Cardiac cell identification and isolation
Författare
Summary, in English
Identification and isolation of cardiomyocytes from the heart, embryonic stem cells, or induced pluripotent stem
cells is a challenging task and will require specific isolation techniques. Less than 30% of the cells within the
murine heart are cardiomyocytes and, what is more, there are multiple sub-populations of cardiomyocytes like
atrial, ventricular and conduction system cardiomyocytes.
In Paper I, we investigated the potential of surface marker isolation of committed cardiomyocytes by using
fluorescence activated cell sorting (FACS). Herein we show, for the first time, that embryonic cardiomyocytes can
be isolated with 98% purity based on their expression of vascular cell adhesion molecule-1 (VCAM-1). Patch
clamp experiments confirmed that the isolated cells are fully functional and are of both atrial and ventricular
subtype.
Classical analysis techniques use population average readouts for the identification of marker expression.
However, these approaches mask cellular heterogeneity and thus single-cell techniques have evolved to combat
this issue. In paper II we use this technique to screen for cardiomyocyte sub-population surface markers. We
found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables the isolation of live and
functional murine trabecular ventricle cells, solid ventricle cells, and atrial cells.
Additionally, the accurate identification of cardiomyocytes and their cell cycle status is an invaluable tool for
cardiac research. In paper III we generate and characterise transgenic mice expressing a fusion protein of human
histone 2B and the red fluorescent protein mCherry under control of the CM specific αMHC promoter. This allows
for the unequivocal identification of cardiomyocytes through their fluorescently labelled nuclei and has enabled
quantification of cardiomyocyte cell fraction in the different parts of the heart. Furthermore, by combining this
transgenic system with the CAG-eGFP-anillin transgene we were able to establish a novel cell-cycle based
screening assay.
Finally, we established a novel use for the commercially available reprograming kit (CytoTune 2.0) in murine cells.
By following the expression of induced human and endogenous mouse pluripotency genes we were able to get a
better insight into the reprogramming process. The generated induced pluripotent stem cells were capable of
differentiating into all three germ layers. Additionally, we investigated the cardiac differentiation potential of
these cells by using single cell technology.
cells is a challenging task and will require specific isolation techniques. Less than 30% of the cells within the
murine heart are cardiomyocytes and, what is more, there are multiple sub-populations of cardiomyocytes like
atrial, ventricular and conduction system cardiomyocytes.
In Paper I, we investigated the potential of surface marker isolation of committed cardiomyocytes by using
fluorescence activated cell sorting (FACS). Herein we show, for the first time, that embryonic cardiomyocytes can
be isolated with 98% purity based on their expression of vascular cell adhesion molecule-1 (VCAM-1). Patch
clamp experiments confirmed that the isolated cells are fully functional and are of both atrial and ventricular
subtype.
Classical analysis techniques use population average readouts for the identification of marker expression.
However, these approaches mask cellular heterogeneity and thus single-cell techniques have evolved to combat
this issue. In paper II we use this technique to screen for cardiomyocyte sub-population surface markers. We
found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables the isolation of live and
functional murine trabecular ventricle cells, solid ventricle cells, and atrial cells.
Additionally, the accurate identification of cardiomyocytes and their cell cycle status is an invaluable tool for
cardiac research. In paper III we generate and characterise transgenic mice expressing a fusion protein of human
histone 2B and the red fluorescent protein mCherry under control of the CM specific αMHC promoter. This allows
for the unequivocal identification of cardiomyocytes through their fluorescently labelled nuclei and has enabled
quantification of cardiomyocyte cell fraction in the different parts of the heart. Furthermore, by combining this
transgenic system with the CAG-eGFP-anillin transgene we were able to establish a novel cell-cycle based
screening assay.
Finally, we established a novel use for the commercially available reprograming kit (CytoTune 2.0) in murine cells.
By following the expression of induced human and endogenous mouse pluripotency genes we were able to get a
better insight into the reprogramming process. The generated induced pluripotent stem cells were capable of
differentiating into all three germ layers. Additionally, we investigated the cardiac differentiation potential of
these cells by using single cell technology.
Avdelning/ar
Publiceringsår
2015
Språk
Engelska
Publikation/Tidskrift/Serie
Lund University Faculty of Medicine Doctoral Dissertation Series
Volym
2015:137
Dokumenttyp
Doktorsavhandling
Förlag
Stem Cell Center, Lund University
Ämne
- Cell and Molecular Biology
Status
Published
Handledare
ISBN/ISSN/Övrigt
- ISSN: 1652-8220
- ISBN: 978-91-7619-217-7
Försvarsdatum
16 december 2015
Försvarstid
09:00
Försvarsplats
Segerfalksalen
Opponent
- Paul Riley (Prof.)