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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

FACS and qPCR analysis reveals that integrin expression can separate atrial and ventricular populations.Flow cytometry plots of wild type mouse heart cells labelled with antibodies to CDH2, FLK1, ITGA5, ITGA6 and ITGA1 (a-e) ED11.5 (n = 8) and (f-j) ED9.5 (n = 4). (a and f) ITGA5 negative and positive cells were isolated and from these (b, g and c, h) CDH2 positive cells were selected for. (d/i) ITGA5-CDH2+ and (e/j) ITGA5+CDH2+ cells were interrogated based on ITGA6 and ITGA1 expression. Doublets, non-viable cells and FLK1 negative cells were gated away and the compensation and gates set based on single stains and FMOs. Populations ITGA6+ITGA1-ITGA5-, ITGA6BrightITGA1+ITGA5-, ITGA6brightITGA1+ITGA5+ and ITGA6-ITGA1+ITGA5+ isolated by FACS were analyzed for (k) Myl2, Hey2, Myl7, Hey1, Mest, Nppa, Tbx3, Hcn4, Tbx5, and Sema3c expression at ED11.5 (n = 3) and for (l) Myl2, Hey2, Myl7, Hey1 and Mest expression at ED9.5 (n = 3). QPCR data depicted as mean relative expression ± s.e.m and p <0.05 was considered statistically significant. (m) Expression of multiple cardiac and non-cardiac genes as detected by Fluidigm analysis in a random selection of integrin population cDNA from wild type ED11.5 and ED9.5 and Nkx2.5-eGFP ED11.5 murine hearts. Delta Cq values for each well were computed relative to Gapdh, and wells negative for Gapdh were excluded from further analysis. For visual purposes, Delta Cq values for wells with no expression detected (Cq = 40) were set to the minimum Delta Cq value (white).
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pone.0135880.g004: FACS and qPCR analysis reveals that integrin expression can separate atrial and ventricular populations.Flow cytometry plots of wild type mouse heart cells labelled with antibodies to CDH2, FLK1, ITGA5, ITGA6 and ITGA1 (a-e) ED11.5 (n = 8) and (f-j) ED9.5 (n = 4). (a and f) ITGA5 negative and positive cells were isolated and from these (b, g and c, h) CDH2 positive cells were selected for. (d/i) ITGA5-CDH2+ and (e/j) ITGA5+CDH2+ cells were interrogated based on ITGA6 and ITGA1 expression. Doublets, non-viable cells and FLK1 negative cells were gated away and the compensation and gates set based on single stains and FMOs. Populations ITGA6+ITGA1-ITGA5-, ITGA6BrightITGA1+ITGA5-, ITGA6brightITGA1+ITGA5+ and ITGA6-ITGA1+ITGA5+ isolated by FACS were analyzed for (k) Myl2, Hey2, Myl7, Hey1, Mest, Nppa, Tbx3, Hcn4, Tbx5, and Sema3c expression at ED11.5 (n = 3) and for (l) Myl2, Hey2, Myl7, Hey1 and Mest expression at ED9.5 (n = 3). QPCR data depicted as mean relative expression ± s.e.m and p <0.05 was considered statistically significant. (m) Expression of multiple cardiac and non-cardiac genes as detected by Fluidigm analysis in a random selection of integrin population cDNA from wild type ED11.5 and ED9.5 and Nkx2.5-eGFP ED11.5 murine hearts. Delta Cq values for each well were computed relative to Gapdh, and wells negative for Gapdh were excluded from further analysis. For visual purposes, Delta Cq values for wells with no expression detected (Cq = 40) were set to the minimum Delta Cq value (white).

Mentions: To confirm these findings on the protein level, ITGA5 and ITGA6 expression was explored in tissue sections (Fig 3 and S3 Fig). MYL2 and Nkx2.5-eGFP expression was used as ventricular and myocardial markers, respectively. At ED11.5, ITGA6 expression was observed in the inflow, primitive ventricular trabecular and all atrial cells (Fig 3A–3C). Conversely, ITGA5 was only expressed in the outflow, compact layer and trabecular ventricular cardiomyocytes ED11.5, leaving the inflow/atria negative (Fig 3D–3F). Additionally, these subpopulations of cells were isolated by FACS and qPCR was used to confirm cardiac identity of the sorted cells. Specifically, we isolated ITGA6+ITGA1-ITGA5- and ITGA6brightITGA1+ITGA5- cells from wild type mice at ED9.5 and ED11.5 with an average purity of 94.9±2.2% (n = 5) (Fig 4A–4J and S4 Fig). These populations had a significantly higher expression of atrial genes Myl7 and Hey1 as well as of the conduction genes Tbx3 and Hcn4 at ED11.5. In addition, they demonstrated lower levels of ventricular markers Myl2 and Hey2 and outflow/pulmonary [19] myocardial marker Sema3c (Fig 4K). Similarly, at ED9.5, these cells had a significantly high expression of Hey1 while expressing low levels of both Myl2 and Hey2 (Fig 4L). When comparing the ITGA6+ITGA1-ITGA5- and ITGA6brightITGA1+ITGA5- populations at ED11.5, ITGA6brightITGA1+ITGA5- cells showed a significantly higher expression of Hey2 and trabecular marker Nppa [20].


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)

FACS and qPCR analysis reveals that integrin expression can separate atrial and ventricular populations.Flow cytometry plots of wild type mouse heart cells labelled with antibodies to CDH2, FLK1, ITGA5, ITGA6 and ITGA1 (a-e) ED11.5 (n = 8) and (f-j) ED9.5 (n = 4). (a and f) ITGA5 negative and positive cells were isolated and from these (b, g and c, h) CDH2 positive cells were selected for. (d/i) ITGA5-CDH2+ and (e/j) ITGA5+CDH2+ cells were interrogated based on ITGA6 and ITGA1 expression. Doublets, non-viable cells and FLK1 negative cells were gated away and the compensation and gates set based on single stains and FMOs. Populations ITGA6+ITGA1-ITGA5-, ITGA6BrightITGA1+ITGA5-, ITGA6brightITGA1+ITGA5+ and ITGA6-ITGA1+ITGA5+ isolated by FACS were analyzed for (k) Myl2, Hey2, Myl7, Hey1, Mest, Nppa, Tbx3, Hcn4, Tbx5, and Sema3c expression at ED11.5 (n = 3) and for (l) Myl2, Hey2, Myl7, Hey1 and Mest expression at ED9.5 (n = 3). QPCR data depicted as mean relative expression ± s.e.m and p <0.05 was considered statistically significant. (m) Expression of multiple cardiac and non-cardiac genes as detected by Fluidigm analysis in a random selection of integrin population cDNA from wild type ED11.5 and ED9.5 and Nkx2.5-eGFP ED11.5 murine hearts. Delta Cq values for each well were computed relative to Gapdh, and wells negative for Gapdh were excluded from further analysis. For visual purposes, Delta Cq values for wells with no expression detected (Cq = 40) were set to the minimum Delta Cq value (white).
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Related In: Results  -  Collection

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pone.0135880.g004: FACS and qPCR analysis reveals that integrin expression can separate atrial and ventricular populations.Flow cytometry plots of wild type mouse heart cells labelled with antibodies to CDH2, FLK1, ITGA5, ITGA6 and ITGA1 (a-e) ED11.5 (n = 8) and (f-j) ED9.5 (n = 4). (a and f) ITGA5 negative and positive cells were isolated and from these (b, g and c, h) CDH2 positive cells were selected for. (d/i) ITGA5-CDH2+ and (e/j) ITGA5+CDH2+ cells were interrogated based on ITGA6 and ITGA1 expression. Doublets, non-viable cells and FLK1 negative cells were gated away and the compensation and gates set based on single stains and FMOs. Populations ITGA6+ITGA1-ITGA5-, ITGA6BrightITGA1+ITGA5-, ITGA6brightITGA1+ITGA5+ and ITGA6-ITGA1+ITGA5+ isolated by FACS were analyzed for (k) Myl2, Hey2, Myl7, Hey1, Mest, Nppa, Tbx3, Hcn4, Tbx5, and Sema3c expression at ED11.5 (n = 3) and for (l) Myl2, Hey2, Myl7, Hey1 and Mest expression at ED9.5 (n = 3). QPCR data depicted as mean relative expression ± s.e.m and p <0.05 was considered statistically significant. (m) Expression of multiple cardiac and non-cardiac genes as detected by Fluidigm analysis in a random selection of integrin population cDNA from wild type ED11.5 and ED9.5 and Nkx2.5-eGFP ED11.5 murine hearts. Delta Cq values for each well were computed relative to Gapdh, and wells negative for Gapdh were excluded from further analysis. For visual purposes, Delta Cq values for wells with no expression detected (Cq = 40) were set to the minimum Delta Cq value (white).
Mentions: To confirm these findings on the protein level, ITGA5 and ITGA6 expression was explored in tissue sections (Fig 3 and S3 Fig). MYL2 and Nkx2.5-eGFP expression was used as ventricular and myocardial markers, respectively. At ED11.5, ITGA6 expression was observed in the inflow, primitive ventricular trabecular and all atrial cells (Fig 3A–3C). Conversely, ITGA5 was only expressed in the outflow, compact layer and trabecular ventricular cardiomyocytes ED11.5, leaving the inflow/atria negative (Fig 3D–3F). Additionally, these subpopulations of cells were isolated by FACS and qPCR was used to confirm cardiac identity of the sorted cells. Specifically, we isolated ITGA6+ITGA1-ITGA5- and ITGA6brightITGA1+ITGA5- cells from wild type mice at ED9.5 and ED11.5 with an average purity of 94.9±2.2% (n = 5) (Fig 4A–4J and S4 Fig). These populations had a significantly higher expression of atrial genes Myl7 and Hey1 as well as of the conduction genes Tbx3 and Hcn4 at ED11.5. In addition, they demonstrated lower levels of ventricular markers Myl2 and Hey2 and outflow/pulmonary [19] myocardial marker Sema3c (Fig 4K). Similarly, at ED9.5, these cells had a significantly high expression of Hey1 while expressing low levels of both Myl2 and Hey2 (Fig 4L). When comparing the ITGA6+ITGA1-ITGA5- and ITGA6brightITGA1+ITGA5- populations at ED11.5, ITGA6brightITGA1+ITGA5- cells showed a significantly higher expression of Hey2 and trabecular marker Nppa [20].

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