Using Peptides to Reset a Diseased Cell


Researchers at the University of California, San Diego School of Medicine have published a series of proof-of-concept experiments that demonstrate the ability to direct medically relevant cell behaviors by artificially manipulating a central hub in cell communication networks. The manipulation of this communication node, which was reported in the journal Proceedings of the National Academy of Sciences, makes it possible to reprogram major parts of a cell’s signaling network instead of targeting only a single receptor or cell signaling pathway.

This discovery could have tremendous clinical value, since it could slow or reverse the progression of diseases, such as cancer, which are driven by abnormal cell signaling along a variety of signaling pathways.

“Our study shows the feasibility of targeting a hub in the cell signaling network to reset aberrant cell signaling from multiple pathways and receptors,” said senior author Pradipta Ghosh, MD, an associate professor of medicine.

The UC San Diego team engineered two small protein fragments, known as peptides, to either turn on or turn off the activity of a family of proteins called G proteins. G protein-coupled receptors, which are embedded into the surfaces of cells, are used by cells to sense and respond to their environments. Approximately 30 percent of all prescription drugs target cells by binding to and affecting G protein-coupled receptors.

G protein coupled receptor cycle

Several laboratories, including those at UC San Diego, have discovered that G proteins can also be activated inside cells, and not simply on cell surfaces. Other receptors can activate the internal components of the G protein-coupled receptor, as can a protein called GIV. GIV has been implicated in cancer metastasis and other disease states. Both the “on” and “off” peptides were made from parts of the GIV protein receptor.

In a series of cell culture experiments, the “on” peptides were shown to accelerate the ability of the cells to migrate after scratch-wounding, which is a process linked to wound healing. The “off” peptide, in contrast, reduced the aggressiveness of cancer cells and decreased the production of collagen by cells associated with liver fibrosis. In experiments with mice, the topical application of the “on” peptides helped skin wounds heal faster.

“The takeaway is that we can begin to tap an emerging new paradigm of G protein signaling,” Ghosh said.

New Type of Stem Cell Discovered by Salk Scientists


Stem cell scientists from the laboratory of Juan Carlos Izpisua Belmonte at the Salk Institute for Biological Studies in La Jolla, California have discovered a new type of stem cell that could potentially provide a model system for early human development, and might even allow human organs to be grown in large animals for therapeutic purposes.

 

Izpisua Belmonte and his colleagues came across these types of cells somewhat serendipitously while transplanting human pluripotent stem cells into mouse embryos.

 

Other types of pluripotent stem cells have already been well-known to stem cell scientists for some time. Stem cells are “pluripotent,” if they have an intrinsic ability to differentiate into any adult cell type. Embryonic stem cells (ESCs), for example, are derived from early human embryos that have yet to implant into the inner layer of the uterus.  However, epiblast stem cells (EpiSCs) have been established from post-implantation embryos and have different properties.  While both are pluripotent, they bear striking differences in molecular signature, signalling dependency, colony morphology, cloning efficiency, metabolic requirements and epigenetic features (see Nichols, J. & Smith, A. Cell Stem Cell 4, 487492 (2009) and Zhou, W. et al. EMBO J. 31, 21032116 (2012)).  Both of these cells have the ability to re-enter embryogenesis but they do so at different developmental time points (pre-implantation versus post-implantation, respectively), which distinguish ESCs and EpiSCs.  Therefore, these two cell types exist in two temporally distinct pluripotent states.  Even though these two types of pluripotent stem cells can be grown into large numbers in the laboratory, differentiating them into specific types of mature, adult cells has proven difficult in some cases. The cells discovered by Izpisua Belmonte and his colleagues are reportedly easier to grow in vitro and engraft into an embryo if they are injected into the right spot. Izpisua Belmonte call these cells “region-selective pluripotent stem cells” (rsPSCs).

 

 

Because rsPSCs grow more quickly and stably than other pluripotent cells, they may be more useful for developing new therapies, according to Paul Tesar, a developmental biologist at Case Western Reserve University in Cleveland, Ohio.

 

Izpisua Belmonte and colleagues originally wanted to transplant various types of human pluripotent stem cells into mouse embryos in the laboratory. They prepared their cells for transplantation by growing them in various blends of culture media that contained different combinations of growth factors and other chemicals. They found that one particular blend was more effective at making the cells grow and proliferate. However, the cells that grew quite well in this concoction displayed different patterns of metabolism and gene expression in comparison to other pluripotent stem cells. These same cells not graft well into the mouse embryo.

 

Thus, Izpisua Belmonte and his colleagues decided to nail down those features that would cause cells to efficiently integrate into mouse embryos. They injected the human cells into three different regions of a 7.5-day-old mouse embryo. Thirty-six hours later, only those cells that had been grafted into the tail, or posterior of the embryo, integrated and differentiated into the correct cell layers to form a “chimeric” or mixed-tissue embryo. Such organisms contain cells with genomes from DNA organisms. Since these cells seemed to prefer one part of the embryo, Izpisua Belmonte and his team called them region-selective pluripotent stem cells.

 

From these data, Izpisua Belmonte has proposed that embryos contain multiple types of pluripotent stem cells, including rsPSCs, during their early development. It is not yet clear whether the rsPSCs play a part in designating which part of the embryo will become the head, the middle, or hind end. Identifying various types of pluripotent cells might provide researchers with the ability to study the early stages of human embryonic development by transplanting rsPSCs into animal embryos.

 

Izpisua Belmonte and his colleagues found that they could easily use enzymes that modify the sequences of DNA to edit the genomes of rsPSCs, which is usually difficult to do in pluripotent cell lines when grown in culture.

Gene editing could help scientists to optimize the ability of human cells to grow within animals, which might allow the creation of transgenic chimeras. Tesar says that the idea of using human pluripotent cells, such as rsPSCs, to create animals with human organs is not unrealistic, but he expects that it will be very difficult. The immune system of the animal might reject the human cells and the growth rates of the two organs might also cause problems.

Izpisua Belmonte’s lab is already starting to implant pig embryos with a different type of stem cells, and this is the only very first step for these techniques.

 

Induced Pluripotent Stem Cells from Bone Cancer Patients Provide Crucial Insights into the Genesis of Bone Cancer


A team of Mount Sinai researchers have utilized induced pluripotent stem cells (iPSCs) to elucidate the genetic changes that seem to convert a well-known anti-cancer signaling gene into a driver bone cancers. When it comes to bone cancers, the survival rate has not improved in 40 years despite advances in treatment. Since this study might provide new targets and suggest new strategies for attacking such cancers. it represents a welcome addition to the cancer literature.

This study, which was published in the journal Cell, revolves around iPSCs, which were discovered in 2006 by Nobel laureate Shinya Yamanaka. iPSCs use genetic engineering and cell culture techniques to reprogram mature, adult cells to become like embryonic stem cells. These iPSCs are “pluripotent,” which means that they are able to differentiate into any adult cell type and can also divide in culture indefinitely.

For therapeutic purposes, iPSCs can be derived from a patient’s own cells, differentiated into the cells the patient needs to be replaced, and then implanted into the patient’s body to augment tissue healing or even organ reconstruction. Since iPSCs can be successfully differentiated into heart muscle, nerve cells, bone, and other cell types, they have the potential advance the field of regenerative medicine by leaps and bounds.

iPSCs have already made their presence known in the clinic by serving as model systems for research and diagnosis. The new Mount Sinai study used iPSCs to construct an accurate model of a genetic disease “in a dish.” The culture dishes contain self-renewing patient-specific iPSCs or a specific cell line that enable in-depth study diseases that are driven by each person’s genetic differences. When matched with patient records, iPSCs and iPSC-derived target cells have the ability to help physicians predict a patient’s prognosis and whether or not a given drug will be effective for him or her.

In this study, skin cells from healthy patients and patients with a genetic disease called Li-Fraumeni syndrome were isolated and reprogrammed into patient-specific iPSC lines. These iPSCs were then differentiated into bone-making cells (osteoblasts), which are the cells where particular rare and common bone cancers start. Li-Fraumeni syndrome greatly predisposes patients to a variety of cancers in several different types of tissues.

The patient-derived osteoblasts were then tested for their tendencies to become tumor cells and to make bone. This particular bone cancer model did a better job of recapitulating the characteristics of bone cancer than previously used mouse or cellular models.

LFS iPSCs for stem cell production

“Our study is among the first to use induced pluripotent stem cells as the foundation of a model for cancer,” said lead author Dung-Fang Lee, PhD, a postdoctoral fellow in the Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai. “This model, when combined with a rare genetic disease, revealed for the first time how a protein known to prevent tumor growth in most cases, p53, may instead drive bone cancer when genetic changes cause too much of it to be made in the wrong place.”

The Mount Sinai disease model research uses a simple fact of human life as its basis: human genes undergo mutations at a certain rate that tends to increase as we age, and the formation of new mutations in relentless and constant. Some mutations make no difference, a few some confer advantages, and others cause disease. Beyond inherited mutations that contribute to cancer risk, the combination of random, accumulated DNA changes in our cells as we age also increase our cancer risk.

The current study focused on those genetic pathways involved in Li-Fraumeni Syndrome or LFS, a rare genetic disease that causes high risk for many cancers in affected families. Osteosarcoma (bone cancer) is a common cancer observed in LFS patients and many of them are diagnosed before the age of 30. Additionally, osteosarcoma is the most common type of bone cancer in all children, and after leukemia, the second leading cause of cancer death for them.

 

Importantly, about 70 percent of LFS families have a mutation in their copy of a genes called TP53, which encodes the p53 protein. P53 is a “the tumor suppressor,” which means that it functions to preserve the integrity of the genome and keep the rate of cell division in check. Common forms of osteosarcoma, which are driven by somatic or inherited mutations, have also been closely linked by past studies to defects in p53 when mutations interfere with the ability of the protein to function properly.

p53

Crystal Structure of p53 protein bound to DNA

 

Rare genetic diseases like LFS provide excellent model systems because they tend to result from a change in a single gene, instead of the diverse and overlapping mutations observed in common diseases, and, in this case, more common, non-inherited bone cancers. The LFS-iPSC based modeling highlights the contribution of p53 alone to osteosarcoma.

By analyzing iPSC lines, and bone cancer driven by p53 mutations in LFS patients, the Munt Sinai research team showed, for the first time, that the LFS bone cancer results from an overactive p53 gene. Too much p53 in osteoblasts dampens the function of a gene, H19, and a related protein, decorin, that would otherwise help stem cells differentiate into normal osteoblasts.

The inability of cells to differentiate makes them vulnerable to genetic mistakes that drive cancer, since more “stemness” means a tendency toward rapid, abnormal growth, like that observed in tumors. One tragic feature of osteosarcoma is the rapid, error-prone production of weaker bone by cancerous bone-making cells, where a young person surprisingly breaks a bone to reveal undiagnosed, advanced cancer.

Dung-Fang Lee and his colleagues discovered that the H19 gene seems to control a network of interconnected genes that fine-tune the balance between cell growth and resistance to growth. Decorin is a protein that is part of connective tissue like bone, but that also plays a signaling role, interacting with growth factors to slow the rate that cells divide and multiply, unless turned off by too much p53.

“Our experiments showed that restoring H19 expression hindered by too much p53 restored “protective differentiation” of osteoblasts to counter events of tumor growth early on in bone cancer,” said co-author, Ihor Lemischka, PhD, Director of The Black Family Stem Cell Institute within the Icahn School of Medicine. “The work has implications for the future treatment or prevention of LFS-associated osteosarcoma, and possibly for all forms of bone cancer driven by p53 mutations, with H19 and p53 established now as potential targets for future drugs.”

Amniotic Fluid Stem Cells Make Robust Blood Vessel Networks


The growth of new blood vessels in culture received in new boost from researchers at Rice University and Texas Children’s Hospital who used stem cells from amniotic fluid to promote the growth of robust, functional blood vessels in healing hydrogels.

These results were published in the Journal of Biomedical Materials Research Part A.

Engineer Jeffrey Jacot thinks that amniotic fluid stem cells are valuable for regenerative medicine because of their ability to differentiate into many other types of cells, including endothelial cells that form blood vessels. Amniotic fluid stem cells are taken from the discarded membranes in which babies are encased in before birth. Jacot and others combined these cells with an injectable hydrogel that acted as a scaffold.

In previous experiments, Jacot and his colleagues used amniotic fluid cells from pregnant women to help heal infants born with congenital heart defects. Amniotic fluids, drawn during standard tests, are generally discarded but show promise for implants made from a baby’s own genetically matched material.

“The main thing we’ve figured out is how to get a vascularized device: laboratory-grown tissue that is made entirely from amniotic fluid cells,” Jacot said. “We showed it’s possible to use only cells derived from amniotic fluid.”

Researchers from Rice, Texas Children’s Hospital and Baylor College of Medicine combined amniotic fluid stem cells with a hydrogel made from polyethylene glycol and fibrin. Fibrin is the proteins formed during blood clots, but it is also used for cellular-matrix interactions, wound healing and angiogenesis (the process by which new vessels are made). Fibrin is widely used as a bioscaffold but it suffers from low mechanical stiffness and is degraded rapidly in the body. When fibrin was combined with polyethylene glycol, the hydrogel became much more robust, according to Jacot.

Additionally, these groups used a growth factor called vascular endothelial growth factor to induce the stem cells to differentiate into endothelial cells. Furthermore, when induced in the presence of fibrin, these cells infiltrated the native vasculature from neighboring tissue to make additional blood vessels.

When mice were injected with fibrin-only hydrogels, thin fibril structures formed. However if those same hydrogels were infused with amniotic fluid stem cells that had been induced with vascular endothelial growth factor, the cell/fibrin hydrogel concoctions showed far more robust vasculature.

In similar experiments with hydrogels seeded with bone marrow-derived mesenchymal cells, once again, vascular growth was observed, but these vessels did not have the guarantee of a tissue match. Interestingly, seeding with endothelial cells didn’t work as well as the researchers expected, he said.

Jacot and others will continue to study the use of amniotic stem cells to build biocompatible patches for the hearts of infants born with birth defects and for other procedures.

Dead Heart Muscle Regrown in Rodents


If you cut a piece of tissue from the heart of a salamander or zebrafish, they wild simply grow new heart tissue. Unfortunately, humans are unable to easily regenerate heart cells, and this males it difficult to recover from the permanent damage caused by heart attacks.

Fortunately, life scientists from the Weizmann Institute of Science in Israel and the Victor Chang Institute in Sydney have discovered a way to stimulate heart muscle cells in mammals to grow. This finding could have major implications for future heart attack sufferers.

Even though human blood, hair and skin cells renew themselves throughout life, cell division in the heart comes to a virtual standstill shortly after birth, according to Prof. Richard Harvey, from the Victor Chang cardiac research institute, and one of the authors of this research. Harvey said, “So there’s always been an intense interest in the mechanism salamanders and fish use which makes them capable of heart regeneration, and one thing they do is send their cardiomyocytes, or muscle cells, into a dormant state, which they then come out of to go into a proliferative state, which means they start dividing rapidly and replacing lost cardiomyocytes.”

Harvey continued: “There are various theories why the human heart can not do that, one being that our more sophisticated immune system has come at a cost, and because human cardiomyocytes are in a deeper state of quiescence, that has made it very difficult to stimulate them to divide.”

Today, for the first time in history, more people in developing countries die from strokes and heart attacks than infectious diseases. Fortunately there are cost-effective ways to save lives

By studying mice, Harvey and his colleagues found a way to overcome that regenerative barrier – at least in the rodents.

Harvey and others found that by stimulating a cell signaling pathway in the heart that is driven by a hormone called neuregulin, heart muscle cells divided in a spectacular way in both adolescent and adult mice. In humans, neuregulin expression is usually muted about one week after birth, and by about 20 weeks after birth in mice.

By triggering of the neuregulin pathway following a heart attack in mice, Harvey and others induced the replacement of lost muscle, which repaired the heart to a level close to that prior to the heart attack. Harvey said that he and other scientists should be able to determine with in the next five years if it is possible to replicate these results in humans.

“This is such a significant finding that it will harness research activities in many labs around the world, and there will be much more attention now on how this neuregulin-response could be maximised,” Harvey said.

“We will now examine what else we can use, other than genes, to activate that pathway, and it could be that there are already drugs out there, used for other conditions and regarded as safe, that can trigger this response in humans.”

When one of the blood vessels that provide blood to the heart muscle becomes blocked, the patient suffers a heart attack. Heart attacks or “myocardial infractions” cause billions of cardiomyocytes to die. Even if you survive a heart attack, you usually experience diminished quality of life because of it.

“The dream is that one day we will be able to regenerate damaged heart tissue, much like a salamander can regrow a new limb if it is bitten off by a predator,” Harvey said.

Molecular biologist Gabriele D’Uva lead this research, which was published in the scientific journal Nature Cell Biology.

Forcing Sugars on the Surfaces of Cord Blood Cell Increases Their Engraftment


When a child or adult needs new bone marrow, a bone marrow transplant from a donor is usually the only way to save their life. Without properly functioning bone marrow, the patient’s blood cells will die off, and there will be too few red blood cells to ferry oxygen to tissues or white blood cells to fight off infections.

An alternative to bone marrow from a bone marrow donor if umbilical cord blood. Umbilical cord blood does not require the rigorous tissue matching that bone marrow requires because the blood making stem cells from cord blood are immature and not as likely to cause tissue rejection reactions.. However, umbilical cord blood cells suffer from two drawbacks: low numbers of stem cells in cord blood and poor engraftment efficiencies.

Fortunately, some progress has been made at expanding blood-making stem cells from umbilical cord blood, and it is likely that such technologies might be ready for common use in the future. As to the poor engraftment efficiencies, a new paper in the journal Blood from the laboratory of Elizabeth J. Shpall at the University of Texas MD Anderson Cancer Center, in Houston, Texas reports a new way to increase cord blood stem cells engraftment efficiencies.

As previously discusses, delayed engraftment is one of the major limitations of cord blood transplantation (CBT). Delayed engraftment seems to be due to the diminished ability of the cord blood stem cells to home to the bone marrow. How are cells channeled to the bone marrow? A protein receptor called P- and E-selectins is expressed on the surfaces of bone marrow blood vessels. Cells that can bind these selectin receptors will pass from the circulation to the bone marrow. Thus binding selectin receptors is kind of like having the “password” for the bone marrow.

What does it take to bind the selectin proteins? Selectins bind to specific sugars that have been attached to proteins. These sugars are called “fucose” sugars. As it turns out, cord blood stem cells do not express robust levels of these fucosylated proteins. Could increasing the levels of fucosylated proteins on the surfaces of cord blood stem cells increase their engraftment? Shpall and her colleagues tested this hypothesis in patients with blood-based cancers.

Patients with blood cancers had their cancer-producing bone marrow stem cells destroyed with drugs and radiation. Then these same patients had their bone marrows refurbished with two units of umbilical cord blood. However, these cells in these cord blood units were treated with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose for 30 minutes before transplantation. This treatment should have increased the content of fucosylated proteins on the surfaces of cells in the hope of enhancing their interaction with Selectin receptors on the surfaces of bone marrow capillaries.

The results of 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. There was a clear decrease in the length of time it took for cells to engraft into the bone marrow.  For example, the median time to neutrophil (a type of white blood cell) engraftment was 17 days (range 12-34) compared to 26 days (range, 11-48) for controls (P=0.0023). Platelet (a cell used in blood clotting) engraftment was also improved: median 35 days (range, 18-100) compared to 45 days (range, 27-120) for controls (P=0.0520).  These are significant differences.

These findings support show that treating cord blood cells with a rather inexpensive cocktail of enzymes for a short period of time before transplantation is a clinically feasible means to improve engraftment efficiency of CBT.  This is a small study.  Therefore, these data, though very hopeful, must be confirmed with larger studies.

Stem Cell Structure and Obesity


New research conducted at Queen Mary University of London (QMUL) has discovered that the regulation of the length of primary cilia, which are small hair-like projections on the surfaces of most cells, can prevent the production of fat cells taken from adult human bone marrow. Such a discovery might be used to develop a way of preventing obesity.

What are primary cilia?  For many years, almost all attention was focused on cilia that moved because their function was readily observable.  However, Alexander Kowalevsky first reported in 1867 the presence of single (nonmotile) cilia in a variety of vertebrate cells.  These solitary and nonmotile cilia are far more widespread than the motile type.  In humans, only a few cell types have motile cilia, namely epithelial cells in the bronchi and oviducts, and ependymal cells that line brain vesicles.  However, virtually all other cells have a primary cilium.

What makes primary cilia different from the motile form? First, they lack the central pair of microtubules, which would explain the lack of motility.  Primary cilia also seem to lack dynein, one of the molecular motors needed for motility.  In addition, some primary cilia do not project beyond the cell surface, and most, but not all, are very short.  What do these organelles do if they are not sticking out of the cell, or motile?

Further work has shown that primary cilia are important in intracellular transport and also in sensory function for cells.  Now it seems that primary cilia are also important in the process of adipogenesis.

Primary cilia

Adipogenesis refers to the differentiation of stem cells into fat cells. The QMUL research team showed that during adipogenesis, the length of primary cilia increases, which increases the movement of specific proteins associated with the cilia. When the QMUL team genetically restricted primary cilia elongation by genetic means, they were able to stop the formation of new fat cells.

One of the lead authors or this study, Melis Dalbay, said that it was the first time that subtle changes in primary cilia structure can influence the differentiation of stem cells into fat.

Since the length of primary cilia can be influenced by various factors including pharmaceuticals, inflammation and even mechanical forces, this study provides new insight into the regulation of fat cell formation and obesity.

This research points toward a new type of treatment known as “cilia-therapy” where manipulation of primary cilia may be used in the future to treat a growing range of conditions including obesity, cancer, inflammation and arthritis.