Histones Might Hold the Key to the Generation of Totipotent Stem Cells

Reprogramming adult cells into pluripotent stem cells remains a major challenge to stem cell research. The process remains relatively inefficient and slow and a great deal of effort has been expended to improve the speed, efficiency and safety of the reprogramming procedure.

Researchers from RIKEN in Japan have reported one piece of the reprogramming puzzle that can increase the efficiency of reprogramming. Shunsuke Ishii and his colleagues from RIKEN Tsukuba Institute in Ibaraki, Japan have identified two variant histone proteins that dramatically enhance the efficiency of induced pluripotent stem cell (iPS cell) derivation. These proteins might be the key to generating iPS cells.

Terminally-differentiated adult cells can be reprogrammed into a stem-like pluripotent state either by artificially inducing the expression of four factors called the Yamanaka factors, or as recently shown by shocking them with sublethal stress, such as low pH or pressure. However, attempts to create totipotent stem cells capable of giving rise to a fully formed organism, from differentiated cells, have failed.  However, a paper recently published in the journal Nature has shown that STAP or stimulus-triggered acquisition of pluripotency cells from mouse cells have the capacity to form placenta in culture and therefore, are totipotent.

The study by Shunsuke Ishii and his RIKEN colleagues, which was published in the journal Cell Stem Cell, attempted to identify molecules in mammalian oocytes (eggs) that induce the complete reprograming of the genome and lead to the generation of totipotent embryonic stem cells. This is exactly what happens during normal fertilization, and during cloning by means of the technique known as Somatic-Cell Nuclear Transfer (SCNT). SCNT has been used successfully to clone various species of mammals, but the technique has serious limitations and its use on human cells has been controversial for ethical reasons.

Ishii’s research group focused on two histone variants named TH2A and TH2B, which are known to be specific to the testes where they bind tightly to DNA and influence gene expression.

Histones are proteins that bind to DNA non-specifically and act as little spool around which the DNA winds.  These little wound spools of DNA then assemble into spirals that form thread-like structures.  These threads are then looped around a protein scaffold to form the basic structure of a chromosome.  This compacted form of DNA is called “chromatin,” and the DNA is compacted some 10,000 to 100,000 times.  Histones are the main arbiters of chromatin formation.  In the figure below, you can see that the “beads on a string” consist of histones with DNA wrapped around them.


There are five “standard” histone proteins: H1, H2A, H2B, H3, and H4.  H2A, H2B, H3 and H4 form the beads and the H1 histone brings the beads together to for the 30nm solenoid.  Variant histones are different histones that assemble into beads that do not wrap the DNA quite as tightly or wrap it differently than the standard histones.  Two variant histones in particular, TH2A and TH2B, tend to allow DNA wrapped into chromatin to form and more loosely packed structure that allows the expression of particular genes.

When members of Ishii’s laboratory added these two variant histone proteins, TH2A/TH2B, to the Yamanaka cocktail (Oct4, c-Myc, Sox2, and Klf4) to reprogram mouse fibroblasts, they increased the efficiency of iPSC cell generation about twenty-fold and the speed of the process two- to threefold. In fact, TH2A and TH2B function as substitutes for two of the Yamanaka factors (Sox2 and c-Myc).

Ishii and other made knockout mice that lacked the genes that encoded TH2A and TH2B. This work demonstrated that TH2A and TH2B function as a pair, and are highly expressed in oocytes and fertilized eggs. Furthermore, these two proteins are needed for the development of the embryo after fertilization, although their levels decrease as the embryo grows.

Graphical Abstract1 [更新済み]

In early embryos, TH2A and TH2B bind to DNA and induce an open chromatin structure in the paternal genome (the genome of sperm cells), which contributes to its activation after fertilization.

These results indicate that TH2A/TH2B might induce reprogramming by regulating a different set of genes than the Yamanaka factors, and that these genes are involved in the generation of totipotent cells in oocyte-based reprogramming as seen in SCNT.

“We believe that TH2A and TH2B in combination enhance reprogramming because they introduce a process that normally operates in the zygote during fertilization and SCNT, and lead to a form of reprogramming that bears more similarity to oocyte-based reprogramming and SCNT” explains Dr. Ishii.

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.

Vascular Progenitors Made from Induced Pluripotent Stem Cells Repair Blood Vessels in the Eye Regardless of the Site of Injection

Johns Hopkins University medical researchers have reported the derivation of human induced-pluripotent stem cells (iPSCs) that can repair damaged retinal vascular tissue in mice. These stem cells, which were derived from human umbilical cord-blood cells and reprogrammed into an embryonic-like state, were derived without the conventional use of viruses, which can damage genes and initiate cancers. This safer method of growing the cells has drawn increased support among scientists, they say, and paves the way for a stem cell bank of cord-blood derived iPSCs to advance regenerative medical research.

In a report published Jan. 20 in the journal Circulation, Johns Hopkins University stem cell biologist Elias Zambidis and his colleagues described laboratory experiments with these non-viral, human retinal iPSCs, that were created generated using the virus-free method Zambidis first reported in 2011.

“We began with stem cells taken from cord-blood, which have fewer acquired mutations and little, if any, epigenetic memory, which cells accumulate as time goes on,” says Zambidis, associate professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. The scientists converted these cells to a status last experienced when they were part of six-day-old embryos.

Instead of using viruses to deliver a gene package to the cells to turn on processes that convert the cells back to stem cell states, Zambidis and his team used plasmids, which are rings of DNA that replicate briefly inside cells and then are degraded and disappear.

Next, the scientists identified and isolated high-quality, multipotent, vascular stem cells that resulted from the differentiation of these iPSC that can differentiate into the types of blood vessel-rich tissues that can repair retinas and other human tissues as well. They identified these cells by looking for cell surface proteins called CD31 and CD146. Zambidis says that they were able to create twice as many well-functioning vascular stem cells as compared with iPSCs made with other methods, and, “more importantly these cells engrafted and integrated into functioning blood vessels in damaged mouse retina.”

Working with Gerard Lutty, Ph.D., and his team at Johns Hopkins’ Wilmer Eye Institute, Zambidis’ team injected these newly iPSC-derived vascular progenitors into mice with damaged retinas (the light-sensitive part of the eyeball). The cells were injected into the eye, the sinus cavity near the eye or into a tail vein. When Zamdibis and his colleagues took images of the mouse retinas, they found that the iPSC-derived vascular progenitors, regardless of injection location, engrafted and repaired blood vessel structures in the retina.

“The blood vessels enlarged like a balloon in each of the locations where the iPSCs engrafted,” says Zambidis. Their vascular progenitors made from cord blood-derived iPSCs compared very well with the ability of vascular progenitors derived from fibroblast-derived iPSCs to repair retinal damage.

Zambidis says that he has plans to conduct additional experiments in diabetic rats, whose conditions more closely resemble human vascular damage to the retina than the mouse model used for the current study, he says.

With mounting requests from other laboratories, Zambidis says he frequently shares his cord blood-derived iPSC with other scientists. “The popular belief that iPSCs therapies need to be specific to individual patients may not be the case,” says Zambidis. He points to recent success of partially matched bone marrow transplants in humans, shown to be as effective as fully matched transplants.

“Support is growing for building a large bank of iPSCs that scientists around the world can access,” says Zambidis, although large resources and intense quality-control would be needed for such a feat. However, Japanese scientists led by stem-cell pioneer Shinya Yamanaka are doing exactly that, he says, creating a bank of stem cells derived from cord-blood samples from Japanese blood banks.

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.

Using Bone Marrow Stem Cells to Reprogram Neurons and Regenerate the Retina

Spanish researchers from the Center for Genomic Regulation (CGR) have regenerated the retina in mice by reprogramming neurons with bone marrow stem cells.

Cell reprogramming normally uses genetic engineering techniques that introduces genes into cells that push them into another cell fate without taking them through an embryonic-like state. One strategy for reprogramming cells fuses those cells with other cells that express genes that drive the fused cell into a different cell fate.

Pia Cosma and her team have used cell fusion to reprogram retinal neurons in mice. The mechanism consisted of introducing bone marrow stem cells into the damaged retina. The transplanted stem cells fused with existing retinal neurons, which conveyed to these retinal neurons the ability to regenerate the retina.

“For the first time we have managed to regenerate the retina and reprogram its neurons through in vivo cell fusion. We have identified a signaling pathway that, once activated, allows the neurons to be reprogrammed through their fusion with bone marrow cells,” said Pia Cosma, who is the head of the Reprogramming and Regeneration group at the CGR and ICREA (Institució Catalana de Recerca i Estudis Avançats) research professor.

Daniela Sanges, first author or the work and postdoctoral researcher in Pia Cosma’s laboratory, said, “This discovery is important not only because of the possible medical applications for retinal regeneration but also for the possible regeneration of other nervous tissues.”

The study demonstrates that the regeneration of nervous tissue by means of cell fusion is possible in mammals and describes this new technique as a potential mechanism for the regeneration of more complex nervous tissue.

This research is in the very early stages but already there are laboratories interested in being able to continue the work and take it to a more applied level.

Daniela Sanges, Neus Romo, Giacoma Simonte, Umberto Di Vicino, Ariadna Diaz Tahoces, Eduardo Fernández, Maria Pia Cosma. Wnt/β-Catenin Signaling Triggers Neuron Reprogramming and Regeneration in the Mouse Retina . Cell Reports – 25 July 2013 (Vol. 4, Issue 2, pp. 271-286)

Directly Programming Skin Cells to Become Blood-Making Stem Cells

Within our bones lies a spongy, ribbon-like material called bone marrow.  Bone marrow is home to several different populations of stem cells, but the star of the stem cell show in the bone marrow are the hematopoietic stem cells or blood-making stem cells.   When a patient receives a bone marrow transplant these are the stem cells that are transferred, take up residence in the new bone marrow, and begin making new red and white blood cells for the patient.  Because bone marrow is such a precious commodity from a clinical standpoint, finding a way to make more of it is essential.

Hematopoiesis from Pluripotent Stem Cell

A new report from scientists at Mt Sinai Hospital in New York suggest that the transfer of specific genes into skin fibroblasts can reprogram mature, adult cells into hematopoietic stem cells that look and function exactly like the ones normally found within our bone marrow.

A research team at the Icahn School of Medicine at Mount Sinai led by Kateri Moore screen a panel of 18 different genes for their ability to induce blood-forming activity when transfected into fibroblasts. Kateri and others discovered that a combination four different genes (GATA2, GFI1B, cFOS, and ETV6) is sufficient to generate blood vessel precursors with the subsequent appearance of hematopoietic stem cells. These cells expressed several known hematopoietic stem cell surface proteins (CD34, Sca1 and Prominin1/CD133).

Reprogramming of fibroblasts to HSCs

“The cells that we grew in a Petri dish are identical in gene expression to those found in the mouse embryo and could eventually generate colonies of mature blood cells,” said Carlos Filipe Pereira, first author of this paper and a postdoctoral research fellow in Moore’s laboratory.

The combination of gene factors that we used was not composed of the most obvious or expected proteins,” said Ihor Lemischka, a colleague of Dr. Moore at Mt. Sinai Hospital.  “Many investigators have been trying to grow hematopoietic stem cells from embryonic stem cells, but this process has been problematic.  Instead, we used mature mouse fibroblasts, pick the right combination of proteins, and it worked.”

According to Pereira, there is a rather critical shortage of suitable donors for blood stem cells transplants.  Bone marrow donors are currently necessary to meet the needs of patients suffering from blood diseases such as leukemia, aplastic anemia, lymphomas, multiple myeloma and immune deficiency disorders.  “Programming of hematopoietic stem cells represents an exciting alternative,” said Pereira.

“Dr. Lemischka and I have been working together for over 20 years in the fields of hematopoiesis and stem cell biology,” said Kateri Moore.  “It is truly exciting to be able to grow these blood forming cells in a culture dish and learn so much from them.  We have already started applying this new approach to human cells and anticipate similar success.”