Summary of 4 papers

Nkx2.5 Based Ventricular Programming of Murine ESC-Derived

Cardiomyocytes

Reason:
Cardiovascular cell replacement therapies require availability of truly maturated
cardiomyocytic subtypes. There is a need for refined efficient method which can be used for
in vitro cardiogenic differentiation of pluripotent stem cells (PSCs).

Methods used:
A pure αMHC+ cell population with augmented maturation toward early ventricular CMs was
achieved by combining forced exogenous overexpression of the early cardiovascular
transcription factor Nkx2.5 with a αMHC-promoter based antibiotic selection using improved
murine ESC forward programming protocols. To enhance cardiomyogenic differentiation
efficiency, small molecules such as ascorbic acid were also added. Then on the mRNA as
well as protein levels cell fate of the resulting cardiomyocytes were evaluated. Furthermore,
we examined the response of the cells` beating activities to pharmacological substance
administration.

Results:
Analysis of early cardiac markers (MesP1, Isl1) revealed that Nkx2.5 overexpression alone is
not sufficient to induce cardiogenic differentiation.

These experiments revealed that puromycin selection under both oxygen conditions
initiated at day 7 of differentiation, followed by an additional dissociation step on day 11, led
to a ~10-fold (20 % O2) larger population showing α-MHC promoter activity in the Nkx2.5
overexpressing clone derived cells.

Resulting single cells exhibited characteristics of early ventricular cardiomyocytes, such as

sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium
channel occurrence.
In vitro differentiation of rat embryonic stem cells into functional
cardiomyocytes

Reason:
For the study of human diseases, use of rat genetic models has been limited because of the
lack of rat embryonic stem cells (rESCs) due to difficulty of in vitro differentiation. The in
vitro differentiation system from rESCs into derivatives of three germ layers will serve as a
powerful tool and resource for the investigation of mammalian development, cell function,
tissue repair, and drug discovery.

Methods used:
In this study, a system for the efficient formation of EBs from rESCs by harnessing the
application of signal inhibitors and hanging-drop technology was developed.

Modified embryoid body (EB) protocol was developed to achieve in vitro differentiation of
rESCs into derivatives of the three germ layers. To instigate EB formation, for the first 2 days
of differentiation three inhibitors (3i, FGF inhibitor: SU5402, MEK inhibitor: PD184352, and
GSK3 inhibitor: CHIR99021) were used followed by incubating with fetal bovine serum (FBS)-
conditioned medium collected from feeders.

Result:
Results demonstrate that the efficient formation of EBs from rESCs requires a specific
environment in which rESCs can survive and differentiate into three embryonic germ layers.

Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes

after plating.

Analysis of molecular, structural, and functional properties revealed that rESC-derived

cardiomyocytes were similar to those derived from fetal rat hearts and mESCs.
Electrophysiological Characteristics of Embryonic Stem Cell-Derived
Cardiomyocytes are Cell Line-Dependent

Reason:
For studies on cardiac development and cell therapy, a reproducible, stable cardiac
differentiation is needed, with comparable properties of ESC-CMs generated from different
passages of an individual ESC line, or from different ESC lines.

Methods used:
The electrophysiological parameters of ESC-CMs derived from two wild-type and two
transgenic murine ESC lines designated as D3, R1, D3/αPIG44 (derived from D3) and
CGR8/AMPIGX-7 (derived from CGR8) cultured under identical conditions were compared.
The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and
puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain
(αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and
multielectrode arrays in beating clusters of ESC-CMs.

Result:
Direct comparison of electrophysiological properties of different ESC lines, cultured and
differentiated according to the same protocol, demonstrated inherent differences of different
ESC lines

Result of this study is summarized as follows

(i)a substantial cell line-dependent variation of AP frequency, maximal upstroke velocity and
APD,

(ii) a cell line-dependent effect of CM purification by antibiotic selection,

(iii) a substantial cell line-dependent difference in beating regularity, and

(iv) differences in the response to the If-blocker CsCl.

Optimized and Simplified System of Mouse Embryonic Stem Cell
Cardiac Differentiation for the Assessment of Differentiation Modifiers

Reason:
For generating cardiomyocytes from embryonic stem cells, numerous methods have been
published but often are technically challenging, complex, and are not easily adapted to
assessment of specific gene contributions to cardiac myocyte differentiation.

Methods used:
Reliable and efficient method for the differentiation of 2i+LIF cultured mES cells into
cardiomyocytes using lentiviral mediated gain or loss of function initiated at the cardiac
precursor stage was used. It utilizes viral transduction after differentiation as an internal
control for differentiation efficiency. Using this approach, the effects of CHF1/Hey2 on
cardiac myocyte differentiation, using both gain and loss of function was assessed.

Result:
Protocol produces a yield of cardiomyocytes in the intermediate range of efficiency (30–
40%) that allows for detecting either an increase or decrease in cardiomyocyte yield.

This system was found to be efficient in testing the effect of CHF1/Hey2, a bHLH
transcription factor expressed in the developing ventricular myocardium, on the cardiac
differentiation of mES cells.

Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on

cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of
atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the
cardiomyocytes.