Producing blood cells from stem cells could yield a purer, safer cell therapy


The journal Stem Cells Translational Medicine has published a new protocol for reprogramming induced pluripotent stem cells (iPSCs) into mature blood cells. This protocol uses only a small amount of the patient’s own blood and a readily available cell type. This novel method skips the generally accepted process of mixing iPSCs with either mouse or human stromal cells. Therefore, is ensures that no outside viruses or exogenous DNA contaminates the reprogrammed cells. Such a protocol could lead to a purer, safer therapeutic grade of stem cells for use in regenerative medicine.

The potential for the field of regenerative medicine has been greatly advanced by the discovery of iPSCs. These cells allow for the production of patient-specific iPSCs from the individual for potential autologous treatment, or treatment that uses the patient’s own cells. Such a strategy avoids the possibility of rejection and numerous other harmful side effects.

CD34+ cells are found in bone marrow and are involved with the production of new red and white blood cells. However, collecting enough CD34+ cells from a patient to produce enough blood for therapeutic purposes usually requires a large volume of blood from the patient. However, a new study outlined But scientists found a way around this, as outlined by Yuet Wai Kan, M.D., FRS, and Lin Ye, Ph.D. from the Department of Medicine and Institute for Human Genetic, University of California-San Francisco has devised a way around this impasse.

“We used Sendai viral vectors to generate iPSCs efficiently from adult mobilized CD34+ and peripheral blood mononuclear cells (MNCs),” Dr. Kan explained. “Sendai virus is an RNA virus that carries no risk of altering the host genome, so is considered an efficient solution for generating safe iPSC.”

“Just 2 milliliters of blood yielded iPS cells from which hematopoietic stem and progenitor cells could be generated. These cells could contain up to 40 percent CD34+ cells, of which approximately 25 percent were the type of precursors that could be differentiated into mature blood cells. These interesting findings reveal a protocol for the generation iPSCs using a readily available cell type,” Dr. Ye added. “We also found that MNCs can be efficiently reprogrammed into iPSCs as readily as CD34+ cells. Furthermore, these MNCs derived iPSCs can be terminally differentiated into mature blood cells.”

“This method, which uses only a small blood sample, may represent an option for generating iPSCs that maintains their genomic integrity,” said Anthony Atala, MD, Editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “The fact that these cells were differentiated into mature blood cells suggests their use in blood diseases.”

Long-Lasting Blood Vessels Regenerated from Reprogrammed Human Cells


Researchers from Massachusetts General Hospital (MGH) in Boston, MA have used human induced pluripotent stem cells to make vascular precursor cells to produce functional blood vessels that lasted as long as nine months.

Rakesh Jain, director of the Steele Laboratory for Tumor Biology at MGH and his team derived human induced pluripotent stem cells (iPSCs) from adult cells of two different groups of patients. One group of individuals were healthy and the second group had type 1 diabetes. Remember that iPSCs are derived from adult cells through the process of genetic engineering. By introducing specific genes into these adult cells, the adult cells are de-differentiated into an embryonic-like state. The embryonic-like cells can be cultured and grown into a cell line that can be differentiated into various cell types in the laboratory. These differentiated cells types can then be transplanted into laboratory animals for regenerative purposes.

“The discovery of ways to bring mature cells back to a ‘stem-like’ state that ca differentiate into many different types of tissue has brought enormous potential to the field of cell-based regenerative medicine, but the challenge of deriving functional cells from these iPSCs still remains,” said Rakesh. “our team has developed an efficient method to generate vascular precursor cells from human iPSCs and used them to create networks of engineered blood vessels in living mice.”

The ability to regenerate or repair blood vessels could be a coup for regenerative medicine. Cardiovascular disease, for example, continues to be the number one cause of death in the United States and other conditions caused by blood vessel damage (e.g., the vascular complications of diabetes) continue to cause a great deal of morbidity and mortality each year. Also, providing a blood supply to newly generated tissue remains one of the greatest barriers to building solid organs through tissue engineering.

Some studies have used iPSCs to build endothelial cells (the cells that line blood vessels), and connective tissue cells that provide structural support. These cells, unfortunately, tend to not produce long-lasting vessels once they are introduced into laboratory animals. A collaborator with Jain, Dai Fukumura, stated, “The biggest challenge we faced during the early phase of this project was establishing a reliable protocol to generate endothelial cell lines that produced great quantities or precursor cells that could generate durable blood vessels.”

Jain’s group adapted a protocol that was originally designed to derived endothelial cells from human embryonic stem cells. They isolated cells based on the presence of more than one cell surface protein that marked out vascular potential. Then they expanded this population of cells using a culture system developed with embryonic stem cells that had been differentiated into endothelial cells. Further experiments confirmed that only those iPSCs that expressed all three cell surface proteins on their surfaces had the potential to form blood vessels.

To test the capacity of those cells to generate blood vessels, they implanted them onto the surface of the brain of mice in combination with mesenchymal precursors that generate smooth muscles that surround blood vessels.

Within two weeks after transplantation, the implanted cells had formed entire networks of blood vessels with blood flowing through them that has also fused with the already existing blood vessels. These engineered blood vessels continued to function for as long as 280 days in the living animal. Implantation under the skin, however, was a different story. It took 5 times the number of cells to get them to form blood vessels and they were short-lived. This is similar to the results observed in other studies.

Because type 1 diabetes can ravage blood vessels, Jain’s team made iPSCs from patients with type 1 diabetes to determine if iPSCs from such patients would generate functional blood vessels. Similarly to the cells generated from healthy individuals, vascular precursors generated from type 1 diabetics were able to form long-lasting blood vessels. However, these same cell lines showed variability in their ability to form vascular precursors. The reason for this is uncertain.

Rekha Samuel, one of the lead authors of this paper, said “The potential applications of iPSC-generated blood vessels are broad – from repairing damaged vessels supplying the heart or brain to preventing the need to amputate limbs because of the vascular complications of diabetes. But first we need to deal with such challenges as the variability of iPSC lines and the long-term safety issues involved in the use of these cells, which are being addressed by researchers around the world. We need better ways of engineering the specific type of endothelial cell needed for specific organs and functions.”

A Step Towards Making Customized Blood Vessels


Johns Hopkins University scientists have directed stem cells to form networks of new blood vessels, and successfully transplanted those laboratory-made blood vessels into laboratory mice.

The stem cells in this experiment were made by reprogramming ordinary cells. Thus this new technique could potentially be used to make blood vessels that are genetically matched to individual patients and have a very low chance of being rejected by the patient’s immune system.

“In demonstrating the ability to rebuild a microvascular bed in a clinically relevant manner, we have made an important step toward the construction of blood vessels for therapeutic use,” said Sharon Gerecht, associate professor in the Johns Hopkins University Department of Chemical and Biomolecular Engineering, Physical Sciences-Oncology Center and Institute for NanoBioTechnology. “Our findings could yield more effective treatments for patients afflicted with burns, diabetic complications and other conditions in which vasculature function is compromised.”

Gerecht’s research group and others have previously grown blood vessels in the laboratory using stem cells, but there are problems with using these blood vessels in human patients. For example, in a paper published by Gerecht’s group in Stem Cells Translational Medicine earlier this year (Stem Cells Trans Med April 2013 vol. 2 no. 4 297-306), ECFCs or endothelial colony-forming cells from human umbilical cords were grown and used to make networks of blood vessels in culture. Those blood vessels were then embedded in blocks of “hyaluronic acid.” Hyaluronic acid is a component of human connective tissue, and when the ECFCs were embedded into it, they were then placed on the skin of mice that had received third-degree burns. On day 3 following transplantation, white blood cells called macrophages degraded the hyaluronic acid gel rather quickly. Between days 5–7, the mouse’s blood vessels infiltrated the implant and connected with the human blood vessels in the wound area. The growth of the human blood vessels peaked at day 7 and then decreased by the end of the proliferation stage. As the wound reached the remodeling period during the second week after transplantation, the blood vessels, including the transplanted human vessels generally regressed, and a few microvessels, wrapped by mouse smooth muscle cells and with a vessel area less than 200 square micrometers (including the human ones), remained in the healed wound.

This is a fascinating experiment, but making blood vessels this way is a heck of a lot of work, and even though the umbilical cord ECFCs are less likely to be rejected by the immune system of the patient, the chances of immune system rejection are still present. is there a better way?

In this current study, Gerecht and her team tried to streamline the new growth process. Where other experiments used chemical cues to differentiate stem cells into the desired cell type, Sravanti Kusuma in Gerecht’s laboratory devised a way to instruct stem cells to exclusively form the two cell types required for blood vessel construction (smooth muscle cells and endothelial cells).

According to Kusuma, “It makes the process quicker and more robust if you don’t have to sort through a lot of cells you don’t need to find the ones you do, or grow two batches of cells,”

Derivation of EVCs from hPSCs. (A) Schema for self-assembled vascular derivatives. (i) hPSCs are differentiated toward EVCs that can be matured into functional ECs and pericytes. (ii) Derived EVCs are embedded within a synthetic HA matrix that facilitates self-organization into vascular networks. (B) VEcad expression in day 12 differentiated hiPSC-MR31 and hESC-H9 cell lines comparing the three differentiation conditions tested (flow cytometry analysis; n = 3). (C and D) Flow cytometry plots (n = 3) of EVC derivatives assessing the expression of pluripotent markers TRA-1-60 and TRA-1-81 (C) and CD105 and CD146 (D). (E) EVC differentiation efficiency from hPSC lines per 1 million input hPSCs. (F) Flow cytometry plots (n = 3) of EVC derivatives assessing expression of VEcad double-labeled with CD105 or PDGFRβ. (Left) Isotype controls. (G) Quantitative RT-PCR analysis of EC and perivascular marker expression by EVCs and sorted VEcad+ and VEcad− cells. # denotes not detected. Data are normalized to EVCs of each specific hPSC type. (H) Flow cytometry plots (n = 3) of hematopoietic marker CD45 (hiPSC-BC1). (I) Quantitative RT-PCR of H9-EVCs for the expression of SMMHC and peripherin, compared with undifferentiated cells (d0) and mature derivatives (13, 37). Isotype controls for flow cytometry are in gray. Flow cytometry results shown are typical of the independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001
Derivation of EVCs from hPSCs. (A) Schema for self-assembled vascular derivatives. (i) hPSCs are differentiated toward EVCs that can be matured into functional ECs and pericytes. (ii) Derived EVCs are embedded within a synthetic HA matrix that facilitates self-organization into vascular networks. (B) VEcad expression in day 12 differentiated hiPSC-MR31 and hESC-H9 cell lines comparing the three differentiation conditions tested (flow cytometry analysis; n = 3). (C and D) Flow cytometry plots (n = 3) of EVC derivatives assessing the expression of pluripotent markers TRA-1-60 and TRA-1-81 (C) and CD105 and CD146 (D). (E) EVC differentiation efficiency from hPSC lines per 1 million input hPSCs. (F) Flow cytometry plots (n = 3) of EVC derivatives assessing expression of VEcad double-labeled with CD105 or PDGFRβ. (Left) Isotype controls. (G) Quantitative RT-PCR analysis of EC and perivascular marker expression by EVCs and sorted VEcad+ and VEcad− cells. # denotes not detected. Data are normalized to EVCs of each specific hPSC type. (H) Flow cytometry plots (n = 3) of hematopoietic marker CD45 (hiPSC-BC1). (I) Quantitative RT-PCR of H9-EVCs for the expression of SMMHC and peripherin, compared with undifferentiated cells (d0) and mature derivatives (13, 37). Isotype controls for flow cytometry are in gray. Flow cytometry results shown are typical of the independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Another difference from previous experiments was the use of induced pluripotent stem cells rather than bone marrow-derived endothelial precursor cells or umbilical cord-derived endothelial colony-forming cells. Gerecht’s team collaborated with Linzhao Cheng from the Institute for Cell Engineering to co-opt her expertise with induced pluripotent stem cells (iPSCs), which are made from adult cells and are de-differentiated through genetic engineering techniques to become embryonic-like stem cells.

Cheng said that this experiment is “an elegant use of human induced pluripotent stem cells that can form multiple cell types within one kind of tissue or organ and have the same genetic background [as the patient].” Cheng continued” “In addition to being able to form blood cells and neural cells as previously shown, blood-derived human induced pluripotent stem cells can also form multiple types of vascular network cells.”

To grow blood vessels, Cheng, Gerecht and others placed the stem cells into a scaffolding made of hydrogel (hyaluronic acid and water). This hydrogel was full of chemical cues that directed the cells to differentiate in to endothelial and smooth muscle cells and form a network of blood vessels. This constitutes the first time human blood vessels had been made from human pluripotent stem cells in a synthetic material.

Self-assembly of EVCs to multicellular networks in a 3D matrix. (A) Network formation from BC1-EVCs in collagen (i) and HA hydrogels (ii). (B) Sorted VEcad+ and VEcad− cells encapsulated within collagen gels were unable to form networks. (Insert) Example of a cell with typical stellate morphology, with phalloidin in green and nuclei in blue. (Scale bars: 100 μm.) (C) Vacuole formation was observed after one day as evidenced by light microscopy (LM) (i) and confocal images (ii) of vacuole vital stain, FM4-64, in red and nuclei in blue. (Scale bar: 10 μm.) (D) On day 2, network formation with enlarged lumen (i and ii) and cell sprouting (iii and iv) were visualized by LM images (i and iii) and confocal images (ii and iv) of FM4-64 in red and nuclei in blue. (Scale bars: 10 μm in i and iii; 20 μm in ii; 50 μm in iv.) (E) On day 3, complex networks were observed with enlarged and open lumen, as indicated by confocal z-stacks and orthogonal sections of FM4-64 in red and nuclei in blue. (Scale bar: 20 μm.) (F) After 3 d, multilayered structures were also detected, as demonstrated by a 3D projection image of NG2 (green), phalloidin (red), and nuclei (blue) showing NG2+ pericytes integrated onto hollow structures. Images shown are typical of the independent experiment. (Scale bars: 50 µm.)
Self-assembly of EVCs to multicellular networks in a 3D matrix. (A) Network formation from BC1-EVCs in collagen (i) and HA hydrogels (ii). (B) Sorted VEcad+ and VEcad− cells encapsulated within collagen gels were unable to form networks. (Insert) Example of a cell with typical stellate morphology, with phalloidin in green and nuclei in blue. (Scale bars: 100 μm.) (C) Vacuole formation was observed after one day as evidenced by light microscopy (LM) (i) and confocal images (ii) of vacuole vital stain, FM4-64, in red and nuclei in blue. (Scale bar: 10 μm.) (D) On day 2, network formation with enlarged lumen (i and ii) and cell sprouting (iii and iv) were visualized by LM images (i and iii) and confocal images (ii and iv) of FM4-64 in red and nuclei in blue. (Scale bars: 10 μm in i and iii; 20 μm in ii; 50 μm in iv.) (E) On day 3, complex networks were observed with enlarged and open lumen, as indicated by confocal z-stacks and orthogonal sections of FM4-64 in red and nuclei in blue. (Scale bar: 20 μm.) (F) After 3 d, multilayered structures were also detected, as demonstrated by a 3D projection image of NG2 (green), phalloidin (red), and nuclei (blue) showing NG2+ pericytes integrated onto hollow structures. Images shown are typical of the independent experiment. (Scale bars: 50 µm.)

While these networks of blood vessels looked like the real thing, would they work within a living creature? The answer that question, Gerecht and her group transplanted them into mice. After two weeks the lab-grown blood vessels had integrated with the mouse’s own blood vessels and the hydrogel had dissolved and been degraded. “That these vessels survive and function inside a living animal is a crucial step in getting them to medical application,” Kusama said.

An important follow-up to these experiments is to examine the three-dimensional structure of these blood vessels to determine if truly have all the characteristics of human blood vessels that can deliver blood to damaged tissues and help those tissues recover from injury or trauma.

Stem Cell-Promoting Gene Also Promotes the Growth of Head and Neck Cancer


Nanog is a very funny name for a gene, but the Nanog gene is an essential part of the cellular machinery that keeps embryonic stem cells from differentiating and maintains them in a pluripotent state. Unfortunately, Nanog also has other roles if it is mis-expressed and that includes in the genesis of cancers of the head and neck.

Nanog function during development
Nanog function during development

This study emerged from work done by researchers at the Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital, and Richard J. Solove Research Institute or OSUCCC-James. Since Nanog has been studied in some depth, understanding Nanog activity might provide vital clues in the design of targeted drugs and reagents for treating particular cancers.

“This study defines a signaling axis that is essential for head and neck cancer progression, and our findings show that this axis may be disrupted at three key steps,” said Quintin Pan, associate professor of otolaryngology at OSUCCC-James and principal investigator in this research effort. “Targeted drugs that are designed to inhibit any or all of these three steps might greatly improve the treatment of head and neck cancer.”

What kind of signaling axis is Dr. Pan referring to? An enzyme called protein kinase C-epsilon or PKC-epsilon can place phosphate groups on the Nanog protein. Phosphate groups are negatively charged and are also quite bulky. Attaching such chemical groups to a protein can effectively change its structure and function. In the case of Nanog, phosphorylation of stabilizes it and activates it.

Phosphorylated Nanog proteins can bind together to form a dimer, which attracts a third protein to it; p300. This third protein, p300, in combination with the paired Nanog proteins acts as a potent activator of gene expression of particular genes, in particular a gene called Bmi1. When expressed at high levels, Bmi1 stimulates the proliferation of cells in an uncontrolled fashion.

Bmi1 - Nanog interaction

“Our work shows that the PKCepsilon/Nanog/Bmi1 signaling axis is essential to promote head and neck cancer,” Pan said. “And it provides initial evidence that the development of inhibitors that block critical points in this axis might yield a potent collection of targeted anti-cancer therapeutics that could be valuable for the treatment of head and neck cancer.”

Making Pancreatic Beta Cells from Embryonic Stem Cells


Type 1 diabetes results from an inability to produce sufficient quantities of the hormone insulin. Without insulin, the body does not receive the signal to take up sugar from the blood, and the result is high blood sugar levels, which are damaging to tissues, and a general wasting of tissues because they cannot take up enough sugar to feed them.

Pancreatic beta cells

The cells in the pancreas that produce insulin are the beta cells, and animal studies have shown that transplantation of new beta cells into diabetic animals can reverse and even in some cases cure the diabetic animals. Therefore researchers have tried to make beta cells from pluripotent stem cells in order to make a source of beta cells for transplantation.

Unfortunately, beta cell production in the laboratory has been fraught with problems. The cells produced by differentiation of embryonic stem cells do not have the characteristics of mature beta cells and they produce little insulin and are not glucose responsive (D’Amour, et al., (2006) Nat Biotechnol 24, 1392-1401).

A different strategy, however, works much better. Instead of differentiating stem cells into beta cells, differentiate them into those cells that will form beta cells and other types of pancreatic cells in the embryo – immature endocrine cell precursors – and then transplant those into the pancreas of diabetic mice. In this case, the endocrine cell precursors differentiate in the bodies of the mice into pancreatic beta cells that greatly resemble normal beta cells.

Why don’t embryonic stem cells for beta cells in culture? This question was pursued by a collaboration between research team led by Maike Sander at UC San Diego and a company called ViaCyte, Inc.

When it comes to endocrine precursors transplanted into mice, Dr. Sander noted that, “We found that the endocrine cells retrieved from transplanted mice are remarkably similar to primary human endocrine cells.” He continued, “This shows that hESCs (human embryonic stem cells) can differentiate into endocrine cells that are almost indistinguishable from their primary human counterparts.”

Well, ESCs can make perfectly fine beta cells in the mouse body, but not in culture. What’s up with that?

Sander and her colleagues examined the gene expression patterns of embryonic stem cells as they were differentiated and compared them with the gene expression patterns in those cells that were transplanted into mice and allowed to differentiate inside the body of the mouse.

What Sander and her team found was astounding. As cells progress through their developmental program, particular genes are brought on-line and expressed, and then turned off as the cells passed through each stage of endocrine cell differentiation. The cellular machinery that shuts off genes after they have been activated consists of a family of proteins that remodel chromatin known as the Polycomb group (PcG). PcG-mediated repression of genes silenced those genes that were only turned on temporarily once they were no longer required.

Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .
Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .

In the transplanted endocrine precursors, Sanders and his team noted an orderly progression of genes that were turned on and then turned off once as needed. However, in the embryonic stem cells that were differentiated into beta cells in culture, they discovered that these cells failed to express the majority of genes critical for endocrine cell function. The main reason for this appeared to be that the PcG-mediated repression of genes was not fully eliminated when particular genes had to be expressed at specific developmental stages. Thus these cultured cells failed to fully eliminate PcG-mediated repression on endocrine-specific genes, which contributes to the abnormality of the culture-derived beta cells.

Sander commented: “This information will help devise protocols to generate functional insulin-producing beta cells in vitro. This will be important not only for cell therapies, but also for identifying disease mechanisms that underlie the pathogenesis of diabetes.”

How Stem Cells Stay Ready


Embryonic stem cells and induced pluripotent stem cells have a characteristic known as “pluripotency,” which simply means that they can become any cell type found in the adult human body. When these cells are given the proper cues, they can differentiate into muscle, skin, heart, blood, brain, or kidney cells, just to name a few. How do they do this?

A new study from the laboratory of Ali Shilatifard a scientist at the Stowers Institute for Medical Research in Kansas City, Mo, has shown that the plasticity of pluripotent stem cells partially comes from the stationing of a protein called EII3 at various stretches of DNA in the genome of these cells.

The vast majority of the cells in our bodies have a nucleus that houses the chromosomes, which are made of DNA. The DNA contains stretches known as genes that encode RNAs that are either translated into protein or have some other function. DNA stores genetic information and this genetic information is accessed by a complex set of enzymes called RNA polymerases and make these RNA molecules.

How do cells know when are where to make these RNA molecules? The signals for when to make a gene resides in sequences in or near the gene called “enhancer” sequences. Enhancers bind particular proteins and the assembly of particular proteins on the enhancers of a gene activate the expression of that gene.

EII3 is a member of a family of proteins known as the EII or (get ready) the “eleven-nineteen-lysine-rich-leukemia gene” (told you) family of elongation factors. ELongation factors increase the rate at which genes are expressed, and in pluripotent stem cells, EII3 parks itself at the enhancers of a variety of developmentally regulated genes, even ones that are silent in pluripotent cells.

According to Shilatifard: “We now know that some enhancer misregulation is involved in the pathogenesis of solid and hematological (that means blood-based) malignancies. But a problem in the field has been how to identify inactive or poised enhancer elements. Our discovery that EII3 interacts with enhancers in ES cells gives us a hand-hold to identify and to study them.”

EII3 was initially thought to be a dud because it was expressed at high levels in testes, which is a notoriously uninteresting tissue to work on from a gene expression perspective. All that changed when a postdoctoral research fellow in Shilatifard’s lab named Chengqi Lin searched all the potential places in the genome that EII3 could potentially bind in mouse embryonic stem cells. For this study, Lin collaborated with Alesander Garruss, another postdoctoral fellow in the Shilatifard lab, who is a specialist in bioinformational technologies. Lin and Garruss showed that EII3 occupies more than 5,000 enhancers, and many of these are affiliated with genes that regulate stem cell differentiation into spinal cord tissues, kidney cell types, blood cells and so on.

Lin put it this way: “What was interesting was that EII3 marked enhancers that are active and inactive, as well as enhancers that are known as “poised.” That indicated that EII3’s major function might be to prime activation of genes that are just about to be expressed during development.”

The fact that EII3 could prime silent genes for immediate expression under the right conditions was not a surprise, since researchers knew that enzymatic machine that copies DNA into RNA – RNA polymerase II, also known as Pol II, often pauses at the start of some genes. Pol II acts very like like a race car that has started its engine and is revving the engine in anticipation of the green light. When researchers in Shilatifard’s lab removed EII3 from the stem cells by means of a genetic trick, they discovered that the paused Pol II molecules disappeared these genes. This shows that EII3 oreferentiually marks stem cells enhancers and its presence there is necessary to keep an idling Pol II molecule there ready for action.

When the conditions are right, the EII3-Pol II complex interacts with the “Super Elongation Complex to give Pol II the green light and transcribe the gene. Without EII3, these genes are never expressed, even under the right conditions.

As a layer of icing on this remarkable research cake, Fengli Guo at the Stowers Institute use electron microscopy to prepare highly magnified images of mouse sperm DNA with Pol II and EII3 bound to this DNA. The significance of this come from development. Once fertilization occurs, the zygotes begins to divide, and the daughter cells are gradually committed to various developmental possibilities. The fact that EII3 is necessary for differentiation and that it is carried into the embryo by the sperm explains how the blastomeres of the early embryo are so exquisitely poised to readily differentiate into the various cell types that they eventually become.

“It is very significant that EII3 and other factors that regulate transcription are found in sperm,” said Lin. He continued to note that that it “would be exciting to further investigate whether transcription factors found in sperm could contribute to the decondensation of sperm chromatin or even further gene activation after fertilization by serving as epigenetic markers.”

Shilatifard is cautious about not overinterpreting these results, but he does think that they have fundamental implications for development and also, perhaps, cancer research. “This work has opened up a whole new area of research in my lab,” said Shilatifard, who has in the last decade focused on aberrant gene expression associated with leukemia. “If we find that transcription factors bind to specific regions of chromatin in germ cells, I may focus on germ cells in the next few decades. This would open a huge door enabling us to determine the role of these factors in early development.”