Afleiding van Cardiac stamcellen uit embryonale stamcellen
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
Hart-en vaatziekten nog steeds de belangrijkste oorzaak in de wereld van vandaag en de sterftecijfers hebben vrijwel onveranderd gebleven in de afgelopen twee decennia (American Heart Association). Er is dringend behoefte aan het ontwikkelen van nieuwe therapeutische strategieën om effectief te voorkomen of om te keren hartfalen. Een veelbelovende strategie-celtherapie na de snelle ontwikkeling van stamcelbiologie 1. In dit opzicht zou multipotente CPC een uitstekende bron cel voor de therapie vanwege hun vermogen tot prolifereren doch slechts gehouden cardiale lineage differentiatie. Daarom efficiënte en robuuste methode te genereren en te isoleren CPC is van groot belang voor hart celtherapie.
Dit protocol is gericht op embryonale CPC's die tijdens de vroege embryogenese en hoe hun generatie van de SER. Verschillende getuigschriften zijn geïsoleerd uit embryonale en volwassen harten, zelfs uit beenmerg 2. Tijdens de ontwikkeling van het embryo, bot morphogesche eiwitten (BMP's), vleugelloze-type MMTV integratieplaats familieleden (Wnt) en Nodal signalen van de inzet van de Mesp1 + multipotente mesoderm 3 induceren. Mesp1 + cellen vervolgens differentiëren in de embryonale CPC 4. Deze CPC's worden doorgaans gekenmerkt door HCN4, NK2- homeobox 5 (Nkx2-5), Isl LIM homeobox 1 (Isl1), T-doos 5 (Tbx5), en myocyte enhancer factor 2C (MEF2C), vormen de primaire en tweede hart velden, en bijdragen aan het grote delen van het hart tijdens cardiogenese 5-10. Zowel Nkx2-5 + en Isl1 + / MEF2C + CPC's zijn in staat om te differentiëren in hartspiercellen, gladde spiercellen (SMC) en endotheelcellen 5-8. Dus deze CPC's zullen leiden tot cardiale bloedvaten evenals hartweefsels geven en zijn een ideale bron van cellen voor cel-gebaseerde hart therapie. Bijgevolg is het genereren van CPC's in vitro heeft een groot onderzoeksproject focus op cardiovasculaire studies geweest. Sinds SER hebben onbeperkt capaciteitsuitbreiding eennd vertegenwoordigen de ICM cellen in het blastocyststadium, is de differentiatie van de SER in embryonale CPC's als gevolg van de natuurlijke embryogenese beschouwd als een logische en effectieve aanpak van CPC's te verkrijgen.
Een grote schaal toegepast aanpak van CPC's te verkrijgen van de SER is het SER aggregeren tot EBS 11. Om de differentiatie efficiëntie, gedefinieerde chemische en groeifactoren gebaseerd op de kennis van hartontwikkeling gebruikt 12-14. Er zijn echter geen definitieve CPC markers, met name niet celoppervlak markers, die wijd geaccepteerd in het veld. Om dit probleem aan te pakken, SER's zijn ontworpen om Isl1 + of MEF2C + CPC's en hun derivaten met TL-verslaggevers met behulp van Cre / loxP systeem te markeren. Het cre recombinase stoot in onder besturing van Isl1 / MEF2C promoter / enhancer. Gemodificeerde fluorescerend eiwit RFP of YFP-gen aangedreven door een constitutieve promoter kan worden geactiveerd door excisie van flox stopcodon met cre-recombinase(ISL1: cre; pCAG-Flox-STOP-Flox-GFP of RFP / Isl1-cre; Rosa26YFP / MEF2C-cre; Rosa26YFP) 5,6. Zodra de SER worden gedifferentieerd in tweede hart veld CPC's, zal Isl1 / MEF2C promotor / enhancer gedreven cre de tl-reporters activeren en CPC's kunnen worden verrijkt door FACS-zuivering. In het kort wordt EB aggregatie gebruikt ESC differentiatie initiëren. Om differentiatie efficiëntie, worden de gedifferentieerde cellen behandeld met ascorbinezuur (AA) en groeifactoren zoals Bmp4, activine A en VEGF 13,15. Dit protocol maakt robuuste en efficiënte CPC differentiatie met behulp van zowel muis en mens SER.
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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