lec 12

Question 1

What were the 2 main theories of therapy for the heart post MI?

Answer 1

Therefore 2 potential modes of therapy would be to induce an increased capacity for regeneration and one to replace the injured cells via cell therapy.

Question 2

How were IPSCs used to form CMs at the start?

Answer 2

Starting off by putting them on a feeder layer which acts as the niche
Movement away from the niche causes spontaneous differentiation, however, the differentiation is random.
Only 1% of generated cells would be cardiomyocytes
This method was called embryoid body approach and was the most widely used method until 2009.

Question 3

How is the Mesodermal lineage specified?

Answer 3

Mesodermal lineage is specified by NODAL, NODAL is activated by the inactivation of cerberus which requires activation of VG1 and Wint8c. Once the cells are committed to the mesodermal lineage it will express Brachury which is unique to the mesoderm

Question 4

How is the cardiac fate set?

Answer 4

The lateral endoderm releases BMP 2 and 4 into the mesoderm
The notochord releases Wnt3a and Wnt8c into the mesoderm
Anterior endoderm releases Wnt inhibitors into the mesoderm

BMP+Wnt inhibitor gives rise to cardiac cells
BMP+Wnt gives rise to blood lineage.

In the cardiac stem cells Nkx2.5 and Tbx5 TFs are expressed.

Question 5

What is needed for CM formation?

Answer 5

Two orthogonal gradients one of Wnt activity along the anterior posterior axis and the other of BMP signals along the dorsal ventral axis. Therefore needs high BMP and low Wnt activity.

Question 6

How were CMs formed from IPSCs?

Answer 6

So we seeded IPSCs on a feeder layer
Removed the feeder layer and activated NODAL and added BMP and VEGF
Cardiac Progenitors were produced
Differentiated into Cardiomyocytes

This gives a 60% efficiency.

Question 7

How do we tell these cells are CM?

Answer 7

We can tell it is cardiomyocytes by:

- Gene expression 
- Spontaneous beating 
- Reporter systems (tag specific genes, if it is expressed we will know it is a cardiomyocyte)

Question 8

What can we do with iCMs?

Answer 8

By seeding the cardiomyocytes using fibronectin to keep the orientation we can see the beating of the cardiomyocytes. They seeded in a flower like jellyfish shape so when supplied with a pulse it would contract in a regular beat and move.
This can also be seen in any shape as long as they are seeded in an anisotrophic fashion.

Now we can test drugs by adding them to the cardiomyocytes.
Epinephrine increased the beating
Lidocaine decreased and so on

We can now use these models to study cardiomyopathies
Timothy Syndrome:
A missense SNP in CACNA1C gene 
Calcium channel defect
Effects contractility 
C.1216G>A

Question 9

How do we study Timothy Syndrome?

Answer 9

We can derive IPSC cells from patients with timothy syndrome to use for studies

We can test that they are stem cells by looking at the pluripotency markers (Nanog and Tra-1-81) teratroma formation.

We know that the specific IPSC derived cardiomyocytes have the pathology as electrophysiological recordings and calcium imaging studies showed irregular contractions and excess Ca2+ influx with prolonged action potentials and irregular electrical activity.