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From Embryo to Beating Heart
Directed Differentiation of hESCs into Mesoderm
Assignment 1: Answer Key
Assignment 1: Planning an experimental procedure to direct differentiation of human ES
cells into cardiomyocytes.
Please fill out the worksheet on your own (TOTAL = 50 points).
Student Name: _________________________________________
1) The following diagram represents the differentiation of ES cells into cardiomyocytes in
culture. Think about the following questions and fill out Table 1 (review the lectures, primers
and additional reading materials to fill in Table 1 and answer the questions) (10 points).



Which experimental procedures or conditions do you use to generate each cell type
during the differentiation depicted in this diagram?
When do these cell types appear in the dish during the directed differentiation protocol?
What do you expect the frequency for each cell type to be at the end of your protocol?
TABLE 1
Human
ES cell
What conditions do you Human ES cell
use to promote each cell proliferation
type in the dish?
media with
KOSR and FGF2
When do these cell types These cells are
appear in the dish?
present at
beginning of the
experiment
1
Embryoid Body
Cardiomyocyte
Differentiation
media with 20%
FBS
Differentiation
media with 20%
FBS
These cells are
prepared around
Day 4-5 of culture
These cells begin
to appear as
beating
cardiomyocytes by
Day 11
From Embryo to Beating Heart
Directed Differentiation of hESCs into Mesoderm
Assignment 1: Answer Key
What is the frequency for
each cell type that you
expect at the end of your
experiment?
0%. All Human
ES cells have
differentiated at
he end of the
experiment
0%. Multipotent
EBs should not be
present at the end
of the experiment.
However
differentiating
EBs should be
present at variable
frequencies.
Variable. Between
1% - 25 % of EBs
beat by Day 8-11
in culture
depending on
various scientific
reports.
2) a) What possible experimental procedures can you use to differentiate human ES cells into
cardiomyocytes? (Please list at least two different procedures) (5 points)
Two conditions can be used to direct human ES cells into beating
cardiomyocytes:
1) DMEM with Glutamax and 20 % Fetal Bovine Serum (FBS)
2) DMEM with Glutamax with 20% Fetal Bovine Serum plus Activin and
BMP-4 at a concentration of 12.5-25 ng/ml in the first 4 days of
differentiation.
b) What are some advantages and disadvantages for each procedure? (5 points)
Experimental procedure #1 (name) :_DMEM with 20 % FBS (serum)
1.
2.
3.
4.
Advantages
Disadvantages
Easy methodology to follow in
the laboratory
Relatively reliable
methodology for directed
differentiation
Undirected differentiation
Low frequency of beating
cardiomyocytes at the end of the
experiment (1-25%)
Low frequency of sinoatrial cells
(heart pacemaker cells)
The quality of the serum or the
human ES cell line will
determine the efficiency of
directed differentiation
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From Embryo to Beating Heart
Directed Differentiation of hESCs into Mesoderm
Assignment 1: Answer Key
Experimental procedure #2: DMEM with 20 % FBS (serum) plus Activin and
BMP-4 at a concentration of 12.5-25 ng/ml in the first 4 days of
differentiation.
Advantages
1.
2.
3.
Disadvantages
Increase the frequency of
More expensive
beating cardiomyocytes (2535% of EBs)
Reduce the number of blood Still somewhat undirected
precursors with this method
differentiation due to the
presence of serum in the
differentiation media
The quality of the serum, BMP-4
and human ES cell line will
determine the success
(frequency) of beating heart
cells.
4.
3) a) Briefly describe which method you would use for your directed differentiation experiment
and why? (5 points)
The easiest method for directing the differentiation of human ES cells into
beating cardiomyocytes is to prepare EBs from the human ES cells after 4-5
days in culture and grow EBs in differentiation media with DMEM with
Glutamax and 20% FBS for 7-10 days (total days in culture will be (11-14
days) until you observe beating cardiomyocytes. This is the cheapest and
easiest method to differentiate ES cells into the heart muscle cells.
b) What reagents and concentrations will you use in your media, to promote differentiation of
human ES cells into cardiomyocytes with your method of choice? (5 points)
Volume = 250 mL
1.
2.
3.
Reagents
Concentrations
DMEM with Glutamax
Fetal Bovine Serum
Penecillin/Streptomicin
79 % (197.5 mL)
20 % (50 mL)
1% (2.5 mL)
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From Embryo to Beating Heart
Directed Differentiation of hESCs into Mesoderm
Assignment 1: Answer Key
4.
5.
6.
4) What fraction of EBs do you expect to become beating heart cells at the end of the
differentiation procedure? Why? Justify your answer based on your readings and research
(10 points).
The fraction of beating EBs that have acquired the cardiomyocyte fate can be
very variable depending on the scientific report. Some studies have reported
that only 1% of EBs become beating cardiomyocytes in the presence of 20%
serum (Laflamme et al., 2007. Nature Biotechnology 25 (9): 1015-1024.
However, other studies have reported that approximately 25 % of EBs beat by
Day 8-11 in culture and 70% by Day 15 in the presence of 20 % FBS (Xu et
al., 2002. Circulation Research).
5) Why do you think cardiomyocytes beat at different rates in the dish? Justify your answer
based on your readings or research (10 points)
The frequency of beating cardiomyocytes in the dish depends on the
presence of a subset of heart cells called the node cells. These cells express a
variety of proteins that are important for beating properties such as:
 Cardiac troponin T: Troponin is a complex of three regulatory proteins
that is integral to muscle contraction in skeletal and cardiac muscle, but
not smooth muscle. Troponin is attached to the protein tropomyosin and
lies within the groove between actin filaments in muscle tissue.
 HCN4 - The HCN4 gene encodes the pore-forming subunit of a
hyperpolarization-activated, cyclic nucleotide-modulated cation channel.
HCN4 channels are the predominant HCN isoform in the sinoatrial node
and contribute to pacemaker current that controls rhythmic activity in the
heart and brain.
 Cav3.2: is an important voltage-dependent Ca++ channel that regulates the
influx of Ca++ inside the cell that stimulates muscle contractions.
The presence of these cells in the dish will determine the rate of beating heart
cells.
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