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Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

Tarnawski L, Xian X, Monnerat G, Macaulay IC, Malan D, Borgman A, Wu SM, Fleischmann BK, Jovinge S - PLoS ONE (2015)

Bottom Line: In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes.Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance.By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.

ABSTRACT
In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

No MeSH data available.


Related in: MedlinePlus

Subtype characterization and hormonal modulation of isolated cardiomyocytes support the phenotypes suggested by genetic profiling.(a) Representative action potentials (APs) of typical atrial and ventricular-like cells. (b) The ITGA6+ITGA1-ITGA5- sorted population reveals a prominent enrichment of atrial-like cells (n = 12), whereas both the (c) ITGA6-ITGA1+ITGA5+ (n = 18) and (d) ITGA6brightITGA1+ITGA5+ (n = 10) sorted populations reveal ventricular-like cardiomyocytes. (e) Analysis of key AP parameters shows clear differences between the atrial- and ventricular-like cardiomyocytes. (f) Representative voltage ramp recordings from an atrial and a ventricular-like cardiomyocyte. Note the functional expression of inward and outward current components, namely voltage dependent K+–(1), Na+–(2) and L-type Ca2+ (3)–currents. (g) Representative AP traces of ventricular-like cardiomyocytes and their response to perfusion with ACh (10 μM) (left traces) or Isoprenaline (1 μM). Note the negative and positive chronotropic response, respectively, and its reversal upon wash-out of the agonists. (h) Statistical analysis of the negative and positive chronotropic effects expressed as % of frequency variation in presence of the respective agonist compared to normal solution. Abbreviations: (APD90) action potential duration at 90% of repolarization; (Max dV/dt) Maximum rate of rise of the action potential; (NS) normal solution, (ISO) Isoprenaline, (ACh) Acetylcholine. The results are expressed as mean ± s.e.m. *** P <0.001 significant difference between the atrial and ventricular-like cells.
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pone.0135880.g005: Subtype characterization and hormonal modulation of isolated cardiomyocytes support the phenotypes suggested by genetic profiling.(a) Representative action potentials (APs) of typical atrial and ventricular-like cells. (b) The ITGA6+ITGA1-ITGA5- sorted population reveals a prominent enrichment of atrial-like cells (n = 12), whereas both the (c) ITGA6-ITGA1+ITGA5+ (n = 18) and (d) ITGA6brightITGA1+ITGA5+ (n = 10) sorted populations reveal ventricular-like cardiomyocytes. (e) Analysis of key AP parameters shows clear differences between the atrial- and ventricular-like cardiomyocytes. (f) Representative voltage ramp recordings from an atrial and a ventricular-like cardiomyocyte. Note the functional expression of inward and outward current components, namely voltage dependent K+–(1), Na+–(2) and L-type Ca2+ (3)–currents. (g) Representative AP traces of ventricular-like cardiomyocytes and their response to perfusion with ACh (10 μM) (left traces) or Isoprenaline (1 μM). Note the negative and positive chronotropic response, respectively, and its reversal upon wash-out of the agonists. (h) Statistical analysis of the negative and positive chronotropic effects expressed as % of frequency variation in presence of the respective agonist compared to normal solution. Abbreviations: (APD90) action potential duration at 90% of repolarization; (Max dV/dt) Maximum rate of rise of the action potential; (NS) normal solution, (ISO) Isoprenaline, (ACh) Acetylcholine. The results are expressed as mean ± s.e.m. *** P <0.001 significant difference between the atrial and ventricular-like cells.

Mentions: In order to prove cellular integrity and physiological function of the FACS-sorted cardiomyocytes and to characterize their cellular subtypes, patch clamp experiments were performed. A vast majority of the ITGA6+ITGA1-ITGA5- cardiomyocytes (83.3%, n = 12) displayed action potential (AP) shape and short AP duration typical for atrial-like cells (Fig 5B), only two cells did not. In contrast, the ITGA6-ITGA1+ITGA5+ and ITGA6brightITGA1+ITGA5+ populations revealed a prominent enrichment (82.30%, n = 18 cells and 70%, n = 10, respectively) for ventricular-like cells showing typical AP shape and longer AP duration (Fig 5C and 5D). Within each of these, only three cells did not display a typical ventricular shape. Interestingly, two cells within the ITGA6brightITGA1+ITGA5+ population displayed pacemaker-like phenotypes (Fig 5D).


Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

Tarnawski L, Xian X, Monnerat G, Macaulay IC, Malan D, Borgman A, Wu SM, Fleischmann BK, Jovinge S - PLoS ONE (2015)

Subtype characterization and hormonal modulation of isolated cardiomyocytes support the phenotypes suggested by genetic profiling.(a) Representative action potentials (APs) of typical atrial and ventricular-like cells. (b) The ITGA6+ITGA1-ITGA5- sorted population reveals a prominent enrichment of atrial-like cells (n = 12), whereas both the (c) ITGA6-ITGA1+ITGA5+ (n = 18) and (d) ITGA6brightITGA1+ITGA5+ (n = 10) sorted populations reveal ventricular-like cardiomyocytes. (e) Analysis of key AP parameters shows clear differences between the atrial- and ventricular-like cardiomyocytes. (f) Representative voltage ramp recordings from an atrial and a ventricular-like cardiomyocyte. Note the functional expression of inward and outward current components, namely voltage dependent K+–(1), Na+–(2) and L-type Ca2+ (3)–currents. (g) Representative AP traces of ventricular-like cardiomyocytes and their response to perfusion with ACh (10 μM) (left traces) or Isoprenaline (1 μM). Note the negative and positive chronotropic response, respectively, and its reversal upon wash-out of the agonists. (h) Statistical analysis of the negative and positive chronotropic effects expressed as % of frequency variation in presence of the respective agonist compared to normal solution. Abbreviations: (APD90) action potential duration at 90% of repolarization; (Max dV/dt) Maximum rate of rise of the action potential; (NS) normal solution, (ISO) Isoprenaline, (ACh) Acetylcholine. The results are expressed as mean ± s.e.m. *** P <0.001 significant difference between the atrial and ventricular-like cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556377&req=5

pone.0135880.g005: Subtype characterization and hormonal modulation of isolated cardiomyocytes support the phenotypes suggested by genetic profiling.(a) Representative action potentials (APs) of typical atrial and ventricular-like cells. (b) The ITGA6+ITGA1-ITGA5- sorted population reveals a prominent enrichment of atrial-like cells (n = 12), whereas both the (c) ITGA6-ITGA1+ITGA5+ (n = 18) and (d) ITGA6brightITGA1+ITGA5+ (n = 10) sorted populations reveal ventricular-like cardiomyocytes. (e) Analysis of key AP parameters shows clear differences between the atrial- and ventricular-like cardiomyocytes. (f) Representative voltage ramp recordings from an atrial and a ventricular-like cardiomyocyte. Note the functional expression of inward and outward current components, namely voltage dependent K+–(1), Na+–(2) and L-type Ca2+ (3)–currents. (g) Representative AP traces of ventricular-like cardiomyocytes and their response to perfusion with ACh (10 μM) (left traces) or Isoprenaline (1 μM). Note the negative and positive chronotropic response, respectively, and its reversal upon wash-out of the agonists. (h) Statistical analysis of the negative and positive chronotropic effects expressed as % of frequency variation in presence of the respective agonist compared to normal solution. Abbreviations: (APD90) action potential duration at 90% of repolarization; (Max dV/dt) Maximum rate of rise of the action potential; (NS) normal solution, (ISO) Isoprenaline, (ACh) Acetylcholine. The results are expressed as mean ± s.e.m. *** P <0.001 significant difference between the atrial and ventricular-like cells.
Mentions: In order to prove cellular integrity and physiological function of the FACS-sorted cardiomyocytes and to characterize their cellular subtypes, patch clamp experiments were performed. A vast majority of the ITGA6+ITGA1-ITGA5- cardiomyocytes (83.3%, n = 12) displayed action potential (AP) shape and short AP duration typical for atrial-like cells (Fig 5B), only two cells did not. In contrast, the ITGA6-ITGA1+ITGA5+ and ITGA6brightITGA1+ITGA5+ populations revealed a prominent enrichment (82.30%, n = 18 cells and 70%, n = 10, respectively) for ventricular-like cells showing typical AP shape and longer AP duration (Fig 5C and 5D). Within each of these, only three cells did not display a typical ventricular shape. Interestingly, two cells within the ITGA6brightITGA1+ITGA5+ population displayed pacemaker-like phenotypes (Fig 5D).

Bottom Line: In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes.Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance.By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes.

View Article: PubMed Central - PubMed

Affiliation: Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden.

ABSTRACT
In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

No MeSH data available.


Related in: MedlinePlus