Transformation of Non-Beating Human Cells into Heart Muscle Cells Lays Foundation for Regenerating Damaged Hearts

After a heart attack, the cells within the damaged part of the heart stop beating and become ensconced in scar tissue. Not only does this region not beat, it does not conduct the signal to beat either and that can not only lead to a slow, sluggish heartbeat, it can also cause irregular heart rates or arrhythmias.

Now, however, scientists have demonstrated that this damage to the heart muscle need not be permanent. Instead there is a way to transform those cells that form the human scar tissue into cells that closely resemble beating heart cells.

Last year, researchers from the laboratory of Deepak Srivastava, MD, the director of Cardiovascular and Stem Cell Research at the Gladstone Institute, transformed scar-forming heart cells (fibroblasts) into beating heart-muscle cells in live mice. Now they report doing the same to human cells in a culture dishes.

“Fibroblasts make up about 50 percent of all cells in the heart and therefore represent a vast pool of cells that could one day be harnessed and reprogrammed to create new muscle,” said Dr. Srivastava, who is also a professor at the University of California, San Francisco. “Our findings here serve as a proof of concept that human fibroblasts can be reprogrammed successfully into beating heart cells.”

In 2012, Srivastava and his team reported that fibroblasts could be reprogrammed into beating heart cells by injecting just three genes (collectively known as GMT, which is short for Gata4, Mef2c, and Tbx5), into the hearts of live mice that had been damaged by a heart attack (Qian L, et al., Nature. 2012 31;485(7400):593-8). From this work, they reasonably concluded that the same three genes could have the same effect on human cells.

“When we injected GMT into each of the three types of human fibroblasts (fetal heart cells, embryonic stem cells and neonatal skin cells) nothing happened—they never transformed—so we went back to the drawing board to look for additional genes that would help initiate the transformation,” said Gladstone staff scientist Ji-dong Fu, Ph.D., the study’s lead author. “We narrowed our search to just 16 potential genes, which we then screened alongside GMT, in the hopes that we could find the right combination.”

The research team began by injecting all candidate genes into the human fibroblasts. They then systematically removed each one to see which were necessary for reprogramming and which were dispensable. In the end, they found that injecting a cocktail of five genes—the 3-gene GMT mix plus the genes ESRRG and MESP1—were sufficient to reprogram the fibroblasts into heart-like cells. They then found that with the addition of two more genes, called MYOCD and ZFPM2, the transformation was even more complete.

To help things along, the team used a growth factor known as Transforming Growth Factor-Beta (TGF-Beta) to induce a signaling pathway during the early stages of reprogramming that further improved reprogramming success rates.

“While almost all the cells in our study exhibited at least a partial transformation, about 20 percent of them were capable of transmitting electrical signals—a key feature of beating heart cells,” said Dr. Fu. “Clearly, there are some yet-to-be-determined barriers preventing a more complete transformation for many of the cells. For example, success rates might be improved by transforming the fibroblasts within living hearts rather than in a dish—something we also observed during our initial experiments in mice.”

The immediate next steps are to test the five-gene cocktail in hearts of larger mammals. Eventually, the team hopes that a combination of small, drug-like molecules could be developed to replace the cocktail, which would offer a safer and easier method of delivery.

This latest study was published online August 22 in Stem Cell Reports.

Reprogramming Skin Cells into Neural Stem Cells By Introducing One Gene

Transforming skin cells into nerve cells that interconnect and send nerve impulses to each other requires an extensive amount of reprogramming. The production of induced pluripotent stem cells is rather labor-intensive and introduces some risks. However, a new procedure designed by Yadong Huang at the Gladstone Institutes has shown that the introduction of a single gene into skin cell can generate nerve cells from skin cells.

This single gene, Sox2, transforms skin cells within days into early-stage brain stem cells known as induced neural stem cells or iNSCs. In culture, iNSCs self-renew and mature into neurons that can connect with each other and then transmit electro-chemical signals between each other. When the iNSCs were cultured for one month, they had already formed a completely new neural network.

An excited Huang made these points: “Many drug candidates, especially those developed for neurodegenerative diseases, fail in clinical trials because current models don’t accurately predict the drug’s effects on the human brain. Human neurons derived from reengineered skin cells could help assess the efficacy and safety of these drugs, thereby reducing risks and resources associated with human trials.”

Huang’s findings build on the work of Japanese research Shinya Yamanaka, who was the first scientist to publish the production of induced pluripotent stem cells. Since that time, other researchers have used genetic engineering techniques to directly reprogram adult cells into other types of adult cells without passing through the embryonic-stem-cell stage. Last year, Sheng Ding managed to use a combination of small molecules and genes to transform skin cells directly into neural stem cells. Huang’s technique now simplifies this technique even more so that only one gene is required to reprogram skin cells into neural stem cells. By avoiding the induced pluripotent stem cell stage, Huang and Ding hope to avoid the risk of tumor formation and the mutations induced by the production of induced pluripotent stem cells.

Karen Ring, a graduate student in Biomedical Sciences at the University of California, San Francisco, who was the lead author on this paper vouched for the safety of the iNSCs: “We wanted to see whether these newly generated neurons could result in tumor growth after transplanting them into mouse brains. Instead, we saw the reprogrammed cells integrate into the mouse’s brain, and not a single tumor developed.”

Huang’s paper also addresses the function Sox2 in the reprogramming of the skin cells. Huang and his research team also want to identify similar regulators that direct the development of specific types of neurons in the brain that tend to degenerate in the case of particular types of neurodegenerative diseases. Huang noted: “If we can pinpoint which genes control the development of each neuron type, we can generate them in the Petri dish from a single sample of human skin cells. We could then test drugs that affect different neuron types, such as those involved in Parkinson’s disease.” Huang added that such a discovery would help drug developers design treatments for neurodegenerative diseases that are much more specific, and the drug design would probably occur much faster.

Alzheimer’s disease still afflicts 5.4 million people in the US alone and this number is thought to triple by 2050. There are still no medications that can reverse the devastation wrought by this disease. Huang’s data might provide the means to test such new drugs.

Neurons from Skin Cells

Can we make nerve cells from skin cells? The answer seems to be yes. Furthermore, it seems to be really easy to do.

Marius Wernig at the Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology at Stanford University School of Medicine had published a remarkable paper. They started from a pool of nineteen candidate genes, but they identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that rapidly and efficiently convert mouse embryonic and adult mouse fibroblasts into functional neurons in vitro. He called these cells induced neuronal (iN) cells. He further showed that they expressed multiple neuron-specific proteins, and were able to generate nerve impulses and form functional connections between other nerve cells (synapses). Because they could make iN cells from non-nerve cells, they might be able to make large quantities studies of neurons for research.

Of even greater importance is the ability to make nerve cells for regenerative medicine. While it is too early to get too excited about this, the ability to form neurons from your own skin cells to repair spinal cord injuries and other neurological disorders without killing embryos is thrilling to say the least.

A recipe for heart cells from amnion

Embryonic stem cells can be made from adult cells. Such cells are called iPSCs or induced embryonic stem cells, and they have all the characteristics of embryonic stem cells made by means of the destruction of embryos.

Lately, scientists have found a way to convert one type of adult cell into another type of adult without going through any embryonic step.

Qi Zhou and his colleagues from the Melton lab at Harvard were able to transform pancreatic enzyme-secreting cells (exocrine cells) into insulin-secreting cells by inserting three transcription factors (Ngn3, also known as Neurog3), Pdx1 and Mafa into the exocrine cells and they reprogrammed themselves into beta-cells (Nature 455, 627–32 (2008)). Also, Yechoor and his colleagues used a similar technique that placed neurogenin into liver cells in a live animal. These animals shows insulin-secreting cells into their livers, which showed that the liver cells had been reprogrammed into beta cells (V. Yechoor et al., Dev. Cell 16, 358–73 (2009)).

This shows that reprogramming is vastly superior and cheaper than making cloned embryos that we subsequently kill and use to make embryonic stem cells. This is the therapeutic way of the future.

Now, Jun Takeuchi and Benoit Bruneau at the Gladstone Institute of Cardiovascular Disease in San Francisco have found that adding cardiac-specific genes to developing mouse embryos can make even some extra-embryonic parts become beating heart cells.  They made the cells from amnion, the thin layer that surrounds the embryo and fetus throughout development.  This is the sac that breaks when we say that a mother’s water breaks.  The amnion is normally medical waste, but can now be used to make heart cells.

See this link for the paper.