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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

Single cell Fluidigm analysis revealed two Myl2 positive populations with distinct expression profiles.(a) Selected log2 fold-change estimates between Myl2 negative and positive cells at ED11.5 and ED9.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest. (b) Rank values at ED9.5 and ED11.5 for Myl2 expression revealed two different Myl2 populations ED11.5 by Mann-Whitney U test. (c) Prominent differences in median Cq values at ED11.5 for the Myl2High and Myl2Low populations could be identified for Itga6, Myl2, Hey2, Myl7 and Hey1 by with the Mann–Whitney U test where *** P<0.001. (d) Proportion of positive cells ED11.5 between the Myl2High and Myl2Low group showed to be statistically significant for genes Itga6, Itga5, Itga1, Hey2 and Cx45, *** P<0.001. (e) Selected log2 fold-change estimates between Myl2Low and the Myl2High group at ED11.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest.
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pone.0135880.g002: Single cell Fluidigm analysis revealed two Myl2 positive populations with distinct expression profiles.(a) Selected log2 fold-change estimates between Myl2 negative and positive cells at ED11.5 and ED9.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest. (b) Rank values at ED9.5 and ED11.5 for Myl2 expression revealed two different Myl2 populations ED11.5 by Mann-Whitney U test. (c) Prominent differences in median Cq values at ED11.5 for the Myl2High and Myl2Low populations could be identified for Itga6, Myl2, Hey2, Myl7 and Hey1 by with the Mann–Whitney U test where *** P<0.001. (d) Proportion of positive cells ED11.5 between the Myl2High and Myl2Low group showed to be statistically significant for genes Itga6, Itga5, Itga1, Hey2 and Cx45, *** P<0.001. (e) Selected log2 fold-change estimates between Myl2Low and the Myl2High group at ED11.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest.

Mentions: We then stratified cardiomyocytes based on Myl2 expression for subsequent gene expression analysis. Myl2 is a well-established marker for commitment to the ventricular cardiomyocyte lineage, and provides earlier discrimination than Myl7 [4, 5] (which, notably was expressed in all cells at ED11.5). Gene expression analysis of Myl2 positive and negative cells at both ED9.5 and ED11.5 showed the Myl2+ cells had a higher expression of Myl7 (Log2 Fold-change = 6.17, 95% CI [4.7–7.6]), cTropT (Log2 Fold-change = 4.8, 95% CI [3.7–5.9]) and Cdh2 (Log2 Fold-change = 2.3, 95% CI [1.7–2.9]) and as well other numerous genes critical for cardiomyocyte function, including Atp1b1, Cav3.2, Tbx18, Cx40 and Cx45 (Fig 2A). The Myl2- population exhibited a number of gene expression shifts relevant to both lineage (cardiac vs. non-cardiac) and degree of differentiation. These included higher expression of Twist1 (Log2 Fold-change = 7.98, 95% CI [5.4–10.6]) [18], Vim (Log2 Fold-change = 2.4, 95% CI [1.9–2.9]), Pecam (Log2 Fold-change = 11.4, 95% CI [9.5–13.8]), Pdgfra (Log2 Fold-change = 4.4, 95% CI [2.7–7.5]), Pdgfrb (Log2 Fold-change = 4.4, 95% CI [3.0–5.8]), ckit (Log2 Fold-change = 2.2, 95% CI [0.9–3.4]) and Flk1 (Log2 Fold-change = 5.2, 95% CI [3.8–6.6]) (Fig 2A).


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)

Single cell Fluidigm analysis revealed two Myl2 positive populations with distinct expression profiles.(a) Selected log2 fold-change estimates between Myl2 negative and positive cells at ED11.5 and ED9.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest. (b) Rank values at ED9.5 and ED11.5 for Myl2 expression revealed two different Myl2 populations ED11.5 by Mann-Whitney U test. (c) Prominent differences in median Cq values at ED11.5 for the Myl2High and Myl2Low populations could be identified for Itga6, Myl2, Hey2, Myl7 and Hey1 by with the Mann–Whitney U test where *** P<0.001. (d) Proportion of positive cells ED11.5 between the Myl2High and Myl2Low group showed to be statistically significant for genes Itga6, Itga5, Itga1, Hey2 and Cx45, *** P<0.001. (e) Selected log2 fold-change estimates between Myl2Low and the Myl2High group at ED11.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556377&req=5

pone.0135880.g002: Single cell Fluidigm analysis revealed two Myl2 positive populations with distinct expression profiles.(a) Selected log2 fold-change estimates between Myl2 negative and positive cells at ED11.5 and ED9.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest. (b) Rank values at ED9.5 and ED11.5 for Myl2 expression revealed two different Myl2 populations ED11.5 by Mann-Whitney U test. (c) Prominent differences in median Cq values at ED11.5 for the Myl2High and Myl2Low populations could be identified for Itga6, Myl2, Hey2, Myl7 and Hey1 by with the Mann–Whitney U test where *** P<0.001. (d) Proportion of positive cells ED11.5 between the Myl2High and Myl2Low group showed to be statistically significant for genes Itga6, Itga5, Itga1, Hey2 and Cx45, *** P<0.001. (e) Selected log2 fold-change estimates between Myl2Low and the Myl2High group at ED11.5 with accompanying 95% confidence intervals. Comparisons were included in figure if 95% confidence interval did not overlap zero and at least 3 cells in each group expressed the gene of interest.
Mentions: We then stratified cardiomyocytes based on Myl2 expression for subsequent gene expression analysis. Myl2 is a well-established marker for commitment to the ventricular cardiomyocyte lineage, and provides earlier discrimination than Myl7 [4, 5] (which, notably was expressed in all cells at ED11.5). Gene expression analysis of Myl2 positive and negative cells at both ED9.5 and ED11.5 showed the Myl2+ cells had a higher expression of Myl7 (Log2 Fold-change = 6.17, 95% CI [4.7–7.6]), cTropT (Log2 Fold-change = 4.8, 95% CI [3.7–5.9]) and Cdh2 (Log2 Fold-change = 2.3, 95% CI [1.7–2.9]) and as well other numerous genes critical for cardiomyocyte function, including Atp1b1, Cav3.2, Tbx18, Cx40 and Cx45 (Fig 2A). The Myl2- population exhibited a number of gene expression shifts relevant to both lineage (cardiac vs. non-cardiac) and degree of differentiation. These included higher expression of Twist1 (Log2 Fold-change = 7.98, 95% CI [5.4–10.6]) [18], Vim (Log2 Fold-change = 2.4, 95% CI [1.9–2.9]), Pecam (Log2 Fold-change = 11.4, 95% CI [9.5–13.8]), Pdgfra (Log2 Fold-change = 4.4, 95% CI [2.7–7.5]), Pdgfrb (Log2 Fold-change = 4.4, 95% CI [3.0–5.8]), ckit (Log2 Fold-change = 2.2, 95% CI [0.9–3.4]) and Flk1 (Log2 Fold-change = 5.2, 95% CI [3.8–6.6]) (Fig 2A).

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