Afledning af Cardiac progenitorceller af 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
Hjertesygdom er den hyppigste årsag i verden i dag, og dødeligheden er forblevet stort set uændret i de seneste to årtier (American Heart Association). Der er et kritisk behov for udvikling af nye terapeutiske strategier til effektivt forebygge eller reversere hjertesvigt. En lovende strategi er celle-baseret behandling efter den hurtige udvikling af stamceller biologi 1. I denne henseende kunne multipotente CPC være en fremragende cellekilde til terapi på grund af deres evne til at formere sig, men kun forpligtet til hjerte- afstamning differentiering. Derfor er effektiv og robust metode generere og isolere CPC'er er af stor betydning for studier hjerte celleterapi.
Denne protokol fokuserer på embryonale CPC'er identificeret under tidlig embryogenese og hvordan deres produktion fra økonomiske og sociale råd. Forskellige CPC'er er blevet isoleret fra embryonale og voksne hjerter, selv fra knoglemarv 2. Under udviklingen embryo, ben morphogetiske proteiner (BMP'er), vingeløse-type MMTV integrationssted familiemedlemmer (Wnts) og Nodal signaler inducerer engagement Mesp1 + multipotent mesoderm 3. Mesp1 + celler derefter differentiere til de embryonale CPC'er 4. Disse CPC'er typisk præget af HCN4, NK2 homeobox 5 (Nkx2-5), Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5), og myocyt forstærker faktor 2C (Mef2c), danner primære og andet hjerte felter, og bidrage til de store dele af hjertet under cardiogenese 5-10. Både Nkx2-5 + og Isl1 + / Mef2c + CPC er i stand til at differentiere til cardiomyocytter, glatte muskelceller (SMC'er), og endotelceller 5-8. Således disse CPC'er vil give anledning til hjerte-kar samt hjertevæv og er en ideel celle kilde til cellebaserede hjerte terapi. Derfor genererer CPC'er in vitro har været et stort forskningsfokus i hjerte-kar-studier. Da økonomiske og sociale råd har ubegrænset ekspansion kapacitet and repræsenterer ICM celler ved blastocyststadiet, er differentiering af økonomiske og sociale råd i embryonale CPC'er efter den naturlige embryogenese betragtes som en logisk og effektiv tilgang til at opnå priser pr.
En almindelig anvendt metode til at opnå CPC'er fra økonomiske og sociale råd er at samle økonomiske og sociale råd i EB'er 11. For at forbedre differentiering effektivitet, har defineret kemiske og vækstfaktorer baseret på viden om hjerte-udvikling blevet brugt 12-14. Men der er ingen endelige CPC markører, især ingen celle-overflade markører, der er almindeligt accepteret i marken. For at løse dette problem, er økonomiske og sociale råd manipuleret til at markere Isl1 + eller Mef2c + CPC'er og deres derivater med fluorescerende reportere ved hjælp Cre / loxP-systemet. Cre-rekombinase er slået i under kontrol af Isl1 / Mef2c promotor / enhancer. Modificeret fluorescerende protein RFP eller YFP-genet drevet af en konstitutiv promotor kan aktiveres ved udskæring af flox stopkodon med Cre-rekombinase(ISL1: cre; pCAG-FLOX-STOP-FLOX-GFP eller RFP / Isl1-cre; Rosa26YFP / Mef2c-CRE; Rosa26YFP) 5,6. Når økonomiske og sociale råd er differentieret i andet hjerte felt CPC'er vil Isl1 / Mef2c promotor / enhancer drevet CRE aktivere de fluorescerende reportere og CPC'er kan beriges ved FACS-oprensning. Kort fortalt EB sammenlægning metode anvendes til at initiere ESC differentiering. For at øge differentiering effektivitet er de differentierede celler behandlet med ascorbinsyre (AA) og vækstfaktorer, såsom BMP4, Activin A og VEGF 13,15. Denne protokol giver robust og effektivt CPC differentiering ved hjælp af både mus og menneskelige økonomiske og sociale råd.
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 …
Disclosures
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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