Forbedring af engraftment af humane inducerede pluripotente stamcelle afledte Kardiomyocytter via en forbigående hæmning af Rho Kinaseaktivitet
Summary
I denne protokol viser og uddyber vi, hvordan man bruger menneskeskabte pluripotente stamceller til differentiering og rensning af kardiomyocytter, og yderligere om, hvordan man kan forbedre sin transplantations effektivitet med Rho-associeret proteinkinasehæmmer forbehandling i en mus myokardieinfarkt model.
Abstract
En afgørende faktor for at forbedre cellulære terapi effektivitet for Myokardie regenerering er at sikkert og effektivt øge cellen engraftment sats. Y-27632 er en meget potent hæmmer af Rho-associeret, coiled-spole-holdige protein kinase (RhoA/ROCK) og bruges til at forhindre dissociation-induceret celle apoptose (anoikis). Vi viser, at Y-27632 forbehandling for menneskeskabte pluripotente stamcelle-afledte kardiomyocytter (hiPSC-CMs+ Ri) forud for implantation resulterer i en celle engraftment rate forbedring i en musemodel af akut MYOKARDIEINFARKT (MI). Her beskriver vi en komplet procedure af hiPSC-CMs differentiering, rensning, og celle forbehandling med Y-27632, samt den resulterende celle sammentrækning, calcium forbigående målinger, og transplantation i mus MI modeller. Den foreslåede metode giver en enkel, sikker, effektiv og billig metode, som i væsentlig grad øger cellengraftment sats. Denne metode kan ikke kun anvendes i forbindelse med andre metoder til yderligere at forbedre celletransplantation effektivitet, men også giver et gunstigt grundlag for studiet af mekanismerne i andre hjertesygdomme.
Introduction
Stamcelle baserede terapier har vist et betydeligt potentiale som en behandling for hjerte skader forårsaget af MI1. Brugen af differentierede hiPSCs giver en uudtømmelig kilde til hiPSC-CMs2 og åbner døren for en hurtig udvikling af banebrydende behandlinger. Men, mange begrænsninger for terapeutisk oversættelse forbliver, herunder udfordringen med den alvorligt lave engraftment sats af implanterede celler.
Dissocierende celler med trypsin initierer anoikis3, som kun accelereres, når disse celler injiceres i barske miljøer som den iskæmiske myokardiet, hvor det hypoxiske miljø accelererer kursen mod celledød. Af de resterende celler, en stor del vaskes ud fra implantationsstedet ind i blodbanen og spredes i hele periferien. En af de vigtigste apoptotiske veje er RhoA/ROCK pathway4. Baseret på tidligere forskning, rhoa/rock pathway regulerer actin cytoskelet organisation5,6, som er ansvarlig for celle dysfunktion7,8. Rock inhibitor Y-27632 er meget udbredt under somatisk og stamcelle dissociation og passaging, at øge celle vedhæftning og reducere celle apoptose9,10,11. I dette studie anvendes Y-27632 til behandling af hiPSC-CMs før transplantation i et forsøg på at øge cellernes engraftment.
Der er fastlagt flere metoder til forbedring af celle engraftment, såsom varmechok og kælder membran matrix belægning12. Bortset fra disse metoder kan genteknologi også fremme kardiomyocyt proliferation13 eller vende nonmyokardial celler til kardiomyocytter14. Fra Bioengineering perspektiv, cardiomyocytter seedet på en biomaterialet stilladset for at forbedre transplantationen effektivitet15. Desværre er de fleste af disse metoder komplicerede og dyre. Tværtimod er den metode, der foreslås her, enkel, omkostningseffektiv og effektiv, og den kan bruges som basal behandling før transplantation, samt i konjugering med andre teknologier.
Protocol
Representative Results
Discussion
De vigtigste trin i denne undersøgelse omfatter at opnå ren hiPSC-CMs, forbedre aktiviteten af hiPSC-CMs gennem Y-27632 forbehandling, og endelig, transplantere en præcis mængde hiPSC-CMs i en mus MI model.
De centrale spørgsmål, der blev behandlet her, var, at vi først optimerede de glucosefri rensningsmetoder19 og etablerede et nyt effektivt rensningssystem. System proceduren omfattede anvendelse af celle dissociations enzymer, genbeplantning af celler i gelati…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Forfatterne takker Dr. Joseph C. Wu (Stanford University) for venligt at give Fluc-GFP konstruere og Dr. Yanwen Liu for fremragende teknisk assistance. Denne undersøgelse er støttet af National Institutes of Health RO1 tilskud HL95077, HL114120, HL131017, HL138023, UO1 HL134764 (til J.Z.), og HL121206A1 (til L.Z.), og en R56 Grant HL142627 (til W.Z.), en amerikansk hjerte forening videnskabsmand udvikling Grant 16SDG30410018, og University of Alabama på Birmingham Faculty Development Grant (til W.Z.).
Materials
| Reagent | |||
| Accutase (stem cell detachment solution) | STEMCELL Technologies | #07920 | |
| B27 minus insulin | Fisher Scientific | A1895601 | |
| B27 Supplement | Fisher Scientific | 17-504-044 | |
| CHIR99021 | Stem Cell Technologies | 72054 | |
| DMEM (1x), high glucose, HEPES, no phenol red | Thermofisher | 20163029 | |
| Fetal bovine serum | Atlanta Biologicals | S11150 | |
| Fluo-4 AM (calcium indicator) | Invitrogen/Thermofisher | F14201 | |
| Glucose-free RPMI 1640 | Fisher Scientific | 11879020 | |
| IWR1 | Stem Cell Technologies | 72562 | |
| Matrigel (extracellular matrix ) | Fisher Scientific | CB-40230C | |
| mTeSR (human pluripotent stem cells medium) | STEMCELL Technologies | 85850 | |
| Pen-strep antibiotic | Fisher Scientific | 15-140-122 | |
| Pluronic F-127 (surfactant polyol) | Sigma-Aldrich | P2443 | |
| Rho activator II | Cytoskeleton | CN03 | |
| RPMI1640 | Fisher Scientific | 11875119 | |
| Sodium DL-lactate | Sigma-Aldrich | L4263 | |
| TrypLE (cell-dissociation enzymes) | Fisher Scientific | 12-605-010 | |
| Verapamil | Sigma-Aldrich | V4629 | |
| Y-27632 | STEMCELL Technologies | 72304 | |
| Name | Company | Catalog Number | Comments |
| Equipment and Supplies | |||
| IVIS Lumina III Bioluminescence Instruments | PerkinElmer | CLS136334 | |
| 15 mm Coverslips | Warner | CS-15R15 | |
| Centrifuge | Eppendorf | 5415R | |
| Confocal Microscope | Olympus | IX81 | |
| Cryostat | Thermo Scientific | NX50 | |
| Dual Automatic Temperature Controller | Warner Instruments | TC-344B | |
| Electrophoresis Power Supply | BIO-RAD | 1645050 | |
| Fluoresence Microscope | Olympus | IX83 | |
| High Speed Camera | pco | 1200 s | |
| Laser Scan Head | Olympus | FV-1000 | |
| Low Profile Open Bath Chamber (mounts into above microincubation system) | Warner Instruments | RC-42LP | |
| Microincubation System | Warner Instruments | DH-40iL | |
| Minivent Mouse Ventilator | Harvard Apparatus | 845 | |
| NOD/SCID mice | Jackson Laboratory | 001303 | |
| Precast Protein Gels | BIO-RAD | 4561033 | |
| PVDF Transfer Packs | BIO-RAD | 1704156 | |
| Trans-Blot System | BIO-RAD | Trans-Blot Turbo | |
| Hot bead sterilizer | Fine Science Tools | 18000-45 | |
| Name | Company | Catalog Number | Comments |
| Antibody | |||
| Anti-human Nucleolin (Alexa Fluor 647) | Abcam | ab198580 | |
| Cardiac Troponin T | R&D Systems | MAB1874 | |
| Cardiac Troponin C | Abcam | ab137130 | |
| Cardiac Troponin I | Abcam | ab47003 | |
| Cy5-donkey anti-mouse | Jackson ImmunoResearch Laboratory | 715-175-150 | |
| Cy3-donkey anti-rabbit | Jackson ImmunoResearch Laboratory | 711-165-152 | |
| Fitc-donkey anti-mouse | Jackson ImmunoResearch Laboratory | 715-095-150 | |
| GAPDH | Abcam | ab22555 | |
| Human Cardiac Troponin T | Abcam | ab91605 | |
| Integrin β1 | Abcam | ab24693 | |
| Ki67 | EMD Millipore | ab9260 | |
| N-cadherin | Abcam | ab18203 | |
| Phospho-Myosin Light Chain 2 | Cell Signaling Technology | 3671s | |
| Name | Company | Catalog Number | Comments |
| Software | |||
| Matlab | MathWorks | R2016A | |
| Image J | NIH | 1.52g |
Riferimenti
- Menasche, P., et al. Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience. European Heart Journal. 36 (12), 743-750 (2015).
- Burridge, P. W., Keller, G., Gold, J. D., Wu, J. C. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell. 10 (1), 16-28 (2012).
- Frisch, S. M., Francis, H. Disruption of epithelial cell-matrix interactions induces apoptosis. Journal of Cell Biology. 124 (4), 619-626 (1994).
- Haun, F., et al. Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin. Nature Communications. 9 (1), 3524 (2018).
- Ohashi, K., et al. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. Journal of Biological Chemistry. 275 (5), 3577-3582 (2000).
- Katoh, K., Kano, Y., Noda, Y. Rho-associated kinase-dependent contraction of stress fibres and the organization of focal adhesions. Journal of The Royal Society Interface. 8 (56), 305-311 (2011).
- Paoli, P., Giannoni, E., Chiarugi, P. Anoikis molecular pathways and its role in cancer progression. Biochimica et Biophysica Acta. 1833 (12), 3481-3498 (2013).
- Legate, K. R., Fassler, R. Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails. Journal of Cell Science. 122 (Pt 2), 187-198 (2009).
- Watanabe, K., et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology. 25 (6), 681-686 (2007).
- Emre, N., et al. The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers. PLoS One. 5 (8), e12148 (2010).
- Ni, Y., Qin, Y., Fang, Z., Zhang, Z. ROCK Inhibitor Y-27632 Promotes Human Retinal Pigment Epithelium Survival by Altering Cellular Biomechanical Properties. Current Molecular Medicine. 17 (9), 637-646 (2017).
- 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).
- Zhu, W., Zhao, M., Mattapally, S., Chen, S., Zhang, J. CCND2 Overexpression Enhances the Regenerative Potency of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Remuscularization of Injured Ventricle. Circulation Research. 122 (1), 88-96 (2018).
- Song, K., et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 485 (7400), 599-604 (2012).
- Ye, L., et al. Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells. Cell Stem Cell. 15 (6), 750-761 (2014).
- Tohyama, S., et al. Glutamine Oxidation Is Indispensable for Survival of Human Pluripotent Stem Cells. Cell Metabolism. 23 (4), 663-674 (2016).
- Ong, S. G., et al. Microfluidic Single-Cell Analysis of Transplanted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes After Acute Myocardial Infarction. Circulation. 132 (8), 762-771 (2015).
- Zhao, M., et al. Y-27632 Preconditioning Enhances Transplantation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Myocardial Infarction Mice. Cardiovascular Research. , (2018).
- Tohyama, S., et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell. 12 (1), 127-137 (2013).
- Silginer, M., Weller, M., Ziegler, U., Roth, P. Integrin inhibition promotes atypical anoikis in glioma cells. Cell Death & Disease. 5, e1012 (2014).
- Lelievre, E. C., et al. N-cadherin mediates neuronal cell survival through Bim down-regulation. PLoS One. 7 (3), e33206 (2012).
- Murata, K., et al. Increase in cell motility by carbon ion irradiation via the Rho signaling pathway and its inhibition by the ROCK inhibitor Y-27632 in lung adenocarcinoma A549 cells. Journal of Radiation Research. 55 (4), 658-664 (2014).
- Srivastava, K., Shao, B., Bayraktutan, U. PKC-beta exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway. Journal of Cerebral Blood Flow & Metabolism. 33 (12), 1928-1936 (2013).
