Article by Nicole Julia & Elizabeth Cordovez
Rethinking Immunosuppression for Stem Cell-derived Heart Patches
Emiko Ito, et al., Shigeru Miyagawa Lab
Heart disease is a leading cause of death globally. Myocardial infarctions, commonly known as heart attacks, are a form of heart disease that occurs when a section of the myocardium (heart muscle tissue) undergoes ischemia (which is a lack of blood flow and oxygen). This causes the muscle cells, known as cardiomyocytes, to undergo cell death, reducing the heart muscle function. Allogeneic (same species) human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches provide a possible option for improving cardiac function in heart attack patients. This can be achieved by attaching a patch of healthy, hiPSC-CMs onto the diseased, non-functioning components of the patient’s heart. Although promising, this approach can be associated with an adverse immune response against the implanted patch. Therefore, patients who receive hiPSC-CM patches are expected to require immunosuppression – a medication that weakens the patient’s immune system. Although this is anticipated to improve the lifetime of the implanted patch, long-term immunosuppressive regimens are associated with increased risk of infection and malignancy. With this in mind, in this paper, a group from Osaka University reassessed the timeline of immunosuppressive regimens required following hiPSC-CM patch transplantation. They aimed to discern whether shorter immunosuppressive regimens can enable hiPSC-CM patches to perform just as well as the traditional, longer-term regimens.
What did these researchers do?
The researchers derived human induced pluripotent stem cells (hiPSCs) from mononuclear cells (e.g. a skin biopsy or cells isolated from a blood sample). Then, they differentiated the hiPSCs into cardiomyocytes and cultured them to form a patch of healthy heart muscle tissue. Ten male Sprague-Dawley rats underwent left anterior descending coronary artery ligation – which is a common model used to cut off a key source of blood to the heart muscle – inducing a heart attack. Two weeks later, hiPSC-CM patches were implanted on the infarcted area (the section where ischemia and impaired function occurred) and the rats were split into three groups to test different immunosuppressive regimens. Group 1 received no immunosuppression, group 2 received two months of immunosuppression and group 3 received four months of immunosuppression. The immunosuppressants used were: tacrolimus (TAC), mycophenolate mofetil (MMF) and prednisolone (PSL). These are all typically used in clinical practice. Subjects were studied over a six-month period to see whether heart function improved with the patch transplant and whether this changed with alteration to the immunosuppressive regimen.
Why is this important?
Allogeneic hiPSC-CM patches are promising as a commercially-available approach to improving cardiac function in patients that have had a heart attack. A primary drawback to hiPSC-CM patch transplantation is the need for subsequent immunosuppression, which has serious side effects including infection and cancer. The short-term immunosuppression regimen explored in this paper may help make hiPSC-CM transplantation more clinically feasible, by reducing the risk of adverse effects associated with long-term immunosuppression.
How did the researchers do this?
As researchers studied the hiPSC-CM patch and the effect of the immunosuppressive regimens, they conducted the following assays:
To confirm that the hiPSCs differentiated into cardiomyocytes, the researchers used flow cytometry to test for cardiac markers. They also used RNA from the differentiated cardiomyocytes to evaluate the expression of stem cell and cardiomyocyte markers. Moreover, the authors carried out immunofluorescent staining to investigate the expression of cardiac-specific structural proteins. Finally, isoproterenol – a drug used to improve the strength and rate of heart muscle contractility – was administered to see if the differentiated cardiomyocytes would behave functionally as primary cardiomyocytes do in the human body.
To assess heart function after hiPSC-CM patch transplantation, the authors visualized the rat hearts using echocardiography. They assessed the movement of the left ventricle, and evaluated the heart’s features during systole (when the heart contracts to push blood throughout the body) and diastole (when the heart relaxes to allow blood to fill the heart). They quantified the strength of contractility, and looked for signs of hypertrophy (overexertion of muscle causing tissue enlargement) as well as fibrosis (thickening of tissue).
To quantify whether hypertrophy was present, Periodic acid Schiff staining was done on sections of the rat heart tissue and the diameter of the cardiomyocytes was measured. Here, increased cardiomyocyte diameter is indicative of hypertrophy.
To evaluate the extent of fibrosis in the ischemic area of the hearts, Masson’s trichrome staining was done to directly stain fibrotic tissue.
To evaluate the potential for tumor formation from the implanted patches, researchers use a hematoxylin and eosin (H&E) stain. This was done to address the risk of tumor formation from any implanted stem cells which did not differentiate into cardiomyocytes.
To observe angiogenesis (formation of new blood vessels) in the infarcted myocardium, the tissue was stained with Von Willebrand factor (vWF) antibody and the capillary densities were quantified.
Lastly, to assess arteriogenesis (development of arteries) in the infarcted myocardium, the tissue was double stained with vWF antibody and α-smooth muscle actin. The arteriole densities were quantified.
Diameter of cardiomyocytes in infarct with no immunosuppresion (Left), short-term immunosuppresion (middle), and long-term immunosuppresion (right)
What comes next?
Ultimately, the researchers found that there was no difference in the performance of hiPSC-CM patches when rats were treated with two months versus four months of immunosuppression. In both groups with immunosuppression, cardiac function following hiPSC-CM transplantation improved. The paper’s results may therefore have important implications for defining an immunosuppressive regimen for hiPSC-CM transplanted patches.
However, to fully explore the effects of immunosuppression, the following limitations will have to be overcome:
Although hiPSC-CM patch transplantation is anticipated to be allogeneic (clinically, human-to-human), in this research they use a xenogeneic model (human-to-rat) to test different immunosuppressive regimens. Using an allogenic model (in this case, rat-to-rat) to test the effect of immunosuppression may be valuable in future studies.
The researchers indicate that long term engraftment of the hiPSC-CM patch does not occur. This suggests that the patch’s effect on cardiac function likely occurs through cellular signals released by the patch, as opposed to long-term integration of hiPSC-CMs into the patient’s heart muscle tissue. In order to fully assess the persistence of patch cells, the researchers can tag the hiPSC-CMs with a fluorescent marker such as green fluorescent protein (GFP) and track the presence of the transplanted hiPSC-CMs over time.
This research brings up important questions. For example: If the hiPSC-CM patch disappears after two months, could this then explain why two months of immunosuppression was just as effective as four in improving cardiac function? How do the direct and secondary (signaling-based) effects of the hiPSC-CM patch differ in their contributions to improving cardiac function, and which method is more effective?
Taken together, this research investigates the appropriate immunosuppressive regimen required for hiPSC-CM patch transplantation – a novel method for possibly treating heart failure. Its results bring up important questions regarding the mechanism underlying hiPSC-CM patch transplantation’s therapeutic effects, and the role that immunosuppression may play in enabling these effects.