kan multipotente CPC-være en utmerket cellekilde for terapi på grunn av deres evne til å proliferere, men opptatt bare for hjerte avstamning differensiering. Derfor, effektiv og robust metode generere og isolere CPC-er av stor betydning for hjertecelle terapistudier.
Denne protokollen fokuserer på embryonale CPC identifisert under tidlig embryo og hvordan deres generasjon fra ESCs. Ulike CPC har blitt isolert fra embryonale og voksne hjerter, selv fra benmarger to. Under embryo utvikling, bein morphogemagnetiske proteiner (BMPs), vingeløse-type MMTV integrering nettstedet familiemedlemmer (Wnts) og nodal signaler indusere engasjementet til Mesp1 + multipotent mesoderm tre. Mesp1 + celler deretter differensieres til de embryonale CPC 4. Disse CPC er vanligvis preget av HCN4, NK2 homeobox 5 (Nkx2-5), Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5), og myocyte Enhancer faktor 2C (Mef2c), danner primære og andre hjerte felt, og bidra til de store deler av hjertet under cardiogenesis 5-10. Både Nkx2-5 + og Isl1 + / Mef2c + CPC er i stand til å differensiere i kardiomyocytter, glatte muskelceller (SMCS), og endotelceller 5-8. Dermed disse CPC vil gi opphav til hjerte blodkar samt hjerte vev, og er en ideell celle kilde for celle-baserte hjerteterapi. Derfor har generere CPC in vitro vært en stor forskningsfokus i hjerte-studier. Siden ESCs har ubegrenset kapasitetsutvidelse ennd representerer ICM celler på blastocyststadiet, er differensiering av ESCs til embryonale CPC følge den naturlige embryogenese betraktet som en logisk og effektiv tilnærming for å oppnå CPC.
En mye brukt metode for å skaffe CPC fra ESCs er å aggregere ESCs inn EBS 11. For å bedre differensiering effektivitet, har definert kjemiske og vekstfaktorer basert på kunnskap om hjerte utvikling blitt brukt 12-14. Det finnes imidlertid ingen definitive CPC markører, spesielt ikke noen celleoverflatemarkører som er vidt akseptert på området. For å løse dette problemet, er ESCs konstruert for å markere Isl1 + eller Mef2c + CPC og deres derivater med fluorescerende reportere bruker Cre / loxP system. Den ere rekombinase blir slått på under kontroll av Isl1 / Mef2c promoter / enhancer. Modifisert fluorescerende protein RFP eller YFP genet drevet av en konstitutiv promoter kan aktiveres ved fjerning av FLOX stoppkodon med ere rekombinase(ISL1: CRE; pCAG-FLOX-STOP-FLOX-GFP eller RFP / Isl1-cre; Rosa26YFP / Mef2c-cre; Rosa26YFP) 5,6. Når ESCs er differensiert i andre hjerte felt CPC, vil Isl1 / Mef2c promoter / enhancer drevet cre aktivere fluorescerende reportere og CPC kan bli beriket av FACS-rensing. I korthet er EB aggregering metode som brukes for å initiere ESC differensiering. For å forbedre effektiviteten differensiering, er de differensierte cellene behandlet med askorbinsyre (AA), og vekstfaktorer som Bmp4, aktivin A og VEGF 13,15. Denne protokollen tillater robust og effektiv CPC differensiering ved hjelp av både mus og menneske ESCs.
Protocol
Representative Results
Discussion
This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.
Materials
| Name | Company | Catalog Number |
| FBS | Thermo scentific | SH30070.03E |
| Knockout SR | Life technology | 10828028 |
| Knockout DMEM | Life technology | 10829018 |
| DMEM | Life technology | 11965118 |
| NEAA | Life technology | 11140050 |
| GlutaMAX | Life technology | 35050061 |
| N2 | Life technology | 17502048 |
| B27 | Life technology | 12587010 |
| Ham’s F12 | Life technology | 11765062 |
| IMDM | Life technology | 12440061 |
| Pen/Strep | Life technology | 15140122 |
| Pyruvate | Life technology | 11360070 |
| Dispase | Life technology | 17105041 |
| Stempro-34 | Life technology | 10639011 |
| DMEM/F12 | Life technology | 11330032 |
| BSA | Life technology | 15260037 |
| Trypsin | Life technology | 25200056 |
| Ascobic Acid | Sigma | A5960 |
| 1-Thioglycerol | Sigma | M1753 |
| 2-Mercaptoethanol | Sigma | M3148 |
| VEGF | R&D | 293-VE |
| Bmp4 | R&D | 314-BP |
| ActivinA | R&D | 338-AC |
| bFgf | R&D | 233-FB |
| Fgf10 | R&D | 345-FG |
| mTeSR | Stemcell technologies | 5850 |
| Matrigel | BD Biosciences | 354277 |
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