The Speed of the Cell Cycle Makes Aging Cells Young Again

When Shinya Yamanaka and his colleagues at the RIKEN Institute discovered a way to reprogram adult cells into embryonic stem cell-like cells, known as induced pluripotent stem cells (iPSCs), they overthrew a core understanding of cell and developmental biology; namely that once cells become committed to a particular cell fate, they irreversibly remain committed to that cell fate.

Most of the work on iPSCs has examined how to increase the efficiency and safety of this reprogramming procedure. The slowness and inefficiency of this process has frustrated stem cell scientists for some time. Even though some progress has been made at increasing the efficiency of the reprogramming process, the “nuts and bolts” of why this procedure is so slow has remained unclear.

However a recent paper from the laboratory of Shangqin Guo at the Yale School of Medicine has revealed a key component of why this procedure is so slow. That component is the speed of the cell cycle or the length of time the cell takes to divide.

Fast-growing cells have lower barriers to keeping the cell committed to a particular cell fate. Thus faster-growing cells are more easily coaxed into being reprogrammed into pluripotency (the ability to differentiate into all adult cell types).

Guo’s research team examined blood cell-forming stem cells in bone marrow. Normally these stem cells are multipotent, which means that they can differentiate into a limited number of adult cell types. The particular type of blood cells that the progeny of these stem cells differentiate into depends on the particular types of growth factors available to the cells.

Guo and others found that these fast growing bone marrow stem cells could be reprogrammed in as little as four cell divisions.  Ultrafast cell cycle is a key feature of these “privileged cells” that can be reprogrammed to efficiently.  Slower-growing stem cells could not be reprogrammed nearly as fast. Thus the length of the cell cycle seemed to be the key to the speed with which cells could be reprogrammed to iPSCs.

This study also has implications for several other applications, besides making individualized iPSCs for patients. Several human diseases are associated with abnormalities in the establishment of proper cell fates and abnormalities in the cell cycle. Therefore, Guo’s paper could provide insights into why certain genetic diseases affect cells the way they do.

An Even Better Way to Make Induced Pluripotent Stem Cells

Researchers from the Centre for Genomic Regulation in Barcelona, Spain, have discovered an even faster and more efficient way to reprogram adult cells to make induced pluripotent stem cells (iPSCs).

This new discovery decreases the time it takes to derived iPSCs from adult cells from a few weeks to a few days. It also elucidated new things about the reprogramming process for iPSCs and their potential for regenerative medical applications.

iPSCs behave similarly to embryonic stem cells, but they can be created from terminally differentiated adult cells. The problem with the earlier protocols for the derivation of iPSCs is that only a very small percentage of cells were successfully reprogrammed (0.1%-2%). Also this reprogramming process takes weeks and is a rather hit-and-miss process.

The Centre for Genomic Regulation (CRG) research team have been able to reprogram adult cells very efficiently and in a very short period of time.

“Our group was using a particular transcription factor (C/EBPalpha) to reprogram one type of blood cells into another (transdifferentiation). We have now discovered that this factor also acts as a catalyst when reprogramming adult cells into iPS,” said Thomas Graf, senior group leader at the CRG and ICREA research professor.

“The work that we’ve just published presents a detailed description of the mechanism for transforming a blood cell into an iPS. We now understand the mechanics used by the cell so we can reprogram it and make it become pluripotent again in a controlled way, successfully and in a short period of time,” said Graf.

Genetic information is compacted into the nucleus like a wadded up ball of yarn. In order to access genes for gene expression, that ball of yarn has to be unwound so that the cell can find the information it needs.

The C/EBPalpha (CCAAT/Enhancer Binding Protein alpha) protein temporarily unwinds that region of DNA that contains the genes necessary for the induction of pluripotency. Thus, when the reprogramming process begin, the right genes are activated and they enable the successful reprogramming all the cells.

“We already knew that C/EBPalpha was related to cell transdifferentiation processes. We now know its role and why it serves as a catalyst in the reprogramming,” said Bruno Di Stefano, a PhD student. “Following the process described by Yamanaka the reprogramming took weeks, had a very small success rate and, in addition, accumulated mutations and errors. If we incorporate C/EBPalpha, the same process takes only a few days, has a much higher success rate and less possibility of errors, said Di Stefano.

This discovery provides a remarkable insight into stem cell-forming molecular mechanisms, and is of great interest for those studies on the early stages of life, during embryonic development. At the same time, the work provides new clues for successfully reprogramming cells in humans and advances in regenerative medicine and its medical applications.

100% Reprogramming Rates

For the first time, stem cell scientists have reprogrammed cultured skin cells into induced pluripotent cells (iPSCs) with near-perfect efficiency.

Even several laboratories have examined protocols to increase the efficiency of cellular reprogramming, a research team at the Weizmann Institute of Science in Rehovot, Israel has managed to increase the conversion rate to almost 100%, ten times the rate normally achieved, by removing a single proteins called Mbd3. This discovery can potentially allow scientists to generate large volumes of stem cells on demand, which would accelerate the development of new treatments.

In 2006, scientists from the laboratory of Shinya Yamanaka showed that mature cells could be reprogrammed to act like embryonic stem cells (ESCs). These reprogrammed adult cells could grow in culture indefinitely and differentiate into any type of cell in the body. However the creation of iPSc lines was notoriously inefficient and labor-intensive. Low cell-conversion rates have slowed the study of the reprogramming process itself. It has also discouraged the development of protocols for producing iPSCs under GMP or “Good Manufacturing Practice” conditions for use in human patients.

However, in a series of experiments that were published in the journal Nature, Weizmann Institute stem-cell researcher Jacob Hanna and his team have reprogrammed cells with nearly 100% efficiency. Moreover, Hanna and his group showed that reprogrammed cells transition to pluripotency on a synchronized schedule.

“This is the first report showing that you can make reprogramming as efficient as anyone was hoping for,” says Konrad Hochedlinger, a stem-cell scientist at Harvard Medical School in Boston, Massachusetts. “It is really surprising that manipulating a single molecule is sufficient to make this switch, and make essentially every single cell pluripotent within a week.”

To make iPSCs from adult cells, scientists typically transfect them with a set of four genes. These genes turn on the cells’ own pluripotency program, which converts them into iPSCs. But even established techniques convert less than 1% of cultured cells. Many cells get stuck in a partially reprogrammed state, and some become pluripotent faster than others, which makes the whole reprogramming process difficult to monitor.

Hanna and his team investigated the potential roadblocks to reprogramming by working with a line of genetically-engineered mouse cells. In these cells, the reprogramming genes were already inserted into the genomes of the cells and could be activated with a small molecule. Such cells normally reprogram at rates below 10%. But when a gene responsible for producing the protein Mbd3 was repressed, reprogramming rates soared to nearly 100%.

Hanna says that the precise timing of embryonic development led him to wonder whether it is possible to “reprogram the reprogramming process.” Cells in an embryo do not remain pluripotent indefinitely, explained Hanna. Usually, Mbd3 represses the pluripotency program as an embryo develops, and mature cells maintain their expression of Mbd3. However, during cellular reprogramming, those proteins expressed from the inserted pluripotency genes induce Mbd3 to repress the cells’ own pluripotency genes.

This hamstrings reprogramming, says Hanna. “It creates a clash, and that’s why the process is random and stochastic. It’s trying to have the gas and brakes on at the same time.” Depleting the cells of Mbd3 allows reprogramming to proceed unhindered.

The team also reprogrammed cells from a human, using a method that does not require inserting extra genes. This technique usually requires daily doses of RNA over more than two weeks. With Mbd3 repressed, only two doses were required.