JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes
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PROVIDER: PRJNA970232 | ENA |
REPOSITORIES: ENA
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Project description:The generation and purification of cardiomyocyte sub-population are critical for precise modelling of cardiac diseases, drug screening, and regenerative medicine due to their distinct phenotypic and functional characteristics. Therefore, identifying surface markers for cardiomyocyte subtypes that facilitate live cell sorting of homogenous population of cardiomyocytes is of utmost importance for successful translation of cardiovascular research and therapeutic applications. Using MARIS method coupled with RNA sequencing analysis on MLC2A- and MLC2V-expressing cardiomyocytes, we identified JAK2 as a potential cell surface marker for purifying ventricular cardiomyocytes. We demonstrated that that SIRPA+/JAK2+ population sorted expressed high levels of ventricular-specific marker and had electrophysiological properties that corresponded to ventricular-like cardiomyocytes. Functionally, SIRPA+/JAK2+ cardiomyocytes do not response to vernakalant, an atrial-selective anti-arrhythmic agent, confirming their identity as ventricular-like cells.
2023-12-01 | GSE231913 | GEO
Project description:Adult ventricular cardiomyocytes
| PRJNA556434 | ENA
Project description:BackgroundHuman pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for cardiac disease modelling, drug discovery and regenerative medicine. Despite the advancement in various differentiation protocols, the heterogeneity of the generated population composed of diverse cardiac subtypes poses a significant challenge to their practical applications. Mixed populations of cardiac subtypes can compromise disease modelling and drug discovery, while transplanting them may lead to undesired arrhythmias as they may not integrate and synchronize with the host tissue's contractility. It is therefore crucial to identify cell surface markers that could enable high purity of ventricular CMs for subsequent applications.MethodsBy exploiting the fact that immature CMs expressing myosin light chain 2A (MLC2A) will gradually express myosin light chain 2 V (MLC2V) protein as they mature towards ventricular fate, we isolated signal regulatory protein alpha (SIRPA)-positive CMs expressing intracellular MLC2A or MLC2V using MARIS (method for analysing RNA following intracellular sorting). Subsequently, RNA sequencing analysis was performed to examine the gene expression profile of MLC2A + and MLC2V + sorted CMs. We identified genes that were significantly up-regulated in MLC2V + samples to be potential surface marker candidates for ventricular specification. To validate these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA + CMs that were either positive or negative for the putative surface markers, JAK2 (Janus kinase 2) or CD200. We then characterized the electrophysiological properties of surface marker-sorted CMs, using fluo-4 AM, a green-fluorescent calcium indicator, to measure the cellular calcium transient at the single cell level. For functional validation, we investigated the response of the surface marker-sorted CMs to vernakalant, an atrial-selective anti-arrhythmic agent.ResultsIn this study, while JAK2 and CD200 were identified as potential surface markers for the purification of ventricular-like CMs, the SIRPA+/JAK2+ population showed a higher percentage of MLC2V-expressing cells (~ 90%) compared to SIRPA+/CD200+ population (~ 75%). SIRPA+/JAK2+ sorted CMs exhibited ventricular-like electrophysiological properties, including slower beating rate, slower calcium depolarization and longer calcium repolarization duration. Importantly, vernakalant had limited to no significant effect on the calcium repolarization duration of SIRPA+/JAK2+ population, indicating their enrichment for ventricular-like CMs.ConclusionOur study lays the groundwork for the identification of cardiac subtype surface markers that allow purification of cardiomyocyte sub-populations. Our findings suggest that JAK2 can be employed as a cell surface marker for enrichment of hPSC-derived ventricular-like CMs.
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Project description:We addressed the question of which protein kinases are expressed in cardiomyocytes. We assessed the changes during postnatal development, comparing profiles in rat neonatal ventricular cardiomyocytes (NVMs) with adult ventricular cardiomyocytes (AVMs). Neonatal and adult rat ventricular cardiomyocytes prepared according to established procedures (Marshall et al. PLoS ONE 2010 5(4):e10027; Fuller and Sugden, FEBs Lett. 1989 247:209-12; Rodrigues and Severson In Biochemical Techniques in the Heart (McNeill, J. H., Ed.) pp 101-115, CRC Press, New York.). mRNA expression profiles compared using Affymetrix rat genome 230 2.0 arrays.
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2009-12-10 | E-GEOD-15856 | biostudies-arrayexpress
Project description:Central questions like cardiomyocyte subtype emergence during cardiogenesis or availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, characterization and implementation of pure cardiomyocyte subtypes is still challenging due to technical limitations. Our aim was to identify surface markers enabling the selective detection and purification of atrial and ventricular cardiomyocytes from mouse hearts. In a surface marker screen we found differential expression of CD49f in atrial and ventricular embryonic cardiomyocytes (E13.5). By flow cytometry we could correlate a high CD49f expression with MLC-2a on the single cell level; a low CD49f expression corresponded to MLC-2v. Based on the persisting differential CD49f expression we developed purification protocols for cardiomyocytes subtypes from the developing mouse heart. Flow sorting of E15.5 hearts into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cells led to a selective depletion (CD49flow) or enrichment of MLC-2a+ cells (CD49fhigh). We found a corresponding CD49f-dependent distribution of MLC-2a when pre-enriched neonatal cardiomyocytes (P2) were flow-sorted into CD49flow and CD49fhigh. Atrial and ventricular identity was confirmed by expression profiling and patch clamp analysis of sorted embryonic hearts, which unequivocally demonstrated that the sorted cells were viable and functional. For the first time, we introduce a non-genetic, antibody-based approach to specifically isolate atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This newly gained capability of obtaining highly pure, viable cells will facilitate in-depths characterization of the individual cellular subsets and will aid translational research and therapeutic applications. The dataset comprises four different cardiomyocytes subtypes from the developing mouse heart. Embryonic (E15.5) hearts were dissociated and flow-sorted into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cardiomyocytes. Neonatal (P2) hearts were dissociated, contaminating non-myocytes were removed by MACS depletion, and the purified cardiomyocytes were flow-sorted into CD49flow and CD49fhigh cardiomyocytes. Four biological replicates were available for each sample groups. Microarray analysis was conducted on the Agilent Whole Mouse Genome Oligo Microarray 8x60K platform.
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