Stimulus-Triggered Acquisition of Pluripotency Cells: Embryonic-Like Stem Cells Without Killing Embryos or Genetic Engineering

Embryonic stem cells have been the gold standard for pluripotent stem cells. Pluripotent means capable of differentiating into one of many cell types in the adult body. Ever since James Thomson isolated the first human embryonic stem cell lines in 1998, scientists have dreamed of using embryonic stem cells to treat diseases in human patients.

However, deriving human embryonic stem cell lines requires the destruction or molestation of a human embryo, the smallest, youngest, and most vulnerable member of our community. In 2006, Shinya Yamanaka and his colleges used genetic engineering techniques to make induced pluripotent stem (iPS) cells, which are very similar to embryonic stem cells in many ways. Unfortunately, the derivation of iPSCs introduces mutations into the cells.

Now, researchers from Brigham and Women’s Hospital (BWH), in Boston, in collaboration with the RIKEN Center for Developmental Biology in Japan, have demonstrated that any mature adult cell has the potential to be converted into the equivalent of an embryonic stem cell. Published in the January 30, 2014 issue of the journal Nature, this research team demonstrated in a preclinical model, a novel and unique way to reprogram cells. They called this phenomenon stimulus-triggered acquisition of pluripotency (STAP). Importantly, this process does not require the introduction of new outside DNA, which is required for the reprogramming process that produces iPSCs.

“It may not be necessary to create an embryo to acquire embryonic stem cells. Our research findings demonstrate that creation of an autologous pluripotent stem cell – a stem cell from an individual that has the potential to be used for a therapeutic purpose – without an embryo, is possible. The fate of adult cells can be drastically converted by exposing mature cells to an external stress or injury. This finding has the potential to reduce the need to utilize both embryonic stem cells and DNA-manipulated iPS cells,” said senior author Charles Vacanti, MD, chairman of the Department of Anesthesiology, Perioperative and Pain Medicine and Director of the Laboratory for Tissue Engineering and Regenerative Medicine at BWH and senior author of the study. “This study would not have been possible without the significant international collaboration between BWH and the RIKEN Center,” he added.

The inspiration for this research was an observation in plant cells – the ability of a plant callus, which is made by an injured plant, to grow into a new plant. These relatively dated observations led Vacanti and his collaborators to suggest that any mature adult cell, once differentiated into a specific cell type, could be reprogrammed and de-differentiated through a natural process that does not require inserting genetic material into the cells.

“Could simple injury cause mature, adult cells to turn into stem cells that could in turn develop into any cell type?” hypothesized the Vacanti brothers.

Vacanti and others used cultured, mature adult cells. After stressing the cells almost to the point of death by exposing them to various stressful environments including trauma, a low oxygen and acidic environments, researchers discovered that within a period of only a few days, the cells survived and recovered from the stressful stimulus by naturally reverting into a state that is equivalent to an embryonic stem cell. With the proper culture conditions, those embryonic-like stem cells were propagated and when exposed to external stimuli, they were then able to redifferentiate and mature into any type of cell and grow into any type of tissue.

To examine the growth potential of these STAP cells, Vacanti and his team used mature blood cells from mice that had been genetically engineered to glow green under a specific wavelength of light. They stressed these cells from the blood by exposing them to acid, and found that in the days following the stress, these cells reverted back to an embryonic stem cell-like state. These stem cells then began growing in spherical clusters (like plant callus tissue). The cell clusters were introduced into developing mouse embryos that came from mice that did not glow green. These embryos now contained a mixture of cells (a “chimera”). The implanted clusters were able to differentiate into green-glowing tissues that were distributed in all organs tested, confirming that the implanted cells are pluripotent.

Thus, external stress might activate unknown cellular functions that set mature adult cells free from their current commitment to a particular cell fate and permit them to revert to their naïve cell state.

“Our findings suggest that somehow, through part of a natural repair process, mature cells turn off some of the epigenetic controls that inhibit expression of certain nuclear genes that result in differentiation,” said Vacanti.

Of course, the next step is to explore this process in more sophisticated mammals, and, ultimately in humans.

“If we can work out the mechanisms by which differentiation states are maintained and lost, it could open up a wide range of possibilities for new research and applications using living cells. But for me the most interesting questions will be the ones that let us gain a deeper understanding of the basic principles at work in these phenomena,” said first author Haruko Obokata, PhD.

If human cells can be made into embryonic stem cells by a similar process, then someday, a simple skin biopsy or blood sample might provide the material to generate embryonic stem cells that are specific to each individual, without the need for genetic engineering or killing the smallest among us. This truly creates endless possibilities for therapeutic options.

Injected microparticles shown to greatly reduce heart attack damage

Reducing inflammation in the heart after a heart by injecting biodegradable microparticles.

Leaders in Pharmaceutical Business Intelligence (LPBI) Group

See on Scoop.itCardiovascular and vascular imaging

After a heart attack has occurred, inflammatory cells known as monocytes rush to the damaged tissue. This causes the heart to swell, further damaging the ti…

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Human Stem Cell Gene Therapy Appears Safe and Effective

Two recent studies in the journal Science have reported the outcome of virally-mediated gene correction in hematopoietic stem cells (HSCs) to treat human patients. These studies may usher in a new era of safe and effective gene therapy. These exciting new clinical findings both come from the laboratory of Luigi Naldini at the San Raffaele Scientific Institute, Milan, Italy. The first experiment examined the treatment of metachromatic leukodystrophy (MLD), which is caused by mutations in the arylsulfatase A (ARSA) gene, and the second, investigated treatments for Wiskott-Aldrich syndrome (WAS), which is caused by mutations in the gene that encodes WASP.

MLD is one of several diseases that affects the lysosome; a structure in cells that acts as the garbage disposal of the cell. So called “lysosomal storage diseases” result from the inability of cells to degrade molecules that come to the lysosome for degradation. Without the ability to degrade these molecules, they build up to toxic levels and produce progressive motor and cognitive impairment and death within a few years of the onset of symptoms.

To treat MLD, workers in Naldini’s laboratory isolated blood-making stem cells from the bone marrow of three pre-symptomatic MLD patients (MLD01, 02 and 03). These stem cells were infected with genetically engineered viruses that encoded the human ARSA gene. After expanding these stem cells in culture, they were re-introduced into the MLD patients after those same patients had their resident bone marrow wiped out. The expression of the ARSA gene in the reconstituted bone marrow was greater than 10 fold the levels measured in healthy controls and there were no signs of blood cancers or other maladies. One month after the transplant, the implanted cells showed very high-level and stable engraftment. Between 45%-80% of cells isolated and grown from bone marrow samples harbored the fixed ARSA gene. AS expected, the levels of the ARSA protein rose to above-normal levels in therapeutically relevant blood cells and above normal levels of ARSA protein were isolated from hematopoietic cells after one month and cerebrospinal fluid (CSF) one to two years after transfusion. This is remarkable when you consider that one year before, no ARSA was seen. This shows that the implanted cells and their progeny properly homed to the right places in the body. The patient evaluations at time points beyond the expected age of disease onset was even more exciting, since these treat patients showed normal, continuous motor and cognitive development compared to their siblings who had MLD, but were untreated. The sibling of the patient designated “MLD01” was wheelchair-bound and unable to support their head and trunk at 39 months, but excitingly, after treatment, patient MLD01 was able to stand, walk and run at 39 months of age and showed signs of continuous motor and cognitive development. Lastly, and perhaps most importantly, there was no evidence of implanted cells becoming cancerous, even though they underwent self-renewal, like all good stem cells. This is the first report of an MLD patient at 39 months displaying such positive clinical features.

The second study treated WAS, which is an inherited disease that affects the immune system and leads to infections, abnormal platelets, scaly skin (eczema), blood tumors, and autoimmunity. In this second study, blood-making stem cells were collected from three patients infected with genetically engineered viruses that expressed the WASP gene. These stem cells were then reinfused intravenously (~11 million cells ) three days after collection. Blood tests and bone marrow biopsies showed evidence of robust engraftment of gene-corrected cells in bone marrow and peripheral blood up to 30 months later. WASP expression increased with time in most blood cells. Although serious adverse infectious events occurred in two patients, overall clinical improvement resulted in reduced disease severities in all patients. None of the three patients demonstrated signs of blood cancers and the platelet counts rose, but, unfortunately, not to normal levels. Again, no evidence for adverse effects were observed.

Simply put, these authors have presented a strategy for ex vivo gene correction in HSCs for inherited disorders which works and appears safe in comparison to previous strategies. Long-term analyses will undoubtedly need to be intensely scrutinized, but this research surely represents a huge step forward in the safe treatment of these and similar genetic disorders.

Stem Cell-based Baldness Cure One Step Closer

Scientists might be able to offer people with less that optimal amounts of hair new hope when it comes to reversing baldness. Researchers from the University of Pennsylvania say they’ve moved closer to using stem cells to treat thinning hair — at least in mice.

This group said that the use of stem cells to regenerate missing or dying hair follicles is considered a potential way to reverse hair loss. However, the technology did not exist to generate adequate numbers of hair-follicle-generating stem cells.

But new findings indicate that this may now be achievable. “This is the first time anyone has made scalable amounts of epithelial stem cells that are capable of generating the epithelial component of hair follicles,” Dr. Xiaowei Xu, an associate professor of dermatology at Penn’s Perelman School of Medicine, said in a university news release.

According to Xu, those cells have many potential applications that extend to wound healing, cosmetics and hair regeneration.

In their new study, Xu’s team converted induced pluripotent stem cells (iPSCs) – reprogrammed adult stem cells with many of the characteristics of embryonic stem cells – into epithelial stem cells. This is the first time this has been done in either mice or people.

The epithelial stem cells were mixed with certain other cells and implanted into mice. They produced the outermost layers of the skin and hair follicles that are similar to human hair follicles. This study was published in the Jan. 28 edition of the journal Nature Communications.

This suggests that these cells might eventually help regenerate hair in people.

Xu said this achievement with iPSC-derived epithelial stem cells does not mean that a treatment for baldness is around the corner. Hair follicles contain both epithelial cells and a second type of adult cells called dermal papillae.

“When a person loses hair, they lose both types of cells,” Xu said. “We have solved one major problem — the epithelial component of the hair follicle. We need to figure out a way to also make new dermal papillae cells, and no one has figured that part out yet.”

Experts also note that studies conducted in animals often fail when tested in humans.

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.

Synthetic Matrices that Induce Stem Cell-Mediated Bone Formation

Biomimetic matrices resemble living structures even though they are made from synthetic materials. Researchers in the laboratory of Shyni Varghese at the UC San Diego Jacobs School of Engineering have used calcium phosphate to direct mesenchymal stem cells to form bone. In doing so, Varghese and his colleagues have identified a surprising pathway from biomaterials to bone.

Varghese and his colleagues think that their work may point out new targets for treating bone defects, such as major fractures, and bone metabolic disorders such as osteoporosis.

The first goal of this research was to use materials to build something that looked like bone. This way, stem cells harvested from bone marrow (the squishy stuff inside our bones) could sense the presence of bone and differentiate into osteoblasts, the cells in our bodies that build bone.

“We knew for years that calcium phosphate-based materials promote osteogenic differentiation of stem cells, but none of use knew why.” said Varghese. “As engineers, we want to build something that is reproducible and consistent, so we need to know how building factors contribute to this end.”

Varghese and co-workers discovered that phosphate ions dissolved from calcium phosphate-based materials and these stray phosphate ions are taken up by the stem cells and used for the production of adenosine triphosphate or ATP. ATP is the energy currency of the cell, and it is the way cells store energy in a form that is readily usable for powering other reactions.

In stem cells, the generation of ATP eventually increases the intracellular concentration of the ATP breakdown product adenosine, and adenosine signals to stem cells to differentiate into osteoblasts and make bone.

Varghese said that she was surprised that “the biomaterials were connected to metabolic pathways. And we didn’t know how these metabolic pathways could influence stem cells,” and their commitment to bone formation.

These results also explain another clinical observation. Plastic surgeons have been using fat-based stem cells for eyelid lifts, breast augmentation, and other types of reconstructive surgeries. In once case, a plastic surgeon injected a dermal filler that contained calcium hydroxyapatite with the fat-based stem cells into a woman’s eyelid to provide an eye lift. However, the stem cells formed bone, and the poor lady’s lid painfully clicked every time she blinked and she had to have surgery to remove the ectopic bone. These results from Varghese’s laboratory explains why these fat-based stem cells formed bone in this case, and great care should be taken to never use such fillers in fat-based transplantation procedures.

Micro-Grooved Surfaces Influence Stem Cell Differentiation

Martin Knight and his colleagues from the Queen Mary’s School of Engineering and Materials Science and the Institute of Bioengineering in London, UK have shown that growing adult stem cells on micro-grooved surfaces disrupts a particular biochemical pathway that specified the length of a cellular structure called the “primary cilium.” Disruption of the primary cilium ultimately controls the subsequent behavior of these stem cells.

Primary cilia are about one thousand times narrower than a human hair. They are found in most cells and even though they were thought to be irrelevant at one time, this is clearly not the case.

Primary Cilium

The primary cilium acts as a sensory structure that responds to mechanical and chemical stimuli in the environment, and then communicates that external signal to the interior of the cell.  Most of the basic research on this structure was done using a single-celled alga called Chlamydomonas.

Martin Knight and his team, however, are certain that primary cilia in adult stem cells play a definite role in controlling cell differentiation.  Knight said, “Our research shows that they [primary cilia] play a key role in stem cell differentiation.  We found it’s possible to control stem cell specialization by manipulating primary cilia elongation, and that this occurs when stem cells are grown on these special grooved surfaces.”

When mesenchymal stromal cells were grown on grooved surfaces, the tension inside the cells was altered, and this remodeled the cytoskeleton of the cells.  Cytoskeleton refers to a rigid group of protein inside of cells that act as “rebar.” for the cell.  If you have ever worked with concrete, you will know that structural use of concrete requires the use of reinforcing metal bars to prevent the concrete from crumbling under the force of its own weight.  In the same way, cytoskeletal proteins reinforce the cell, give it shape, help it move, and help it resist shear forces.  Remodeling of the cytoskeleton can greatly change the behavior of the cell.

The primary cilium is important for stem cell differentiation.  Growing mesenchymal stromal cells on micro-grooved surfaces disrupts the primary cilium and prevents stem cell differentiation.  This simple culture technique can help maintain stem cells in an undifferentiated state until they have expanded enough for therapeutic purposes.

Once again we that there are ways to milk adult stem cells for all they are worth.  Destroying embryos is simply not necessary to save the lives of patients.