Patient-Specific Heart Muscle Cells Before the Baby Is Born

Prenatal ultrasound scans can detect congenital heart defects (CHDs) before birth. Some 1% of all children born per year have some kind of CHD. Most of these children will require some kind of rather invasive, albeit life-saving surgery but an estimated 25% of these children will die before their first birthday. This underscores the need for netter therapies of children with CHDs.

To that end, Shaun Kunisaka from C.S. Mott Children’s Hospital in Ann Arbor, Michigan and his colleagues have used induced pluripotent stem cell (iPSC) technology to make patient-specific heart muscle cells in culture from the baby’s amniotic fluid cells. Because these cells can be generated in less than 16 weeks, and because the amniotic fluid can be harvested at about 20-weeks gestation, this procedure can potentially provide large quantities of heart muscle cells before the baby is born.

In this paper, which was published in Stem Cells Translational Medicine, Kunisaki and others collected 8-10 milliliter samples of amniotic fluid at 20 weeks gestation from two pregnant women who provided written consent for their amniocentesis procedures. The amniotic fluid cells from these small samples were expanded in culture, and between passages 3 and 5, cells were selected for mesenchymal stem cell properties. These amniotic fluid mesenchymal stem cells were then infected with genetically engineered non-integrating Sendai viruses that caused transient expression of the Oct4, Sox2, Klf4, and c-Myc genes in these cells. The transient expression of these four genes drove the cells to dedifferentiate into iPSCs that were then grown and then differentiated into heart muscle cells, using well-worked out protocols that have become rather standard in the field.

Not only were the amniotic fluid mesenchymal stem cells very well reprogrammed into iPSCs, but these iPSCs also could be reliably differentiated into cardiomyocytes (heart muscle cells, that is) that had no detectable signs of the transgenes that were used to reprogram them, and, also, had normal karyotypes. Karyotypes are spreads of a cell’s chromosomes, and the chromosome spreads of these reprogrammed cells were normal.

As to what kinds of heart muscle cells were made, these cells showed the usual types of calcium cycling common to heart muscle cells. These cells also beat faster when they were stimulated with epinephrine-like molecules (isoproterenol in this case). Interestingly, the heart muscle cells were a mixed population of ventricular cells that form the large, lower chambers of the heart, atrial cells, that form the small, upper chambers of the heart, and pacemaker cells that spontaneously form their own signals to beat.

This paper demonstrated that second-trimester human amniotic fluid cells can be reliably reprogrammed into iPSCs that can be reliably differentiated into heart muscle cells that are free of reprogramming factors. This approach does have the potential to produce patient-specific, therapeutic-grade heart muscle cells for treatment before the child is even born.

Some caveats do exist. The use of the Sendai virus means that cells have to be passaged several times to rid them of the viral DNA sequences. Also, to make these clinical-grade cells, all animal produces in their production must be removed. Tremendous advances have been made in this regard to date, but those advancements would have to be applied to this procedure in order to make cells under Good Manufacturing Practices (GMP) standards that are required for clinical-grade materials. Finally, neither of these mothers had children who were diagnosed with a CHD. Deriving heart muscle cells from children diagnosed with a CHD and showing that such cells had the ability to improve the function of the heart of such children is the true test of whether or not this procedure might work in the clinic.

Heart Muscle in Young Children May Be Capable of Regeneration

The heart of young children might possess untapped potential for regeneration, according to new research. For decades, scientists believed that after a child’s first few days of life, cardiac muscle cells did not divide. Heart growth was thought to occur by means of enlargement of muscle cells.

This view, however, has been seriously challenged in the last few years. New findings in mice that have recently been published in the journal Cell seriously question this dogma. These results have serious implications for the treatment of congenital heart disorders in humans.

Researchers at Emory University School of Medicine have discovered that in 15-day old mice, cardiac muscle cells undergo a precisely timed spurt of cell division that lasts about a day. The total number of cardiac muscle cells in the heart increases by about 40% during this time when the child’s body is growing rapidly. To give you some perspective, a 15-day-old mouse is roughly comparable to a child in kindergarten, and puberty occurs at day 30-35 in mice.

This burst of cell division is driven by a surge of thyroid hormone. This suggests that thyroid hormone might be able to aid in the treatment of children with congenital heart defects. Small trials have even tested thyroid hormone in children with congenital heart defects.

These findings also have broader hints for researchers who are developing regenerative heart therapies. Activating the regenerative potential of the muscle cells themselves is a strategy that is an alternative to focusing on the heart’s stem cells, according to senior author Ahsan Husain, PhD, professor of medicine (cardiology) at Emory University School of Medicine.

“It’s not as dramatic as in fish or amphibians, but we can show that in young mice, the entire heart is capable of regeneration, not just the stem cells,” he says.

This Emory group collaborated with Robert Graham, MD, executive director of the Victor Change Cardiac Research Institute in Australia.

One test conducted by these groups was to determine how well 15-day old mice can recover from the blockage of a coronary artery. Consistent with previous research, newborn (day 2) mice showed a high level of repair after such an injury, but at day 21, endogenous heart repair was quite poor. The 15-day old mice recovered better than the day 21 mice, indicating that some repair is still possible at day 15.

This discovery was an almost accidental finding while Naqvi and Husain were investigating the role of the c-kit gene. The c-kit gene is an important marker for stem cells in cardiac muscle growth. Adult mice that lack a functional c-kit gene in the heart have more cardiac muscle cells. When do these differences appear?

“We started counting the cardiomyocyte cell numbers from birth until puberty,” Naqvi says. “It was a fascinating thing, to see the numbers increasing so sharply on one day.” According to Naqvi, c-kit-deficient and wild-type mice both have a spurt of proliferation early in life but the differences in cardiac muscle cells between the c-kit+ and c-kit- mice appear later.

“Probably, previous investigators did not see this burst of growth because they were not looking for it,” Husain says. “It occurs during a very limited time period.” Even if in humans, the proliferation of cardiac muscle cells does not take place in such a tight time period as it does in mice, the finding is still relevant for human medicine, he says. “Cardiomyocyte proliferation is happening long after the immediate postnatal period,” Husain says. “And cells that were once thought incapable of dividing are the ones doing it.”

Naqvi and Husain plan to continue to investigate the relationships between thyroid hormone, nutrition during early life, and cardiac muscle growth.

Reference: N. Naqvi et al. A Proliferative Burst during Preadolescence Establishes the Final Cardiomyocyte Number. Cell 157, 795-807, 2014.