Isolation, karakterisering og differentiering af Cardiac stamceller fra voksne mus hjerte
Summary
Det overordnede mål med denne artikel er at standardisere protokol for isolation, karakterisering og differentiering af cardiac stamceller (CSCs) fra voksne mus hjerte. Her, beskriver vi en tæthed gradient centrifugering metode til at isolere murine CSCs og omstændige metoder til CSC kultur, spredning og differentiering i cardiomyocytes.
Abstract
Myokardieinfarkt (MI) er en førende årsag til sygelighed og dødelighed i hele verden. Et vigtigt mål for regenerativ medicin er at genopbygge døde myokardiet efter MI. Selv om flere strategier har været brugt til at regenerere myokardiet, forbliver stamcelleterapi en større tilgang til at genopbygge en MI hjerte døde myokardiet. Akkumulere beviser tyder på tilstedeværelsen af fastboende hjerte stamceller (CSCs) i den voksne hjerte og deres endokrine eller paracrine påvirkning af cardiac regenerering. CSC isolation og deres karakterisering og differentiering mod Myokardie celler, især cardiomyocytes, er imidlertid fortsat en teknisk udfordring. I den foreliggende undersøgelse forudsat vi en simpel metode til isolation, karakterisering og differentiering af CSCs fra voksne mus hjerte. Her, beskriver vi en tæthed gradient metode til isolering af CSCs, hvor hjertet er fordøjet af en 0,2% collagenase II løsning. For at karakterisere de isolerede CSCs, evalueret vi udtryk for CSCs/hjertestop markører Sca-1, NKX2-5 og GATA4 og pluripotency/stemness markører OCT4, SOX2 og Nanog. Vi har også besluttet spredning potentiale af isolerede CSCs ved dyrkning dem i en petriskål og vurdere udtryk for spredning markør Ki-67. Til evaluering af CSCs differentiering potentiale, valgte vi syv – til 10 – dage kulturperler CSCs. Vi overført dem til en ny plade med en cardiomyocyte differentiering medium. De er udruget i en celle kultur inkubator for 12 dage, mens differentiering medium er ændret hver tre dage. De differentierede CSCs express cardiomyocyte-specifikke markører: actinin og troponin jeg. Således, CSCs isoleret med denne protokol har stemness og hjerte markører, og de har et potentiale for spredning og differentiering mod cardiomyocyte afstamning.
Introduction
Iskæmisk hjertesygdom, herunder myokardieinfarkt (MI), er en væsentlig årsag til død omkring verden1. Stamcelleterapi for regenererende døde myokardiet forbliver en større tilgang til at forbedre hjertefunktion en MI hjerte2,3,4,5. Forskellige typer af stamceller har været brugt til at genopbygge døde myokardiet og forbedre hjertefunktion en MI hjerte. De kan groft inddeles i embryonale stamceller6 og voksne stamceller. I voksne stamceller, har forskellige typer af stamceller været brugt, såsom knoglemarv-afledte mononukleære celler7,8, mesenkymale stamceller fra knoglemarven9,10, fedtvæv 11,12, og navlestrengen13og CSCs14,15. Stamceller kan fremme hjerte regenerering gennem endokrine og/eller paracrine handlinger16,17,18,19,20. Imidlertid en stor begrænsning af stamcelleterapi er at opnå et tilstrækkeligt antal stamceller, der kan formere sig og/eller differentiere mod en bestemt hjerte lineage21,22. Autolog og allogen transplantation af stamceller er en vigtig udfordring i stamcelle terapi9. CSCs kunne være en bedre tilgang til hjerte regenerering, fordi de stammer fra hjertet og de lettere kan opdeles i hjertets lineages end ikke-hjerte stamceller. Det nedsætter risikoen for teratom. Derudover kunne de endokrin og paracrine effekter af CSCs, såsom exosomes og miRNAs afledt af CSCs, være mere effektiv end andre typer af stamceller. Således, CSCs stadig en bedre løsning for kardiale regenerering23,24.
Selv om CSCs er en bedre kandidat til hjerte regenerering i en MI hjertet på grund af deres hjerte oprindelse, er en stor begrænsning med CSCs mindre udbytte på grund af manglen på en effektiv isolation metode. En anden begrænsning kunne være nedsat differentiering af CSCs mod cardiomyocytes lineage2,25,26,27. For at omgå disse begrænsninger, er det vigtigt at udvikle en effektiv protokol til CSC isolation, karakterisering og differentiering mod hjerte afstamning. Der er ingen enkelt acceptable markør for CSCs og en bestemt celle-overflade markør-baserede isolering af CSCs udbytter mindre CSCs. Her, standardiserer vi en simpel gradient centrifugering tilgang for at isolere CSCs fra mus hjertet, der er omkostningseffektive og resulterer i en øget afkast på CSCs. Disse isolerede CSCs kan vælges for specifikke celle-overflade markører ved fluorescens-aktiveret celle kortslutning. Ud over CSCs isolation forudsat vi en protokol til CSC kultur, karakterisering og differentiering mod cardiomyocyte afstamning. Dermed, vi præsenterer en elegant metode til at isolere, karakterisere, kultur, og differentiere CSCs fra voksne mus hjerter (figur 5).
Protocol
Representative Results
Discussion
Kritiske trin i protokollens CSC isolation er som følger. 1) en steriliseret tilstand skal opretholdes for udvinding af hjerter fra mus. Enhver forurening under hjertet udvinding kan forringe kvaliteten af CSCs. 2) blodet skal fjernes helt, før hakningen hjerte, der er udført af flere skylninger af hele hjertet og hjertet stykker med HBSS løsning. 3) hjerte stykker skal være helt mængden i en enkelt celle suspension med collagenase løsning. 4) den polysucrose og natrium diatrizoate gradient løsning til separation…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde er i dele, støttet af National Institutes of Health tilskud HL-113281 og HL116205 til Paras Kumar Mishra.
Materials
| Mice | The Jackson laboratory, USA | Stock no. 000664 | |
| Antibodies: | |||
| OCT4- | Abcam | ab18976 (rabbit polyclonal) | OCT4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| SOX2 | Abcam | ab97959 (rabbit polyclonal) | SOX2-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Nanog | Abcam | ab80892 (rabbit polyclonal) | Nanog-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Ki67 | Abcam | ab16667 (rabbit polyclonal) | Ki67-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Sca I | Millipore | AB4336 (rabbit polyclonal) | Sca I Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| NKX2-5 | Santa Cruz | sc-8697 (goat polyclonal) | NKX2-5-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| GATA4 | Abcam | ab84593 (rabbit polyclonal) | GATA4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| MEF2C | Santa Cruz | sc-13268 (goat polyclonal) | MEF2C-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Troponin I | Millipore | MAB1691 (mouse monoclonal) | Troponin I-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Actinin | Millipore | MAB1682 (mouse monoclonal) | Actinin-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| ANP | Millipore | AB5490 (mouse polyclonal) | ANP-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Alex Fluor-488 checken anti-rabbit | Life technology | Ref no. A21441 | |
| Alex Fluor-594 goat anti-rabbit | Life technology | Ref no. A11012 | |
| Alex Fluor-594 rabbit anti-goat | Life technology | Ref no. A11078 | |
| Alex Fluor-488 checken anti-mouse | Life technology | Ref no. A21200 | |
| Alex Fluor-594 checken anti-goat | Life technology | Ref no. A21468 | |
| Name | Company | Catalog Number | Comments |
| Culture medium: | |||
| CSC maintenance medium | Millipore | SCM101 | Note: For CSC culture, PBS or incomplete DMEM medium was used for washing the cells |
| cardiomyocytes differentiation medium | Millipore | SCM102 | |
| DMEM | Sigma-Aldrich | D5546 | |
| Name | Company | Catalog Number | Comments |
| Cell Isolation buffer: | |||
| polysucrose and sodium diatrizoate solution (Histopaque1077) | Sigma | 10771 | |
| HBSS | Gibco | 2018-03 | |
| Collagenase I | Sigma | C0130 | |
| Dispase solution | STEMCELL Technologies | 7913 | |
| PBS | LONZA | S1226 | |
| StemPro Accutase Cell Dissociation Reagent | Thermoscientific | A1110501 | |
| Other reagents: | |||
| BSA | Sigma | A7030 | |
| Normal checken serum | Vector laboratory | S3000 | |
| DAPI solution | Applichem | A100,0010 | Dapi, working concentration-1 µg/mL |
| Trypan blue | Biorad | 145-0013 | |
| Trypsin | Sigma | T4049 | |
| StemPro Accutase Cell Dissociation Reagent | Thermo Fisher Scientific | A1110501 | |
| Formaldehyde | Sigma | 158127 | |
| Triton X-100 | ACROS | Cas No. 900-293-1 | |
| Tween 20 | Fisher Sceintific | Lot No. 160170 | |
| Ethanol | Thermo Scientific | ||
| Name | Company | Catalog Number | Comments |
| Tissue culture materials: | |||
| 100 mm petri dish | Thermo Scientific | ||
| 6-well plate | Thermo Scientific | ||
| 24-well plate | Thermo Scientific | ||
| T-25 flask | Thermo Scientific | ||
| T-75 flask | Thermo Scientific | ||
| 15 ml conical tube | Thermo Scientific | ||
| 50 mL conical tube | Thermo Scientific | ||
| 40 µm cell stainer | Fisher Scientific | 22363547 | |
| 100 µm cell stainer | Fisher Scientific | 22363549 | |
| 0.22 µm filter | Fisher Scientific | 09-719C | |
| 10 mL syring | BD | Ref no. 309604 | |
| 10 µL, 200 µL, 1000 µL pipette tips | Fisher Scientific | ||
| 5 mL, 10mL, 25 mL disposible plastic pipette | Thermo Scientific | ||
| Name | Company | Catalog Number | Comments |
| Instruments | |||
| Centrufuge machine | Thermo Scientific | LEGEND X1R centrifuge | |
| EVOS microscope | Life technology | ||
| Automated cell counter | Biorad | ||
| Cell counting slide | Biorad | 145-0011 | |
| Pippte aid | Thermo Scientific | S1 pipet filler | |
| Name | Company | Catalog Number | Comments |
| Surgical Instruments: | |||
| Surgical scissors | Fine Scientific Tool | ||
| Fine surgical scissors | Fine Scientific Tool | ||
| Curve shank forceps | Fine Scientific Tool | ||
| Surgical blade | Fine Scientific Tool |
References
- Benjamin, E. J., et al. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation. 135, e146 (2017).
- Nguyen, P. K., Rhee, J. W., Wu, J. C. Adult Stem Cell Therapy and Heart Failure, 2000 to 2016: A Systematic Review. The Journal of the American Medical Association Cardiology. 1, 831-841 (2000).
- Emmert, M. Y., et al. Safety and efficacy of cardiopoietic stem cells in the treatment of post-infarction left-ventricular dysfunction – From cardioprotection to functional repair in a translational pig infarction model. Biomaterials. 122, 48-62 (2017).
- Silvestre, J. S., Menasche, P. The Evolution of the Stem Cell Theory for Heart Failure. EBioMedicine. 2, 1871-1879 (2015).
- Terzic, A., Behfar, A. Stem cell therapy for heart failure: Ensuring regenerative proficiency. Trends in Cardiovascular Medicine. 26, 395-404 (2016).
- Yamada, S., et al. Embryonic stem cell therapy of heart failure in genetic cardiomyopathy. Stem Cells. 26, 2644-2653 (2008).
- Sadek, H. A., Martin, C. M., Latif, S. S., Garry, M. G., Garry, D. J. Bone-marrow-derived side population cells for myocardial regeneration. Journal of Cardiovascular Translational Research. 2, 173-181 (2009).
- Vrtovec, B., et al. Effects of intracoronary CD34+ stem cell transplantation in nonischemic dilated cardiomyopathy patients: 5-year follow-up. Circulation Research. 112, 165-173 (2013).
- Hare, J. M., et al. Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. The Journal of American Medical Association. 308, 2369-2379 (2012).
- Guijarro, D., et al. Intramyocardial transplantation of mesenchymal stromal cells for chonic myocardial ischemia and impaired left ventricular function: Results of the MESAMI 1 pilot trial. International Journal of Cardiology. 209, 258-265 (2016).
- Bobi, J., et al. Intracoronary Administration of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction. Journal of the American Heart Association. 6, (2017).
- Suzuki, E., Fujita, D., Takahashi, M., Oba, S., Nishimatsu, H. Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease. World Journal of Cardiology. 7, 454-465 (2015).
- Gao, L. R., et al. Intracoronary infusion of Wharton’s jelly-derived mesenchymal stem cells in acute myocardial infarction: double-blind, randomized controlled trial. BMC Medicine. 13, 162 (2015).
- Simpson, D. L., et al. A strong regenerative ability of cardiac stem cells derived from neonatal hearts. Circulation. , S46-S53 (2012).
- Kazakov, A., et al. C-kit(+) resident cardiac stem cells improve left ventricular fibrosis in pressure overload. Stem Cell Research. 15, 700-711 (2015).
- Ong, S. G., et al. Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer. Circulation. 130, S60-S69 (2014).
- Sahoo, S., Losordo, D. W. Exosomes and cardiac repair after myocardial infarction. Circulation Research. 114, 333-344 (2014).
- Zhang, Z., et al. Pretreatment of Cardiac Stem Cells With Exosomes Derived From Mesenchymal Stem Cells Enhances Myocardial Repair. Journal of the American Heart Association. 5, (2016).
- Ibrahim, A. G., Cheng, K., Marban, E. Exosomes as critical agents of cardiac regeneration triggered by cell therapy. Stem Cell Reports. 2, 606-619 (2014).
- Emanueli, C., Shearn, A. I., Angelini, G. D., Sahoo, S. Exosomes and exosomal miRNAs in cardiovascular protection and repair. Vascular Pharmacology. 71, 24-30 (2015).
- Menasche, P. Cardiac cell therapy: lessons from clinical trials. Journal of Molecular and Cellular Cardiology. 50, 258-265 (2011).
- Trounson, A., McDonald, C. Stem Cell Therapies in Clinical Trials: Progress and Challenges. Cell Stem Cell. 17, 11-22 (2015).
- Takamiya, M., Haider, K. H., Ashaf, M. Identification and characterization of a novel multipotent sub-population of Sca-1(+) cardiac progenitor cells for myocardial regeneration. PLoS One. 6, e25265 (2011).
- Cambria, E., et al. Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types. NPJ Regenerative Medicine. 2, 17 (2017).
- Bruyneel, A. A., Sehgal, A., Malandraki-Miller, S., Carr, C. Stem Cell Therapy for the Heart: Blind Alley or Magic Bullet?. Journal of Cardiovascular Translational Research. 9, 405-418 (2016).
- Garbern, J. C., Lee, R. T. Cardiac stem cell therapy and the promise of heart regeneration. Cell Stem Cell. 12, 689-698 (2013).
- Oh, H., Ito, H., Sano, S. Challenges to success in heart failure: Cardiac cell therapies in patients with heart diseases. Journal of Cardiology. 68, 361-367 (2016).
- Smith, A. J., et al. Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart. Nature Protocols. 9, 1662-1681 (2014).
- Rutering, J., et al. Improved Method for Isolation of Neonatal Rat Cardiomyocytes with Increased Yield of C-Kit+ Cardiac Progenitor Cells. Journal of Stem Cell Research and Therapy. 5, 1-8 (2015).
- Saravanakumar, M., Devaraj, H. Distribution and homing pattern of c-kit+ Sca-1+ CXCR4+ resident cardiac stem cells in neonatal, postnatal, and adult mouse heart. Cardiovascular Pathology. 22, 257-263 (2013).
- Monsanto, M. M., et al. Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy. Circulation Research. 121, 113-124 (2017).
- Vidyasekar, P., Shyamsunder, P., Santhakumar, R., Arun, R., Verma, R. S. A simplified protocol for the isolation and culture of cardiomyocytes and progenitor cells from neonatal mouse ventricles. European Journal of Cell Biology. 94, 444-452 (2015).
- Dergilev, K. V., et al. Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs. Tissue Cell. 49, 64-71 (2017).
- Zaruba, M. M., Soonpaa, M., Reuter, S., Field, L. J. Cardiomyogenic potential of C-kit(+)-expressing cells derived from neonatal and adult mouse hearts. Circulation. 121, 1992-2000 (1992).
- Wang, H., et al. Isolation and characterization of a Sca-1+/CD31- progenitor cell lineage derived from mouse heart tissue. BMC Biotechnology. 14, 75 (2014).
- Smits, A. M., et al. Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nature Protocols. 4, 232-243 (2009).
