A new study has shown that it is possible to reverse the scarring that forms after a heart attack, turning it back into healthy heart tissue. The researchers demonstrated that they can change fibroblasts (scar tissue cells) into cardiomyocytes (heart muscle cells), though getting to the bottom of how this happens has been a challenge.
Changing cells into new heart tissue
The new study published in Nature saw the researchers using single-cell RNA sequencing technology combined with mathematical modeling, genetic and chemical approaches to work out how fibroblasts become cardiomyocytes.
The team of scientists, led by Dr. Li Qian from the UNC School of Medicine, successfully reconstructed the routes that a single cell takes during this transformative process while identifying the underlying molecular pathways that are key to these changes taking place. They used cardiac reprogramming as an example in this study, but the methods the team has established could be used for any other reprogramming process.
This means that we could use the technique to change a variety of cells from one type to another, as the situation requires, by guiding them down certain paths. When we are born, embryonic stem cells in our bodies change into various types of specialized cell types, such as blood cells, heart cells, liver cells and neurons through a process called differentiation.
For a long time, it was believed that once these differentiated cells had specialized, there was no turning back. However, recent research shows that it is possible to revert these differentiated cells back to a developmental state where they can once again become other cell types; this state is known as pluripotency. Pluripotent cells can be thought of as “Swiss army knife” cells that can become any cell type.
Researchers have also recently worked out how to change one differentiated cell type into another without reverting the cell back to the pluripotent stage. This means that cells can be changed from one type to another more easily and quickly than previously thought possible. This discovery has been a major breakthrough for regenerative medicine, but working out how these processes work and finding ways to use them for clinical research has been a challenge.
During direct cardiac reprogramming, the conversion of cardiac non-myocytes into induced cardiomyocytes (iCMs) happens in an asynchronous manner; the transformations happen at different intervals during the process. So in a cell population at any given time, you have a mixture of unconverted, partially reprogrammed and fully converted cells. Just like other cell reprogramming processes, the various cells are being reprogrammed at different rates and so do not change at the same time; this means cell populations are heterogeneous. This has made it difficult to use traditional approaches to study the processes and pathways involved.
Using microfluidic single-cell RNA sequencing techniques, the research team addressed the two problems of asynchronous programming and heterogeneous cell populations. The team analyzed changes to global gene expression when the cells were converting from fibroblasts to iCMs. With the aid of mathematical algorithms, the researchers identified molecularly distinct subpopulations of cells in the reprogramming process. They then re-constructed how iCMs form based on simulation and through experimental validation. This allowed the team to create a detailed roadmap showing the mechanisms of cell conversion.
During their study, they found that after a heart attack, cardiac fibroblasts at the injury site become activated immediately and are highly proliferative, although this proliferative capacity decreases over time. Being able to manipulate this following a heart attack could make cellular reprogramming even more useful and help optimize patient outcomes.
Cells have epigenetic memories
The researchers also discovered that in some subpopulations of fibroblasts, some molecular features were differentially suppressed during reprogramming; this suggests that the ease with which different cells can be reprogrammed varies. This coincides with the timing of cardiomyocyte differentiation during heart development. They found that the signatures in intermediate populations of cells that appear earlier in heart development resist reprogramming more so than newer cells.
This suggests that more recent epigenetic memories in cells are easier to erase than older ones; thus, fibroblast subpopulations with such epigenetic features are more malleable and are more easily converted into cardiomyocytes. This means that manipulating epigenetic memories as well as changing their epigenetic status could be important in manipulating a cell to accept cellular reprogramming.
As they analyzed global gene expression changes during the reprogramming process, they noticed an unexpected down-regulation of the factors involved in mRNA processing and splicing. This was a surprise to the researchers; they found that some of the basic cell machinery changed dramatically, protein production, transportation, and degradation processes in the cell changed, as did mRNA splicing.
The team continued by doing a functional analysis of the splicing factor called ptbp1. Research suggests that Ptbp1 is a barrier to fibroblasts acquiring cardiomyocyte-specific splicing patterns. The research showed that the depletion of Ptbp1 promotes the formation of more iCMs.
This means that a single splicing factor regulates the conversion of a fibroblast to a cardiomyocyte. Further analysis showed a strong correlation between the expression of each reprogramming factor and the progress of cells through the reprogramming process and has led to the discovery of new surface markers to help enrich iCMs.
This is an important step in not only helping heart disease patients but also potentially helping patients with diabetes, neurological diseases, cancer and other ailments. The more we learn about reprogramming cells, the more potential there is for reprogramming cells in situ to combat disease as well as better understanding the nature of cells and disease progression.
 Qian, Li et al. (2017) Single-cell transcriptomics reconstructs fate conversion from fibroblast to cardiomyocyte. nature 10.1038/nature24454