Dr. NewmanMSNT Practicum Report Louisiana Tech University

Identification of Cardiac Genes in Embryoid Bodies

Chris Miller

Introduction:

Cardiac cells are a unique type of cell found in the body with vital importance. They

cannot divide once fully differentiated making them much more significant than other cells.

Once damage is sustained by the heart, the cells can no longer function properly in the damaged

area. This leaves the heart vulnerable to further attacks or problems. New cells cannot go through

cell division in the damaged area due to tight methylation patterns found at cell cycle genes of

cardiac cells (1). These cardiac cells, or cardiomyocytes, are vitally important to understand, so

future medical treatments for heart associated tissue damage can one day be achieved.

Stem cells can potentially provide the answer to people suffering from heart issues such

as infarctions, stroke, and birth defects. There are many types of stem cells though including

embryonic, mesenchymal, and tissue specific adult stem cells. Embryonic stem cells (ESCs) are

cells obtained from the blastocyst very early after the zygote has formed (2). ESCs have shown

the ability to differentiate into many cell types including that of cardiomyocytes. Placed in

suspension, ESCs collectively form Embryoid Bodies (EBs). This mass of ESCs can then form

cardiomyocytes in a spontaneous manner (3).

ESCs are extremely sensitive to the environment they reside in though. Working with

stem cells in culture creates an issue as the environment inside and outside the body are very

different. Understanding the growth of stem cells outside the body is crucial for future

therapeutic treatments. As long as the right environmental conditions are met along with other

cell specific transcription factors, ESCs allow researchers to generate a particular cell of interest.

The stem cells ability to differentiate into cardiomyocytes is a huge discovery for people

suffering from heart damage and disease. This makes them crucial for future tissue engineering

and therapeutic medical procedures; specifically in the area of developing treatment options for

cardiac tissue damage (4).

Methods:

RNA Extraction

ESCs were obtained from a line previously cultured by Dr. Newman’s lab where TRIzol

(Catalog #15596-026) was added to the cells, the cells were collected, and then stored at -80⁰C

until processed. The samples examined were from embryoid bodies collected at Day 5 and Day 8

post differentiation. Each sample was incubated in TRIzol at room temperature for five minutes

in an Eppendorf tube. Next, .1 mL of chloroform was added to the samples. Each sample was

shaken vigorously for 15 seconds, followed by 2 minutes of incubation at room temperature.

Samples were centrifuged at 12,000 rpms for 15 minutes at 4⁰C. Three layers were formed

leaving the aqueous solution on top with the desired RNA. The aqueous solution was pipetted

out and placed in a new tube labeled RNA extraction. The remaining layers were discarded.

Next, the RNA was isolated. There was .5 μL of RNase free glycogen added to the RNA

extract as a carrier to the aqueous phase. The samples were then washed with 0.5 mL of 100%

isopropanol solution. The solution was allowed to incubate for 10 minutes at room temperature.

Samples were centrifuged at 12,000 rpm for 10 minutes at 4⁰C. The isolated RNA formed a

cloudy-white pellet. All remaining liquid in the tube was discarded. The pellet was washed with

0.5 mL of 75% ethanol followed by a brief vortex. Samples were placed in a centrifuge at 7500

rpms for five minutes at 4⁰C. A small pellet formed where the remaining solution was again

discarded. The sample was allowed to air dry once all liquid was removed. 15 μL of RNase free

water was added allowing the pellet to be resuspended. A cytation 5 image reader was used to

analyze the concentration of the two RNA samples from EB day #5 and EB day #8. Samples

were then stored at -70⁰C overnight.

Synthesis of cDNA

The cDNA can now be formed from the RNA extraction. The values of the

concentrations given by the Cytation 5 image reader were averaged together for both day 5 and

day 8. An average of 155.207 ng/μL for EB day 5 and 176.529 ng/μL for EB day 8 were

collected. From these values, 0.5 μg of RNA was used to synthesize cDNA for each sample. EB

day 5 contained: 4 μL qScript cDNA super mix (Catalog #95048-025), 3.2 μL RNA, and 12.8 μL

water. EB day 8 contained: 4 μL qScript cDNA super mix, 2.8 μL RNA, and 13.2 μL water.

Duplicates were also made for comparison for a total of four PCR tubes. Samples were then

placed in the thermocycler for 45 minutes. The samples first went 5 minutes at 25⁰C, followed

by 30 minutes at 42⁰C, then five minutes at 85⁰C, and finally held at 4 C for the remaining ⁰5 minutes. The samples were placed in the fridge overnight.

Reverse-Transcriptase PCR Amplification of cDNA and Gel Electrophoresis

Six different primers were used in the PCR amplification of the cDNA which included:

gapdh, pou5f1, nanog, gata4, α-mhc, cxn40. Each primer was used for both day 5 and day 8 for a

total of 12 PCR tubes. 12.5 μL GoTaq master mix, 2 μL each 10 μM primer pair (1 μL forward

primer and 1 μL reverse primer), 11.5 μL RNase free water, and 1 μL cDNA were added to each

tube. Samples were then placed in a thermocycler for 2 hours allowing the genes to be amplified

with its associated primer. Denaturing took place at 95⁰C, annealing at 60⁰C, and extension at

70⁰C.

The samples were amplified and now ready for gel electrophoresis. A 1.5% agarose gel

with 5 μL of ethidium bromide was used for the electrophoresis. A ladder was pipetted in column

1 to identify band size. The following 12 columns were filled with each PCR sample. The gel

was allowed to run for 45 minutes at 120 volts initially confirming the presence of bands. It was

then allowed to run again for another hour at 80 volts to further see the ladder develop down the

gel.

Real Time RT-PCR

Three different genes were used for the Real-Time PCR. Gapdh, cxn40, and nanog made up the

three genes to be analyzed. Six Eppendorf tubes was obtained to make a master mix for each

primer and each day. 20 μL PowerUp SYBR green mix (A25742), 4 μL cDNA, 4 μL F/R

primers, and 4 μL RNase free water were pipetted in each of the 6 Eppendorf tubes. 10 μL was

aliquoted into 3 PCR tubes for each different primer. This was done for all 6 master mix set ups

for a total of 18 PCR tubes each with 10 μL. The tubes were spun down and placed in the RT-

PCR machine for 45 minutes. Stage 1 experienced an initial denaturation for 20 seconds at 95⁰C

for one cycle. Stage 2 consisted of 35 cycles at 95⁰C for three seconds followed by 30 seconds at

60⁰C. The results were then analyzed.

Results:

Cardiac genes were analyzed from cells obtained from EBs at five days and at eight days

post differentiation. Confirming the presence of genes found only in the heart is a crucial step in

confirming the presence of cardiomyocytes. After the cells were acquired, the extraction of the

RNA then took place from each cell culture. Once the RNA had been extracted and purified,

cDNA of the mRNA was synthesized using a thermocycler and super mix set up. Specific

cardiac primers were then used to test for the presence of cardiac genes α-mhc, gata4, and cxn40

as well as other non-cardiomyocyte genes known as pou5f1 and nanog. Pou5f1 and nanog are

highly expressed in only ESCs and were used to demonstrate differentiation of the embryoid

bodies. The final primer to be tested was gapdh. This is present in nearly every cell in the body

as this is a housekeeping gene for the cell and a loading control for the experiments.

Real-Time PCR was also run looking at each samples ability to amplify over time. This

allowed for the gene to be amplified while displaying a growth curve in real time. Real-Time

PCR is much more efficient, sensitive, and quantitative. A gel does not need to be run in order to

confirm the presence of amplification taking place of a particular gene.

The RNA extraction is the first step in identifying the selected genes. After the RNA was

extracted, the cytation 5 image reader showed strong purity levels for each sample as a number

as close to 2 is ideal in the column labeled 260/280. The reader also gave the concentration of the

samples in ng/µL. These two values were then averaged for each day. This helped to give us the

value for the amount of RNA to add for the synthesis of the cDNA.

Cytation 5 Image Reader Results

Name 260/280 ng/µL Mean

EB Day 5 #1 1.948 157.876 155.207

EB Day 5 #2  1.959 152.537  

EB Day 8 #1 1.899 177.21 176.529

 EB Day 8 #2 1.88 175.848  

Figure 1. Values from the Cytation 5 image reader and the averages of the concentration

readings from each sample.

After the cDNA was formed, the cDNA was amplified using the six different primers in a

RT-PCR reaction. A gel was then run to check the presence of the cardiac genes. Strong visible

bands were shown for α-mhc at the correct amount of base pairs as well as cxn40 and gata4.

However, cxn40 produced a second band unlike α-mhc or gata4. This was an unexpected result,

but it still did produce a band in the desired area for cxn40. Gapdh should also be expressed

highly due to its expression exhibited in all cells of the body. It was highly expressed in both day

5 and 8 samples. Nanog and pou5f1 showed faint bands unlike the others. This was also expected

as both are only highly expressed in ESCs rather than cardiac cells. The band on day 5 for nanog

was quite pronounced though compared to that of day 8 indicating more primitive characteristics

for the EB day 5. The table from figure 2 helps to show that each band is in the corresponding

area for its particular gene. This indicates good quality of the cDNA as well as the presence of

cardiac genes.

Gene Product size

(bp)

gapdh 216

α-mhc 704

oct4/pou5f1 568

cxn40 180

nanog 613

gata4 126

Figure 2. Results of PCR and

the 6 primers used for EB day #5 and EB day #8. The corresponding table refers to the

actual length of each gene.

Real-time PCR was used to test genes gapdh, nanog, and cxn40. Gapdh was found with a

normal curve consistent with high expression of the gene. Nanog was quite the opposite

indicating a very sporadic curve that was found undetermined in most of the samples it was

involved in. Cxn40 also experienced this as well, but produced a much better curve compared to

that of nanog. Cxn40 did produce an unexpected double band recorded on the agarose gel

previously described though. This was likely interfering with the Real Time-PCR as well. The

last column addresses each sample’s ability to normalize to gapdh. Since we know gapdh is a

common housekeeping gene found in all cells, we can use this value to compare the

amplification levels in our samples with what we would expect. We can then look at that value

for expression levels in the samples. However, the real time PCR didn’t allow for proper

identification due to many undetermined readings.

Sample Name Target Name Cт Cт Mean Cт SD

Normalize to

GAPDH

EB day 5 GAPDH 33.5036 33.21571 0.375545 1

EB day 5 GAPDH 33.35262 33.21571 0.375545

EB day 5 GAPDH 32.79092 33.21571 0.375545

EB day 8 GAPDH 33.68551 33.75679 0.164089 1

EB day 8 GAPDH 33.94447 33.75679 0.164089

EB day 8 GAPDH 33.6404 33.75679 0.164089

EB day 5 Nanog Undetermined 37.01889 0.897264

EB day 5 Nanog 37.01889 37.01889

EB day 5 Nanog Undetermined 37.01889

EB day 8 Nanog Undetermined

EB day 8 Nanog Undetermined

EB day 8 Nanog Undetermined

EB day 5 CXN40 35.16364 36.11965 1.351998 0.919602

EB day 5 CXN40 37.07566 36.11965 1.351998

EB day 5 CXN40 Undetermined 36.11965 1.351998

EB day 8 CXN40 Undetermined 32.20139 1.048302

EB day 8 CXN40 32.20139 32.20139

EB day 8 CXN40 Undetermined 32.20139

Figure 3. Values recorded from the Real-Time PCR performed using the primers cxn40, nanog,

and gapdh. The values were then normalized to gapdh.

Discussion:

The cardiac genes looked at of cxn40, gata4, and α-mhc were found to be expressed in

embryoid bodies from both day 5 and 8. The RNA extraction produced a strong purity level

indicating the presence of only RNA from the embryoid bodies. The cDNA was then able to be

created from the results seen from the image reader. The cDNA was tested for the presence of

cardiac, housekeeping, and ESC genes. The gel was run with many positive results for the

presence of cardiac genes. All three cardiac genes were strongly expressed in both day 5 and day

8 indicating the presence of cardiomyocyte genes. Cxn40 did produce an unexpected double

band for both days though. This may be due to damage taken by some of the DNA or the primers

inability to effectively anneal to the target site. This would cause the DNA to tangle and not

properly extend producing a larger than anticipated band. Human error is unlikely as bands were

still produced in the intended area for cxn40 as well as the other five genes looked at.

Nanog also produced something unexpected as high levels were expressed in day 5.

Nanog and pou5f1 should have produced very faint bands due to their expression being high in

only ESCs. Although expression was higher, we might expect such an event in day 5 versus day

8. The day 5 embryoid bodies may not have differentiated as efficiently yet compared to that of

day 8. This process is spontaneous and many of the cells may still possess more characteristics of

ESCs over cardiomyocytes in the first five days post differentiation. This would allow for such

genes as nanog to be overly expressed.

Real-Time PCR was also looked at to help determine the presence of cardiac genes. The

PCR did not work as intended though. Pou5f1, gapdh, and cxn40 were looked at with mixed

results. While gapdh produced a curve we would expect, cxn40 and pou5f1 produced curves that

were unintended. Another cardiac gene besides cxn40 should have been used due to the double

band produced from the previous ran gel. This likely created the undetermined readings for

cxn40 as we would expect to see a similar curve to that of gapdh. Pou5f1 also produced a curve

that was unlike gapdh, but this would be expected as expression should be quite low in these

particular cells for pou5f1. The Real-Time PCR of pou5f1 should not normalize to gapdh more

so than cxn40. This would indicate ESCs over cardiomyocytes from the RNA collected.

However, the Real-Time PCR did not give us data that can accurately show the presence of

cardiac genes due to the curve and values observed.

Conclusion:

Generating cardiomyocytes from ESCs is a process that needs to be further researched as

no one process allows for an efficient method to date. ESCs are pluripotent and can produce any

cell type in the body such as a cardiomyocyte. This provides huge potential for tissue

engineering. Generating a method to efficiently produce cardiomyocytes in vitro can lead to a

better understanding of the mechanisms for in vivo use. The way cells act in the body are much

different than how they act outside the body. The environment plays a huge role in the

differentiation of these cells.

Techniques to test for your cells are just as important so you know exactly what you are

working with. No one can see a cell from the naked eye and its current molecular processes, so

visualizing your results can sometimes be difficult. RNA extraction methods are useful in

identifying what kind of cell you have exactly. The RNA can then be used to make cDNA for

amplification by PCR. Six different primers were used to help identify genes associated with the

samples from day 5 and day 8. Cardiac genes were highly expressed as expected from the EBs.

Ultimately, the goal of stem cell research is to produce a product to be used within the

human body. The need for new innovative therapies for heart disease remains at a high due to the

ongoing prevalence of increasing heart disease seen across the world. More research needs to be

done in order to grasp the full knowledge of ESCs development in vitro and in vivo. As we move

forward there is hope and promise for such new therapies for people with heart disease and for

future stem cell research. Unlocking the answers can have a monumental impact on the future of

the human race.

References

1. Alberts B, Johnson A, Walter P, et al. Molecular Biology of the cell. 5th edition. 2008. Cell cycle: 1053-1114.

2. Huch M, Koo B. Modeling mouse and human development using organoid cultures. Development for Advances in developmental biology and stem cells. 2015. Available at: http://dev.biologists.org/content/142/18/3113

3. Kehat I, Karsenti D, Gepstein A, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest. 2001;108(3):407–414. doi:10.1172/JCI12131

4. FU J, Jjang P, Li R, et al. Na+/Ca2+ Exchanger is a Determinant of Excitation–Contraction Coupling in Human Embryonic Stem Cell–Derived Ventricular Cardiomyocytes. Stem Cells Dev. 2010. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19719399