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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

Nkx2.5-eGFP expression and CDH2 cardiac specificity is confirmed by flow cytometry and immunofluorescent staining.(a) Immunofluorescent visualization of Nkx2.5-eGFP on heart sections at embryonic day 8.5, 9.5, 10.5 and 11.5. (b) Percentage of eGFP positive (mean ± s.d) cells from mouse embryonic hearts at day 8.5 (n = 3), 9.5 (n = 6) and 11.5 (n = 14). (c) Percentage of cTropT positive cells from the Cdh2+ population of fixed ED11.5 (n = 4) wild type mouse hearts. (d) Immunofluorescent visualization of Cdh2 and cTropT on heart sections of wild type and Nkx2.5-eGFP hearts at embryonic day 11.5. Scale bar; 100 μm. Abbreviations: IN; Inflow, OT; Outflow, V; Ventricle and A; Atria.
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pone.0135880.g001: Nkx2.5-eGFP expression and CDH2 cardiac specificity is confirmed by flow cytometry and immunofluorescent staining.(a) Immunofluorescent visualization of Nkx2.5-eGFP on heart sections at embryonic day 8.5, 9.5, 10.5 and 11.5. (b) Percentage of eGFP positive (mean ± s.d) cells from mouse embryonic hearts at day 8.5 (n = 3), 9.5 (n = 6) and 11.5 (n = 14). (c) Percentage of cTropT positive cells from the Cdh2+ population of fixed ED11.5 (n = 4) wild type mouse hearts. (d) Immunofluorescent visualization of Cdh2 and cTropT on heart sections of wild type and Nkx2.5-eGFP hearts at embryonic day 11.5. Scale bar; 100 μm. Abbreviations: IN; Inflow, OT; Outflow, V; Ventricle and A; Atria.

Mentions: Before the isolation of cells for single cell gene expression analysis, we confirmed the specificity of the markers that are used to isolate cardiomyocytes from developing embryonic hearts. We utilized the Nkx2.5-eGFP mouse [17], where eGFP expression is driven by a cardiac specific element of the Nkx2.5 promoter and will thus only be expressed in the embryonic heart before ED12 [17]. The expression of eGFP was examined by immunofluorescence at embryonic day (ED) 8.5, 9.5, 10.5 and 11.5. From the earliest heart tube, Nkx2.5-eGFP expression was detected throughout cardiac myocytes (Fig 1A). Nkx2.5-eGFP expression diminished after ED9.5, starting in the ventricles (Fig 1A), which is consistent with previous findings [17]. Flow analysis of isolated embryonic hearts confirmed that the eGFP+ percentage was highest at ED9.5 (24.26±11.48%, n = 6) compared to ED8.5 (6.08±0.50%, n = 3) or ED11.5 (5.91±1.42%, n = 14) (Fig 1B). In short, Nkx2.5-eGFP expression specifies cardiomyocytes at the earlier stages of mouse heart development. This was then used as a tool for selection of immature cardiomyocytes for further downstream analysis. Additionally, as we aimed to be less dependent on transgenic mouse models, we confirmed the cardiac expression of CDH2. Nkx2.5-eGFP and wild type ED11.5 hearts were analyzed for CDH2 and cTropT expression using both immunofluorescence and FACS analysis. Our results confirm previous findings [3] as we show that 96.2±0.7% (n = 4) of the wild type CDH2 positive cells express cTropT (Fig 1C and S4 Fig). Furthermore, 90.5±4.3% (n = 3) of the Nkx2.5-eGFP+ cells are positive for both CDH2 and cTropT when analyzed by flow cytometry (S4 Fig). We could also confirm expression of CDH2 in wild type and Nkx2.5-eGFP ED11.5 hearts by immunofluorescence (Fig 1D).


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)

Nkx2.5-eGFP expression and CDH2 cardiac specificity is confirmed by flow cytometry and immunofluorescent staining.(a) Immunofluorescent visualization of Nkx2.5-eGFP on heart sections at embryonic day 8.5, 9.5, 10.5 and 11.5. (b) Percentage of eGFP positive (mean ± s.d) cells from mouse embryonic hearts at day 8.5 (n = 3), 9.5 (n = 6) and 11.5 (n = 14). (c) Percentage of cTropT positive cells from the Cdh2+ population of fixed ED11.5 (n = 4) wild type mouse hearts. (d) Immunofluorescent visualization of Cdh2 and cTropT on heart sections of wild type and Nkx2.5-eGFP hearts at embryonic day 11.5. Scale bar; 100 μm. Abbreviations: IN; Inflow, OT; Outflow, V; Ventricle and A; Atria.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556377&req=5

pone.0135880.g001: Nkx2.5-eGFP expression and CDH2 cardiac specificity is confirmed by flow cytometry and immunofluorescent staining.(a) Immunofluorescent visualization of Nkx2.5-eGFP on heart sections at embryonic day 8.5, 9.5, 10.5 and 11.5. (b) Percentage of eGFP positive (mean ± s.d) cells from mouse embryonic hearts at day 8.5 (n = 3), 9.5 (n = 6) and 11.5 (n = 14). (c) Percentage of cTropT positive cells from the Cdh2+ population of fixed ED11.5 (n = 4) wild type mouse hearts. (d) Immunofluorescent visualization of Cdh2 and cTropT on heart sections of wild type and Nkx2.5-eGFP hearts at embryonic day 11.5. Scale bar; 100 μm. Abbreviations: IN; Inflow, OT; Outflow, V; Ventricle and A; Atria.
Mentions: Before the isolation of cells for single cell gene expression analysis, we confirmed the specificity of the markers that are used to isolate cardiomyocytes from developing embryonic hearts. We utilized the Nkx2.5-eGFP mouse [17], where eGFP expression is driven by a cardiac specific element of the Nkx2.5 promoter and will thus only be expressed in the embryonic heart before ED12 [17]. The expression of eGFP was examined by immunofluorescence at embryonic day (ED) 8.5, 9.5, 10.5 and 11.5. From the earliest heart tube, Nkx2.5-eGFP expression was detected throughout cardiac myocytes (Fig 1A). Nkx2.5-eGFP expression diminished after ED9.5, starting in the ventricles (Fig 1A), which is consistent with previous findings [17]. Flow analysis of isolated embryonic hearts confirmed that the eGFP+ percentage was highest at ED9.5 (24.26±11.48%, n = 6) compared to ED8.5 (6.08±0.50%, n = 3) or ED11.5 (5.91±1.42%, n = 14) (Fig 1B). In short, Nkx2.5-eGFP expression specifies cardiomyocytes at the earlier stages of mouse heart development. This was then used as a tool for selection of immature cardiomyocytes for further downstream analysis. Additionally, as we aimed to be less dependent on transgenic mouse models, we confirmed the cardiac expression of CDH2. Nkx2.5-eGFP and wild type ED11.5 hearts were analyzed for CDH2 and cTropT expression using both immunofluorescence and FACS analysis. Our results confirm previous findings [3] as we show that 96.2±0.7% (n = 4) of the wild type CDH2 positive cells express cTropT (Fig 1C and S4 Fig). Furthermore, 90.5±4.3% (n = 3) of the Nkx2.5-eGFP+ cells are positive for both CDH2 and cTropT when analyzed by flow cytometry (S4 Fig). We could also confirm expression of CDH2 in wild type and Nkx2.5-eGFP ED11.5 hearts by immunofluorescence (Fig 1D).

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