Storstilet produktion af cardiomyocytter fra menneskelige pluripotente stamceller Ved hjælp af en meget reproducerbar lille molekyle-Based Differentiering Protocol
Summary
Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.
Abstract
Maksimering gavn af menneskelige pluripotente stamceller (hPSCs) til forskning, sygdom modellering, farmaceutiske og kliniske anvendelser kræver robuste metoder til produktion i stor skala af funktionelle celletyper, herunder cardiomyocytter. Her demonstrerer vi, at den tidsmæssige manipulation af WNT, TGF-β, og SHH signalveje fører til højeffektive cardiomyocyte differentiering af enkelt-celle passerede hPSC linier i både statiske suspension og omrørt suspension bioreaktorsystemer. Anvender denne strategi resulterede i ~ 100% slå sphæroider, konsekvent indeholder> 80% kardial troponin T-positive celler efter 15 dages dyrkning valideret i flere hPSC linjer. Vi rapporterer også om en variation af denne protokol til brug med cellelinjer i øjeblikket ikke tilpasset encellede passage, hvis succes er blevet verificeret i 42 hPSC linjer. Cardiomyocytter genereres ved hjælp af disse protokoller udtrykker afstamning-specifikke markører og show forventede electrophysiological funktionaliteter. Vores protokol præsenterer en enkel, effektiv og robust platform til produktion i stor skala af humane cardiomyocytter.
Introduction
Humane pluripotente stamceller (hPSCs), herunder humane embryonale stamceller (hESCs) og inducerede pluripotente stamceller (hiPSCs), har mulighed for selv-fornyelse og evnen til at differentiere til celler i de tre embryonale kim lag 1,2. På grund af disse egenskaber, hPSCs en værdifuld og ubegrænset kilde til generering og skalerbar produktion af sygdomsspecifikke relevant celletyper til modellering human sygdom 3-5, til high-throughput lægemiddel-screening og toksicitet assays 6,7 og potentielt til kliniske anvendelser 8 . Generation af cardiomyocytter fra hPSCs giver mulighed for specifikt at undersøge mekanismerne i komplekse menneskelige hjerte-kar-sygdomme og deres mulige behandlinger, der tidligere uden for rammerne af vores kapacitet på grund af mangel på relevante dyremodeller og / eller tilgængeligheden af berørte primære væv.
Alle førnævnte anvendelser af hPSCs necessitate produktionen af massive antal af højt beriget og funktionelle cardiomyocytter. Således tilgængeligheden af en effektiv, reproducerbar og skalerbar in vitro hjerte-differentiering protokol egnet til flere hPSC linjer er afgørende. Konventionelle cardiomyocyte differentiering protokoller har ansat forskellige strategier såsom embryoid krop dannelse 9, co-kultur teknikker 10, induktion med cocktails af cytokiner 11 og protein transduktion metoder 12. På trods af fremskridt i disse teknikker, lider de fleste stadig af dårlig effektivitet, kræver dyre vækstfaktorer, eller tilbyde begrænset universalitet når du forsøger at bruge flere hPSC linjer. Til dato har disse udfordringer sat grænser for produktionen af hPSC-afledte cardiomyocytter for celleterapi studier i dyremodeller, samt i den farmaceutiske industri for drug discovery 13. Derfor er udviklingen af robuste og overkommelige teknikker til store-skala produktion af funktionelle hPSC-afledte cardiomyocytter i skalerbare dyrkningssystemer vil i høj grad lette deres kommercielle og kliniske anvendelser.
I dette manuskript rapporterer vi udviklingen af en omkostningseffektiv og integreret hjertedifferentiering system med høj effektivitet, reproducerbarhed og anvendelighed til hESCs og hiPSCs genereret fra en række forskellige kilder og dyrkningsmetoder, herunder en fremgangsmåde til produktion i stor målestok af højt berigede populationer af hPSC-afledte cardiomyocytter ved anvendelse af en bioreaktor. Derudover har vi optimeret denne protokol for hPSC linjer ikke er tilpasset feeder fri og / eller enkelt cellekultur, såsom nyetablerede hiPSCs eller store kohorter af hPSC linjer er relevante for analysen af sygdom mekanisme.
Protocol
Representative Results
Discussion
Cardiomyocytter afledt hPSCs er en særdeles attraktiv kilde til brug i human sygdom modellering, narkotika screening / toksicitet og måske i fremtiden, regenerative behandlinger. En af de største hindringer til anvendelse af disse celler imidlertid er evnen til at give tilstrækkelig høj kvalitet materiale for deres effektive anvendelse. Ved hjælp af vores beskrevne protokol, tilbyder vi en metode, der overvinder denne begrænsning.
For nylig er syntetiske små molekyler rettet mod spec…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This study was funded by grants provided from Royan Institute, Iranian Council of Stem Cell Research and Technology, the Iran National Science Foundation (INSF), the National Health and Medical Research Council of Australia (NHMRC; 354400), the National Heart Foundation of Australia/Heart Kids Australia (G11S5629), and the New South Wales Cardiovascular Research Network. HF was supported by a University International Postgraduate Scholarship from the University of New South Wales, Australia. RPH was supported by a NHMRC Australia Fellowship. The authors express their gratitude to the human subjects who participated in this research.
Materials
| Knockout DMEM | Life Technologies | 10829018 | |
| Knockout Serum Replacement (KO-SR) | Life Technologies | 10828028 | |
| Glutamax | Life Technologies | 35050061 | |
| MEM Non-essential Amino Acids | Life Technologies | 11140-050 | |
| β-Mercaptoethanol | Life Technologies | 21985-023 | |
| Basic Fibroblast Growth Factor (bFGF) | Miltenyi Biotec | 130-093-843 | |
| RPMI1640 | Life Technologies | 11875093 | |
| DPBS, no calcium, no magnesium | Life Technologies | 14190144 | |
| DPBS | Life Technologies | 14287072 | |
| Attachment Factor (AF) | Life Technologies | S006100 | |
| ECM Gel | Sigma-Aldrich | E1270 | |
| Laminin | Invitrogen | 23017-015 | |
| DMEM | Life Technologies | 11965-092 | |
| Fatal Bovine Serum (FBS) | Life Technologies | 16140-071 | |
| B27 minus insulin | Gibco | A18956-01 | |
| Penicillin/Streptomycin | Life Technologies | 15070063 | |
| 0.05% Trypsin/EDTA | Life Technologies | 25300-054 | |
| Collagenase Type IV | Life Technologies | 17140-019 | |
| Calcium Chloride (CaCl2) | Sigma-Aldrich | C7902 | |
| Mitomycin C | Bioaustralis | BIA-M1183 | |
| CHIR99021 | Miltenyi Biotec | 130-104-172 | |
| IWP2 | Miltenyi Biotec | 130-105-335 | |
| SB431542 | Miltenyi Biotec | 130-095-561 | |
| Purmorphamine | Miltenyi Biotec | 130-104-465 | |
| ROCK inhibitor Y-27632 | Miltenyi Biotec | 130-104-169 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E6758 | |
| Poly Vinyl Alcohol (PVA) | Sigma-Aldrich | 363073 | |
| Gelatin | Sigma-Aldrich | G1890 | |
| Trypan Blue | Bio-Rad | 145-0013 | |
| Accumax | Innovative Cell Technologies Inc. | AM105 | |
| Sigmacote | Sigma-Aldrich | SL2 | |
| CELLSPIN | Integra Biosciences | 183 001 | |
| Spinner flask with 1 pendulum, 100 ml | Integra Biosciences | 182 023 | |
| Mouse Embryonic Fibroblasts (MEF) | Prepared in-house (or commercially available) | ||
| Human pluripotent stem cell (hPSC) lines | Prepared in-house (or commercially available) |
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