Grafted Stem Cell Derivatives Restore Normal Heart Rhythms in Mice

American researchers, in collaboration with technicians from Fujifilm VisualSonics, Inc., have used advanced ultrasonic software to document microscopic, regenerative improvements to heart muscle that has suffered from previous damage.

High-frequency ultrasound and special cardiac-assessment software was developed by FujiFilm VisualSonics, Inc of Toronto, Canada. Scientists from Mayo Clinic implanted engineered cells into the damaged hearts of mice and then used the special software and ultrasound imaging to observe the regeneration of the heart so that it began to contract with normal cardiac rhythms.

After a heart attack, dead heart tissue is replaced with a cardiac scar that consists of scar tissue that neither contracts nor conducts the signals to contract. Depending of the size of the heart scar, the heart can beat abnormally. An abnormal heart beat is known as arrhythmia. Arrhthymias come in three different categories: a heart that beats too fast (tachycardia), a heart that beats too slowly (bradycardia), and a heart that beats erratically. Arrhythmias after a heart attack can be life-threatening, and restoring normal heart rhythm to the heart after a heart attack is very important.

In this experiment, mice were given heart attacks, and then undifferentiated induced pluripotent stem cells (iPSCs) were implanted into these hearts. Those mice that received induced pluripotent stem cells gradually normalized, their heart beat. The resynchronization of the heart beat of these mice was imaged with high-resolution ultrasound.

Satsuki Yamada, first author of this paper, said, “A high-resolution ultrasound revealed harmonized pumping [of the heart] where iPS cells were introduced to be the previously damaged heart tissue.” Yamada also noted that Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, and achieves resynchronization of the heart beat after a heart attack.

This experiment shows, for the first time that undifferentiated iPSCs have the potential to stabilize a patient’s heart after a heart attack. The healing of the heart was documented by ultrasound imaging and by “speckle-tracking echocardiogram.,” Speckle-tracking echocardiography was designed by VevoStrain Advanced Cardiac Analysis Software, which was manufactured by VisualSonics.

This software package provides advanced imaging and quantification capabilities for studying sensitive movements in heart muscles and it is also the only commercial cardiac-strain package optimized for assessing cardiovascular function preclinical rodent studies.

Yamada and her co-researchers utilized this software during the implantation and observation of the iPSCs within the hearts of mice. This software package the motion of the heart wall both at the regional and global levels and from several different perspectives, measurements of these movements, the changes in dimension in the left ventricle during the heart cycle.

The software definitely showed that homogeneous wall movement was restored in those mice that had received implants of iPSCs.

When iPSCs were implanted into mice that had dysfunctional immune systems, they produced tumors, but in mice with normal immune systems, the implanted iPSCs did not produce tumors. What became of those cells is uncertain, but they clearly helped heal the heart and did not cause tumors.

Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.
Immunocompetent status defines cell growth outcome  Immunocompetent infarcted hearts were free from uncontrolled growth following iPS cell implantation as documented in vivo (echocardiography; A and B) and on autopsy (A and C) during the 60-week-long follow-up, in contrast to teratoma formation observed in immunodeficient hosts. In A: M, mass; LV, left ventricle; S, suture for coronary ligation. In B, data represent means ± SEM (n = 8 immunocompetent hearts: n = 7 immunodeficient hosts); *P < 0.05 versus immunocompetent.

This paper is interesting and suggests that undifferentiated cells can also exert healing effects on the heart.

Overexpression of a Potassium Channel in Heart Muscle Cells Made From Embryonic Stem Cells Decreases Their Arrhythmia Risk

Embryonic stem cells have the capacity to differentiate into every cell in the adult body. One cell type into which embryonic stem cells (ESCs) can be differentiated rather efficiently is cardiomyocytes, which is a fancy term for heart muscle cells. The protocol for making heart muscle cells from ESCs is well worked out, and the conversion is rather efficient and the purification schemes that have been developed are also rather effective (for example, see Cao N, et al., Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res. 2013 Sep;23(9):1119-32. doi: 10.1038/cr.2013.102 and Mummery CL et al., Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res. 2012 Jul 20;111(3):344-58).

Using these cells in a clinical setting has two large challenges. The first is that embryonic stem cell derivatives are rejected by the immune system of the recipient, thus setting up the patient for a graft versus host response to the implanted tissue, thus making the patient even sicker than when they started. The second problem is that heart muscle cells made from ESCs are immature and cause the heart to beat abnormally fast thus causing “tachyarrythmias” and died within the first two weeks after the transplant (see Liao SY, et al., Heart Rhythm 2010 7:1852-1859).

Both of these problems are large problems, but the laboratory of Ronald Li at the University of Hong Kong at used a genetic engineering trick to make heart muscle cells from mouse embryonic stem cells to seemingly fix this problem.

Li and his colleagues engineered mouse ESCs with a gene for a potassium rectifier channel that could be induced with drugs. Then they differentiated these genetically ESCs into heart muscle cells. This potassium rectifier channel (Kir2.1) is not present in immature heart muscle cells and putting it into these cells might cause them to beat at a slower rate.

These engineered ESC-derived heart muscle cells were tested for their electrophysiological properties first. Without the drug that induces KIR2.1, the heart muscle cells showed very abnormal electrical properties. However, once the drug was added, their electrical properties looked much more normal.

Then they induced heart attacks in laboratory animals and implanted their engineered ESC-derived heart muscle cells 1 hour after the heart attacks were induced. Animals not given the drug to induce the expression of Kir2.1 faired very poorly and had episodes of tachyarrythmia (really fast heart beat) and over half of them died by 5 weeks after the implantation. Essentially the implanted animals did worse than those animals that had had a heart attack that were not treated. However, those animals that were given the drug that induces the expression of Kir2.1 in heart muscle cells did much better. The survival rate of these animals was higher than the untreated animals after about 7 weeks after the procedure. Survival rates increased by only a little, but the increase was significant. Also, the animals that died did not die of tachyarrythmias. In fact the rate of tachyarrythmias in the animals given the inducing drug (which was doxycycline by the way) had significantly lower levels of tachyarrythmia than the other two groups.

Other heart functions were also significantly affected. The ejection fraction in the animals that ha received the Kir2.1-expression heart muscle cells was 10-20% higher than the control animals. Also the density of blood vessels was substantially higher in both sets of animals treated with ESC-derived heart muscle cells. The echocardiogram of the hearts implanted with the Kir2.1-expressing heart muscle cells was altogether more normal than that of the others.

This paper is a significant contribution to the use of ESC-derived cells to treat heart patients. The induction of heart arrhythmias by ESC-derived heart muscle cells is a documented risk of their use. Li and his colleagues have effectively eliminated that risk in this paper by forcing the expression of a potassium rectifier channel in the ESC-derived heart muscle cells. Also, because these cells were completely differentiated and did not have any interloping pluripotent cells in their culture, tumor formation was not observed.

There are a few caveats I would like to point out. First of all, the increase in survival rate above the control is not that impressive. The improvement in heart function parameters is certainly encouraging, but because the survival rates are not that higher than the control mice that received no treatment, it appears that these benefits were only conferred to those mice who survived in the first place.

Secondly, even though the heart attacks were induced in the ventricles of the heart, Li and his colleagues injected a mixture of heart muscle cells that included atrial, ventricular, nodal and heart fibroblasts. This provides an opportunity for beat mismatches and a “substrate for ventricular tachycardia” as Li puts it. In the future, the transplantation of just ventricular heart muscle cells would be cleaner experiment. Since these mice were not observed long enough to observe potential arrythmias that might have arisen from the presence of a mixed population in the ventricle.

Finally, in adapting this to humans might be difficult, since the hearts of mice beat so much faster than those of humans. It is possible that even if human cardiomyocytes were engineered with Kir2.1-type channels, that arrythmias might still be a potential problem.

Despite all that, Li’s publication is a large step forward.