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.

Controlling Transplanted Stem Cells from the Inside Out

Scientists have worked very hard to understand how to control stem cell differentiation.  However, despite how well you direct stem cell behavior in culture, once those stem cells have been transplanted, they will often do as they wish.  Sometimes, transplanted stem cells surprise people.

Several publications describe stem cells that, once transplanted undergo “heterotropic differentiation.” Heterotropic differentiation refers to tissues that form in the wrong place. For example, one lab found that transplantation of mesenchymal stem cells into mouse hearts after a heart attack produced bone (don’t believe me – see Martin Breitbach and others, “Potential risks of bone marrow cell transplantation into infarcted hearts.” Blood 2007 110:1362-1369).  Bone in the heart – that can’t be good. Therefore, new ways to control the differentiation of cells once they have been transplanted are a desirable goal for stem cell research.

From this motivation comes a weird but wonderful paper from Jeffrey Karp and James Ankrum of Brigham and Women’s Hospital and MIT, respectively, that loads stem cells with microparticles that give the transplanted stem cell continuous cues that tell them how to behave over the course of days or weeks as the particles degrade.

“Regardless of where the cell in the body, it’s going to be receiving its cues from the inside,” said Karp. “This is a completely different strategy than the current method of placing cells onto drug-doped microcarriers or scaffolds, which is limiting because the cells need to remain in close proximity to those materials in order to function. Also these types of materials are too large to be infused into the bloodstream.”

Controlling cells in culture is relatively easy. If cells take up the right molecules, they will change their behavior. This level of control, however, is lost after the cell is transplanted. Sometimes implanted cells readily respond to the environment within the body,. but other times, their behavior is erratic and unpredictable. Karp’s strategy, which her called “particle engineering,” corrects this problem by turning cells into pre-programmable units. The internalized particles stably remain inside the transplanted cell and instruct it precisely how to act. It can direct cells to release anti-inflammatory factors, or regenerate lost tissue and heal lesions or wounds.

“Once those particles are internalized into the cells, which can take on the order of 6-24 hours, we can deliver the transplant immediately or even cryopreserve the cells,” said Karp. “When the cells are thawed at the patient’s bedside, they can be administrated and the agents will start to be released inside the cells to control differentiation, immune modulation or matrix production, for example.”

It could take more than a decade for this type of cell therapy to be a common medical practice, but to speed up the pace of this research, Karp published the study to encourage others in the scientific community to apply the technique to their various fields. Karp’s paper also illustrates the range of different cell types that can be controlled by particle engineering, including stem cells, cells of the immune system, and pancreatic cells.

“With this versatile platform, which leveraged Harvard and MIT experts in drug delivery, cell engineering, and biology, we’ve demonstrated the ability to track cells in the body, control stem cell differentiation, and even change the way cells interact with immune cells, said Ankrum, who is a former graduate student in Karp’s laboratory. “We’re excited to see what applications other researchers will imagine using this platform.”

Growing Intestinal Stem Cells

Researchers from MIT and Brigham and Women’s Hospital in Boston, MA have discovered a protocol that allows them to grow unlimited quantities of intestinal stem cells. These intestinal stem cells can then be induced to differentiate into pure populations of various types of mature intestinal cells. Scientists can used these cultured intestinal cells to develop new drugs and treat gastrointestinal diseases, such as Crohn’s disease or ulcerative colitis.,

The small intestine has a small repository of adult stem cells that differentiate into mature adult cells that have specialized functions. Until recently, there was no good way to grow large numbers of these intestinal stem cells in culture. Intestinal stem cells, you see, only retain their immature characteristics when they are in contact with supportive cells known as Paneth cells.

paneth cells

In order to grow intestinal stem cells in culture, researchers from the laboratories of Robert Langer at the MIT Koch Institute for Integrative Cancer Research and Jeffrey Karp from the Harvard Medical School and Brigham and Women’s Hospital, determined the specific molecules that Paneth cells make that keep the intestinal stem cells in their immature state. Then they designed small molecules that mimic the Paneth cell-specific molecules. When Langer and Karp’s groups grew the intestinal stem cells in culture with those small molecules, the cells remained immature and grew robustly in culture.

Langer said, “This opens the door to doing all kinds of thing, ranging from someday engineering a new gut for patients with intestinal diseases to doing drug screening for safety and efficacy. It’s really the first time this has been done.”

The inner mucosal layer of the intestine has several vital functions: the absorption of nutrients, the secretion of mucus of create a barrier between our own cells and the bacteria and viruses and habitually inhabit our bowels, and alerting the immune system to the presence of potential disease-causing agents in the bowel.

The intestinal mucosa is organized into a collection of folds with small indentations called “intestinal crypts.”  At the bottom of each crypt is a small pool of intestinal stem cells that divide to routinely replace the specialized cells of the intestinal epithelium.  Because the cells of the intestinal epithelium show a high rate of turnover (they only last for about five days), these stem cells must constantly divide to replenish the intestine.


Once these intestinal stem cells divide, they can differentiate into any type of mature intestinal cell type.  Therefore, these intestinal stem cells provide a marvelous example of a “multipotent stem cell.”

Obtaining large quantities of intestinal stem cells could certainly help gastroenterologists  treat gastrointestinal diseases that damage the epithelial layer of the gut.  Fortunately, recent studies in laboratory animals have demonstrated that the delivery of intestinal stem cells can promote the healing of ulcers and regeneration of new tissue, which offers a new way to treat inflammatory bowel diseases like ulcerative colitis.

This, however, is only one of the many uses for cultured intestinal stem cells.  Researchers are literally salivating over the potential of studying things like goblet cells, which control the immune response to proteins in foods to which many people are allergic.  Alternatively, scientists would like to investigate the properties of enteroendocrine cells, which secrete hunger hormones and play a role in obesity.  I think you can see, that large numbers of intestinal stem cells could be a boon to gastrointestinal research.

Karp said, “If we had ways of performing high-throughput screens of large numbers of these very specific cell types, we could potentially identify new targets and develop completely new drugs for diseases ranging from inflammatory bowel disease to diabetes.”

The laboratory of Hans Clevers in 2007 identified a molecule that is specifically made by intestinal stem cells called Lgr5.  Clevers is a professor at the Hubrecht Institute in the Netherlands and he and his co-workers have just identified particular molecules that enable intestinal stem cells to grow in synthetic culture.  In culture, these small clusters of intestinal stem cells differentiate and form small sphere-like structures called “organoids,” because they consist of a ball of intestinal cells that have many of the same organizational properties of our own intestines, but are made in culture.

Clevers and his colleagues tried to properly define the molecules that bind Paneth cells and intestinal stem cell together.  The purpose of this was to mimic the Paneth cells in culture so that the intestinal stem cells would grow robustly in culture.  Clevers’ team discovered that Paneth cells use two signal transduction pathways (biochemical pathways that cells use to talk to each other) to coordinate their “conversations” with the adjacent stem cells.  These two signal transduction pathways are the Notch and Wnt pathways.

Fortunately, two molecules could be used to induce intestinal stem cell proliferation and prevent their differentiation: valproic acid and CHIR-99021.  When Clevers and others grew mouse intestinal stem cells in the presence of these two compounds, they found that large clusters of cells grew that consisted of 70-90 percent pure stem cells.  When they used inhibitors of the Notch and Wnt pathway, they could drive the cells to form particular types of mature intestinal cells.

“We used different combinations of inhibitors and activators to drive stem cells to differentiate into specific populations of mature cells,” said Xiaolei Yin, first author of this paper.  Yin and others were able to get this strategy to work with mouse stomach and colon cells, and that these small molecules also drove the proliferation of human intestinal stem cells.

Presently, Clevers’ laboratory is trying to engineering intestinal tissues for potential transplantation in human patients and for rapidly testing the effects of drugs on intestinal cells.

Ramesh Shivdasani from Harvard Medical School and Dana-Farber Cancer Institute would like to use these cells to investigate what gives stem cells their ability to self-renew and differentiate into other cell types.  “There are a lot of things we don’t know about stem cells,” said Shivdasani.  “Without access to large quantities of these cells, it’s very difficult to do any experiments.  This opens the door to a systematic, incisive, reliable way of interrogating intestinal stem cell biology.”

X. Yi, et al. “Niche-independent high-purity cultures of Lgr5 intestinal stem cells and their progeny.” Nature Methods 2013; DOI:10.1038/nmeth.2737.