Derivação de Cardiac células progenitoras de células estaminais embrionárias
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
A doença cardíaca continua a ser a principal causa no mundo de hoje e as taxas de mortalidade têm permanecido praticamente inalterado nas últimas duas décadas (American Heart Association). Há uma necessidade crítica para o desenvolvimento de novas estratégias terapêuticas para prevenir ou reverter a insuficiência cardíaca de forma eficaz. Uma estratégia promissora é a terapia à base de células após o rápido desenvolvimento da biologia das células estaminais 1. A este respeito, PCC multipotentes poderia ser uma fonte excelente de células para a terapia devido à sua capacidade de proliferar, mas só comprometidas para diferenciação linhagem cardíaca. Portanto, o método eficiente e robusta para gerar e isolar CPCs é de grande importância para os estudos de terapia celular cardíaca.
Este protocolo incide sobre CPCs embrionárias identificadas durante a embriogênese e como a sua geração de CES. Vários CPCs foram isolados de corações embrionárias e adultas, mesmo a partir de medulas ósseas 2. Durante o desenvolvimento embrionário, morphoge ósseaproteínas Netic (BMPs), do tipo sem asas membros da família local de integração MMTV (Wnts) e sinais nodais induzir o compromisso da Mesp1 + mesoderme multipotentes 3. Células Mesp1 + então diferenciar em CPCs 4 embrionárias. Estes CPCs são tipicamente marcado por HCN4, NK2 homeobox 5 (NKX2-5), Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5) e miócitos fator potenciador 2C (MEF2C), formam campos primários do coração e segundo, e contribuir para as principais partes do coração durante cardiogenesis 5-10. Ambos CPCs NKX2-5 + e Isl1 + / + MEF2C são capazes de se diferenciar em cardiomiócitos, células de músculo liso (SMCs), e células endoteliais 5-8. Assim, estas CPCs vai dar origem a vasculatura cardíaca, bem como tecidos cardíacos e são uma fonte ideal de células para a terapia cardíaca com base em células. Consequentemente, gerando CPCs in vitro tem sido um grande foco de pesquisa em estudos cardiovasculares. Desde CES tem expansão ilimitada capacidade de umnd representam as células da MCI em estágio de blastocisto, a diferenciação dos CES em CPCs embrionárias após a embriogênese natural é considerada uma abordagem lógica e eficaz para obter CPCs.
Uma abordagem amplamente aplicada para obter CPCs da CES é agregar CES em EBs 11. Para melhorar a eficiência da diferenciação, crescimento e factores químicos definidos com base no conhecimento de desenvolvimento cardíaco foram utilizados 12-14. No entanto, não há marcadores CPC definitivos, particularmente não há marcadores de superfície celular, que são amplamente aceitos no campo. Para abordar esta questão, os CES são projetados para marcar CPCs Isl1 + ou MEF2C + e seus derivados com repórteres fluorescentes de Cre sistema / loxP. A recombinase cre é batido para dentro sob o controlo de Isl1 / MEF2C promotor / potenciador. Modificado RFP proteína fluorescente ou gene de YFP conduzido por um promotor constitutivo pode ser activado pela excisão de paragem flox codão com a recombinase cre(ISL1: cre; pCAG-Flox-STOP Flox-GFP ou RFP / Isl1-cre; Rosa26YFP / MEF2C-cre; Rosa26YFP) 5,6. Uma vez que os CES são diferenciadas em CPCs segundo campo coração, Isl1 / MEF2C promotor / cre conduzido irá ativar os repórteres fluorescentes e CPCs podem ser enriquecidos por FACS-purificação. Resumidamente, o método de agregação EB é usado para iniciar a diferenciação ESC. Para aumentar a eficiência de diferenciação, as células diferenciadas são tratados com ácido ascórbico (AA) e factores de crescimento, tais como Bmp4, Activina A e VEGF 13,15. Este protocolo permite robusto e eficiente diferenciação CPC usando mouse e do CES humanos.
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 …
Divulgaciones
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 |
Referencias
- Garbern, J. C., Lee, R. T. Cardiac stem cell therapy and the promise of heart regeneration. Cell Stem Cell. 12 (6), 689-698 (2013).
- Passier, R., van Laake, L. W., Mummery, C. L. Stem-cell-based therapy and lessons from the heart. Nature. 453 (7193), 322-329 (2008).
- Bondue, A., Blanpain, C. Mesp1: a key regulator of cardiovascular lineage commitment. Circulation Research. 107 (12), 1414-1427 (2010).
- Bondue, A., et al. Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell. 3 (1), 69-84 (2008).
- Bu, L., et al. Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature. 460 (7251), 113-117 (2009).
- Lei, I., Gao, X., Sham, M. H., Wang, Z. SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development. The Journal of Biological Chemistry. 287 (29), 24255-24262 (2012).
- Lei, I., Liu, L., Sham, M. H., Wang, Z. SWI/SNF in cardiac progenitor cell differentiation. Journal of Cellular Biochemistry. 114 (11), 2437-2445 (2013).
- Wu, S. M., et al. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell. 127 (6), 1137-1150 (2006).
- Spater, D., et al. A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nature Cell Biology. 15 (9), 1098-1106 (2013).
- Verzi, M. P., McCulley, D. J., De Val, S., Dodou, E., Black, B. L. The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field. Biología del desarrollo. 287 (1), 134-145 (2005).
- Boheler, K. R., et al. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circulation Research. 91 (3), 189-201 (2002).
- Wada, R., et al. Induction of human cardiomyocyte-like cells from fibroblasts by defined factors. Proceedings of the National Academy of Sciences of the United States of America. 110 (31), 12667-12672 (2013).
- Kattman, S. J., et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell. 8 (2), 228-240 (2011).
- Takahashi, T., et al. Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation. 107 (14), 1912-1916 (2003).
- Wamstad, J. A., et al. Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage. Cell. 151 (1), 206-220 (2012).
- Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339 (6121), 819-823 (2013).
- Ding, Q., et al. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell. 12 (4), 393-394 (2013).
- Gaj, T., Gersbach, C. S., Barbas, T. A. L. E. N. ZFN, TALEN, CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology. 31 (7), 397-405 (2013).
- Joung, J. K., Sander, J. D. TALENs: a widely applicable technology for targeted genome editing. Nature Reviews. Molecular Cell Biology. 14 (1), 49-55 (2013).
- Yang, H., et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 154 (6), 1370-1379 (2013).
- Mali, P., et al. RNA-guided human genome engineering via Cas9. Science. 339 (6121), 823-826 (2013).
- Laflamme, M. A., et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nature. biotechnology. 25 (9), 1015-1024 (2007).
- Burridge, P. W., et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PloS One. 6 (4), e18293 (2011).
- Kouskoff, V., Lacaud, G., Schwantz, S., Fehling, H. J., Keller, G. Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation. Proceedings of the National Academy of Sciences of the United States of America. 102 (37), 13170-13175 (2005).
- Xu, X. Q., et al. Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. Differentiation: Research in Biological Diversity. 76 (9), 958-970 (2008).
- Lian, X., et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proceedings of the National Academy of Sciences of the United States of America. 109 (27), E1848-1857 (2012).
- Bizy, A., et al. Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes. Stem Cell Research. 11 (3), 1335-1347 (2013).
