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.

Phase 2 Clinical Trial that Tests Stem Cell Treatment for Heart Attack Patients to be Funded by California Institute for Regenerative Medicine

A new stem cell therapy that treats heart attack patients with cells from a donor has been approved to begin a Phase 2 clinical trial.

Capricor Therapeutics Inc. a regenerative medicine company, has developed this treatment, which extracts donor stem cells from the heart called “cardiosphere-derived cells,” and then infuses them into the heart of the heart attack patient by means of a heart catheter procedure, which is quite safe. These stem cells are introduced into the heart to reduce scarring in the heart and potentially replace dead heart muscle cells. One clinical trial called the CADUCEUS trial has already shown that cardiosphere-derived cells can reduce the size of the heart scar.

In a previous phase I study (phase I studies typically only ascertain the safety of a treatment), cardiosphere-derived cells were infused into the hearts of 14 heart attack patients. No major safety issues were observed with these treatments, and therefore, phase 2 studies were warranted.

Alan Trounson, Ph.D., president of the California Institute for Regenerative Medicine (CIRM), which is funding the trial, said this about the phase 2 trial approval: “This is really encouraging news and marks a potential milestone for the use of stem cells to treat heart disease. Funding this type of work is precisely what our Disease Team Awards were designed to do, to give promising treatments up to $20 million dollars to develop new treatments for some of the deadliest diseases in America.”

Capricor was given approval by the National Heart Lung and Blood Institute (NHLBI) Gene and Cell Therapy (GST) to move into the next phase of clinical trials after these regulatory bodies had thoroughly reviewed the safety data from the phase 1 study. After NHLBI and GST determined that the phase 1 study met all the required goals, CIRM also independently reviewed the safety data from the Phase 1 and other aspects of the Phase 2 clinical trial design and operations. Upon successful completion of the independent review, Capricor was given approval to move forward into the CIRM-funded Phase 2 component of the study

Capricor CEO Linda Marbán, Ph.D., said, “Meeting the safety endpoints in the Phase 1 portion of the trial is a giant leap forward for the field and for Capricor Therapeutics. By moving into the Phase 2 portion of this trial, we can now attempt to replicate the results in a larger population.”

For the next phase, an estimated 300 patients who have had heart attacks will be evaluated in a double-blind, randomized, placebo-controlled trial. One group of heart-attack patients will include people 30 to 90 days following the heart attack, and a second group will follow patients 91 days to one year after the incident. Other patients will receive placebos and neither the patients nor the treating physicians know who will receive what.  This clinical trial should definitely determine if an “off-the-shelf” stem cell product can improve the function of a heart attack patient’s heart.

The California Institute for Regenerative Medicine (CIRM) is funding this clinical trial, and for this CIRM should be lauded.  However, when CIRM was brought into existence through the passage of proposition 71, it sold itself as a state-funded entity that would deliver embryonic stem cell-based cures.  Now I know that director Alan Trounson has denied that, but Wesley Smith at the National Review “Human Exceptionalism” blog and the LA times blogger Michael Hiltzik have both documented that Trounson and others said exactly that.  Isn’t ironic that one of the promises intimated by means of embryo-destroying research is now being fulfilled by means of non-embryo-destroying procedures?  If taxpayer money is going to fund research like this, then I’m all for it, but CIRM has to first clean up its administrative act before they deserve a another penny of taxpayer money.

“Noncontroversial” Embryonic Stem Cells?

An article from Bioscience Technology, a working scientist’s rag, has argued that everyone can have their lifetime supply of embryonic stem cells. Below is a summary of the article, after which I will comment on it.

Susan Fisher is the director of the UCSF Human Embryonic Stem Cell program. Last week, her lab reported that they have efficiently created embryonic stem cell lines from the cells removed from early embryos for Preimplantation Genetic Diagnosis (PGD) clinics. PGD takes a single cell from an early embryo that was created by means of in vitro fertilization, and subjects that single cell to genetic analyses to determine if the embryo carries a genetic disease. Because early human embryos have the ability to “regulate,” the removal of a single simply spurs the cells of the embryo to undergo extra cell divisions. The embryos subjected to PGD are then either destroyed, if they harbor a genetic disease, or implanted into the mother’s womb and gestated.

However, these cells removed from embryos could also be used to make an embryonic stem cell culture, since they could be seeded in culture to make an embryonic stem (ES) cell line. Therefore, in theory, cells could now be routinely removed from in vitro fertilization (IVF) clinic embryos, to provide them with a lifetime supply of their own embryonic stem cells. Because these cells were made without destroying embryos, they would be uncontroversial.

“Back in the mid-2000’s, when California was trying to decide whether to fund ES cell research, thousands of interested people would come out to hear us speak about topics like this,” says Fisher, interviewed after her report to the New York Stem Cell Foundation conference last week. “It is possible this particular, refined approach will generate that kind of interest now.”

ES cells have the greatest potency of any human stem cells and they can potentially form every cell type in the adult human body. Because such cells were recently harvested, they would not possess any of the mutations that ES cultures can acquire when they are grown for long periods of time in culture.

Traditionally, ES cell lines have been derived from stored, spare embryos from IVF clinics that were donated by other patients. Therefore, they are not immunologically identical to patients who potentially need them. Patients who receive non-matching tissues must take harsh immunosuppressive drugs for years to avoid rejecting the cells, and even then, over time the immune eventually wins the fight in some cases.

In recent years, scientists have turned to induced Pluripotential Stem Cells (IPSCs). IPSCs are made by genetically engineering adult cells to express four genes that de-differentiate the cells so that they are embryonic-like cells. IPSCs have been a boon to research, since scientists hace used them to make “disease in a dish” models on which to try drugs. But IPSCs are often riddled with mutations, as they come from adults. They have not yet hit the clinic as a result (although trials are upcoming).

However, Fisher, following on the heels of very preliminary work published in the journal Nature by the biotechnology company ACT, has refined the ability to create possibly uncontroversial stem cells—that are immunological matches to patients. By removing one cell from a very young human embryo, Fisher thinks that scientist might be able to produce a veritably unlimited supply of ES cells that are immunologically identical to the embyros from which they came. And as the embryos aren’t destroyed, but implanted into the mothers’ uteruses, the derivation of these tailor-made ES cells should be uncontroversial. “We will see how this is received,” Fisher says.

The process, she reported, is robust, if still not easy to pull off. This procedure, however, is labor-intensive and required a great deal of skill to pull off. In Fisher’s lab at UCSF, they derived ten human ES cell lines from four eight-cell embryos and one 12-cell embryo from a single couple.

When compared to standard ES cells, the UCSF lines were healthy and “formed derivatives of the three germ layers” like standard ES cells. Furthermore, these cells could form trophoblasts (placental cells), and Fisher’s team used them to create the first human trophoblast stem cell line. This is something that standard ES cells cannot do and this could make the UCSF cells useful in the clinic for diseases affecting the placenta.

Will patients begin turning to such cells? A few companies in the mid-2000s started offering designer ES cells like these, but that practice ended due to lack of interest or understanding, Fisher says. Additionally, some technical problems—later fully rectified—associated with the earlier Nature ACT paper may have cast a pall on enthusiasm for the approach, others in the field note.

“It remains to be seen if a place will be found for both iPS and ES cells,” Fisher concludes.

Now follows my comments:

Human embryos are very young human beings.  They do not have the right to vote, own property, or get a driver’s license, but they at least have the right not to be harmed.  By withdrawing cells from the embryo, you are potentially harming it.  “But wait,” proponents will tell you, “there are hundreds or even thousands of children who have been born who grew from embryos that were subjected to PGD and their rates of birth defects are no higher than everyone else’s.”  So their rates of birth defects are lower, but have we followed them for the rest of their lives to establish that removing a blastomere during early development does no harm?

“Oh come on,” you say.  But there are studies in mice that show that removing blastomere from early embryos does not cause higher rates of birth defects, but it does cause higher rates of neurological defects that manifest later in life.  Yu and others found that “mice generated after blastomere biopsy showed weight increase and some memory decline compared with the control group. Further protein expression profiles in adult brains were analyzed by a proteomics approach. A total of 36 proteins were identified with significant differences between the biopsied and control groups, and the alterations in expression of most of these proteins have been associated with neurodegenerative diseases. Furthermore hypomyelination of the nerve fibers was observed in the brains of mice in the biopsied group. This study suggested that the nervous system may be sensitive to blastomere biopsy procedures and indicated an increased relative risk of neurodegenerative disorders in the offspring generated following blastomere biopsy.”  In another paper, Yang and others showed that “blastomere biopsy, increases the rate of embryo death at 4.5-7.5 dpc, but does not affect the development of surviving 7.5 dpc embryos.”  In human embryos, time-lapse photography of biopsied embryos by Kirkegaard K, Hindkjaer JJ and Ingerslev HJ showed that “blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.”  Finally, Sugawara and others showed that “The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.”

There is reason to believe that this procedure potentially hurts the embryo.  Also, not all blastomeres in the early embryo are equally competent to make ES lines (see Lorthongpanich et al., Reproduction. 2008 Jun;135(6):805-1).  Therefore, if more than one blastomere must be taken from the embryo, the risks to it definitely increases (see Groossens et al., Hum. Reprod. (2008) 23 (3): 481-492).  The embryo has a basic right not to be harmed, but PGD potentially harms it without its consent.  This is barbaric.  With any other procedure we would say so, but this seems to be alright because we are dealing with embryos and they are too small and young.  This is ageism and size discrimination.  These are not “uncontroversial stem cells.”  They are anything but.  

Overexpression of a Potassium Channel in Heart Muscle Cells Made From Embryonic Stem Cells Decreases Their Arrhythmia Risk

Embryonic stem cells have the capacity to differentiate into every cell in the adult body. One cell type into which embryonic stem cells (ESCs) can be differentiated rather efficiently is cardiomyocytes, which is a fancy term for heart muscle cells. The protocol for making heart muscle cells from ESCs is well worked out, and the conversion is rather efficient and the purification schemes that have been developed are also rather effective (for example, see Cao N, et al., Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res. 2013 Sep;23(9):1119-32. doi: 10.1038/cr.2013.102 and Mummery CL et al., Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res. 2012 Jul 20;111(3):344-58).

Using these cells in a clinical setting has two large challenges. The first is that embryonic stem cell derivatives are rejected by the immune system of the recipient, thus setting up the patient for a graft versus host response to the implanted tissue, thus making the patient even sicker than when they started. The second problem is that heart muscle cells made from ESCs are immature and cause the heart to beat abnormally fast thus causing “tachyarrythmias” and died within the first two weeks after the transplant (see Liao SY, et al., Heart Rhythm 2010 7:1852-1859).

Both of these problems are large problems, but the laboratory of Ronald Li at the University of Hong Kong at used a genetic engineering trick to make heart muscle cells from mouse embryonic stem cells to seemingly fix this problem.

Li and his colleagues engineered mouse ESCs with a gene for a potassium rectifier channel that could be induced with drugs. Then they differentiated these genetically ESCs into heart muscle cells. This potassium rectifier channel (Kir2.1) is not present in immature heart muscle cells and putting it into these cells might cause them to beat at a slower rate.

These engineered ESC-derived heart muscle cells were tested for their electrophysiological properties first. Without the drug that induces KIR2.1, the heart muscle cells showed very abnormal electrical properties. However, once the drug was added, their electrical properties looked much more normal.

Then they induced heart attacks in laboratory animals and implanted their engineered ESC-derived heart muscle cells 1 hour after the heart attacks were induced. Animals not given the drug to induce the expression of Kir2.1 faired very poorly and had episodes of tachyarrythmia (really fast heart beat) and over half of them died by 5 weeks after the implantation. Essentially the implanted animals did worse than those animals that had had a heart attack that were not treated. However, those animals that were given the drug that induces the expression of Kir2.1 in heart muscle cells did much better. The survival rate of these animals was higher than the untreated animals after about 7 weeks after the procedure. Survival rates increased by only a little, but the increase was significant. Also, the animals that died did not die of tachyarrythmias. In fact the rate of tachyarrythmias in the animals given the inducing drug (which was doxycycline by the way) had significantly lower levels of tachyarrythmia than the other two groups.

Other heart functions were also significantly affected. The ejection fraction in the animals that ha received the Kir2.1-expression heart muscle cells was 10-20% higher than the control animals. Also the density of blood vessels was substantially higher in both sets of animals treated with ESC-derived heart muscle cells. The echocardiogram of the hearts implanted with the Kir2.1-expressing heart muscle cells was altogether more normal than that of the others.

This paper is a significant contribution to the use of ESC-derived cells to treat heart patients. The induction of heart arrhythmias by ESC-derived heart muscle cells is a documented risk of their use. Li and his colleagues have effectively eliminated that risk in this paper by forcing the expression of a potassium rectifier channel in the ESC-derived heart muscle cells. Also, because these cells were completely differentiated and did not have any interloping pluripotent cells in their culture, tumor formation was not observed.

There are a few caveats I would like to point out. First of all, the increase in survival rate above the control is not that impressive. The improvement in heart function parameters is certainly encouraging, but because the survival rates are not that higher than the control mice that received no treatment, it appears that these benefits were only conferred to those mice who survived in the first place.

Secondly, even though the heart attacks were induced in the ventricles of the heart, Li and his colleagues injected a mixture of heart muscle cells that included atrial, ventricular, nodal and heart fibroblasts. This provides an opportunity for beat mismatches and a “substrate for ventricular tachycardia” as Li puts it. In the future, the transplantation of just ventricular heart muscle cells would be cleaner experiment. Since these mice were not observed long enough to observe potential arrythmias that might have arisen from the presence of a mixed population in the ventricle.

Finally, in adapting this to humans might be difficult, since the hearts of mice beat so much faster than those of humans. It is possible that even if human cardiomyocytes were engineered with Kir2.1-type channels, that arrythmias might still be a potential problem.

Despite all that, Li’s publication is a large step forward.

Increased Flexibility in Induced Pluripotent Stem Cell Derivation Might Solve Tumor Concerns

Regenerative medicine depends on stem cells for the promises that it can potentially deliver to ailing patients. Training stem cells to repair injured tissues with custom-grown tissue substitutes and to replace dead cells are some of the goals of regenerative medicine. A major player in regenerative medicine is induced pluripotent stem cells (iPSCs), which are made from a patient’s own tissues. Because iPSCs are derived from a patient’s own cells, their chance of being rejected by the patient’s own immune system is rather low. Unfortunately, Shinya Yamanaka’s formula for making iPSCs, for which he was awarded last year’s Nobel Prize, utilizes a strict recipe that uses a precise combination of genes, some of which increase the risk of cancer risk, and, therefore, restricts their full potential for clinical application.

From left: Emmanuel Nivet and Juan Carlos Belmonte. Seated: Ignacio Sancho Martinez. (Source: Salk Institute for Biological Studies)
From left: Emmanuel Nivet and Juan Carlos Belmonte. Seated: Ignacio Sancho Martinez. (Source: Salk Institute for Biological Studies)

However, the laboratory Juan Carlos Izpisua Belmonte and his colleagues at the Salk Institute have published a paper in the journal Cell Stem Cell that shows that the recipe for iPSCs is much more versatile than originally thought. For the first time, Izpisua Belmonte and his colleague have replaced a gene that was once thought to be impossible to substitute in the production of iPSCs. This creates the potential for more flexible recipes that should speed the adoption of iPSCs for stem cell-based therapies.

Pluripotent stem cells come from two main sources. Embryonic stem cells (ESCs) are derived from early human blastocyst-stage embryos. The cells of the inner cell mass are extracted and these immature cells that have never differentiated into specific cell types, and are cultured, grown, and propagated to form an embryonic stem cell line. Secondly, induced pluripotent stem cells or iPSCs, are derived from mature cells that have been reprogrammed back into an undifferentiated state. In 2006 by Yamanaka introduced four different genes into a mature cell to reprogram the cell to pluripotency. This pluripotent cell can be cultured and grown into an iPSCs line. Because of Yamanaka’s initial success in iPSC production, most stem cell researchers adopted his recipe, even though variations on his protocol have been examined and used.

Izpisua Belmonte and his colleagues used a fresh approach for the derivation of iPSCs. They played around with the Yamanka protocol and in doing do discovered that pluripotency (the stem cell’s ability to differentiate into nearly any kind of adult cell) can also be programmed into adult cells by “balancing” the genes required for differentiation. What genes? Those genes that code for “lineage transcription factors,” which are proteins that direct stem cells to differentiate first into a particular cell lineage, or type, such as a blood cell versus a skin cell, and then finally into a specific cell, such as a white blood cell.

“Prior to this series of experiments, most researchers in the field started from the premise that they were trying to impose an ’embryonic-like’ state on mature cells,” says Izpisua Belmonte, who holds the Institute’s Roger Guillemin Chair. “Accordingly, major efforts had focused on the identification of factors that are typical of naturally occurring embryonic stem cells, which would allow or further enhance reprogramming.”

Despite these efforts, there seemed to be no way to determine through genetic identity alone that cells were pluripotent. Instead, pluripotency was routinely evaluated by functional assays. In other words, if it acts like a stem cell, it must be a stem cell.

That condition led the team to their key insight. “Pluripotency does not seem to represent a discrete cellular entity but rather a functional state elicited by a balance between opposite differentiation forces,” says Izpisua Belmonte.

Once they understood this, they realized the four extra genes weren’t necessary for pluripotency. Instead, adult cells could be reprogrammed by altering the balance of “lineage specifiers,” genes that were already in the cell that specified what type of adult tissue a cell might become.

“One of the implications of our findings is that stem cell identity is actually not fixed but rather an equilibrium that can be achieved by multiple different combinations of factors that are not necessarily typical of ESCs,” says Ignacio Sancho-Martinez, one of the first authors of the paper and a postdoctoral researcher in Izpisua Belmonte’s laboratory.

Izpisua Belmonte’s laboratory showed that more than seven additional genes can facilitate reprogramming adult cells to iPSCs. Most importantly, for the first time in human cells, they were able to replace a gene from the original recipe called Oct4, which had been replaced in mouse cells, but was still thought indispensable for the reprogramming of human cells. Their ability to replace it, as well as SOX2, another gene once thought essential that had never been replaced in combination with Oct4, demonstrated that stem cell development must be viewed in an entirely new way. In point of fact, Belmonte’s group showed that genes that specify mesendodermal lineage can replace OCT4 in human iPSC generation, and ectodermal lineage specifiers are able to replace SOX2 in hiPSC generation. Simultaneous replacement of OCT4 and SOX2 allows human cell reprogramming to iPSCs

“It was generally assumed that development led to cell/tissue specification by ‘opening’ certain differentiation doors,” says Emmanuel Nivet, a post-doctoral researcher in Izpisua Belmonte’s laboratory and co-first author of the paper, along with Sancho-Martinez and Nuria Montserrat of the Center for Regenerative Medicine in Barcelona, Spain.

Instead, the successful substitution of both Oct4 and SOX2 shows the opposite. “Pluripotency is like a room with all doors open, in which differentiation is accomplished by ‘closing’ doors,” Nivet says. “Inversely, reprogramming to pluripotency is accomplished by opening doors.”

This work should help to overcome one of the major hurdles in the widespread adoption of iPSC-based therapies; namely, that the original four genes used to reprogram stem cells had been implicated in cancer. “Recent studies in cancer, many of them done by my Salk colleagues, have shown molecular similarities between the proliferation of stem cells and cancer cells, so it is not surprising that oncogenes [genes linked to cancer] would be part of the iPSC recipe,” says Izpisua Belmonte.

With this new method, which allows for a customized recipe, the team hopes to push therapeutic research forward. “Since we have shown that it is possible to replace genes thought essential for reprogramming with several different genes that have not been previously involved in tumorigenesis, it is our hope that this study will enable iPSC research to more quickly translate into the clinic,” says Izpisua Belmonte.

Other researchers on the study were Tomoaki Hishida, Sachin Kumar, Yuriko Hishida, Yun Xia and Concepcion Rodriguez Esteban of the Salk Institute; Laia Miquel and Carme Cortina of the Center of Regenerative Medicine in Barcelona, Spain.

Stress-Resistant Stem Cells From Fat

During liposuction patients lose a fat cells, fat-based mesenchymal stem cells, and now, according to new results from UCLA scientists, stress-enduring stem cells.

This new stem cell population has been called a Multi-lineage Stress-Enduring Adipose Tissue or Muse-AT stem cells. UCLA scientists found Muse-AT stem cells by accident when a particular machine in the laboratory malfunctioned, killing all the cells found in cells from human liposuction, with the exception on the Muse-AT stem cells.

Gregorio Chazenbalk from the UCLA Department of Obstetrics and Gynecology and his research team discovered, after further tests on Muse-AT stem cells, that they not only survive stress, but might be activated by it.

The removal of Muse-AT stem cells from the human body by means of liposuction revealed cells that express several embryonic stem cell-specific proteins (SSEA3, TR-1-60, Oct3/4, Nanog and Sox2). Furthermore, Muse-AT stem cells were able to differentiate into muscle, bone, fat, heart muscle, liver, and neuronal cells. Finally, when Chazenbalk and his group examined the properties of Muse-AT stem cells, they discovered that these stem cells could repair and regenerate tissues when transplanted back into the body after having been exposed to cellular stress.

Muse-ATs express pluripotent stem cell markers. Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, [16] were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X. doi:10.1371/journal.pone.0064752.g002
Muse-ATs express pluripotent stem cell markers.
Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, [16] were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X.
“This population of cells lies dormant in the fat tissue until it is subjected to very harsh conditions. These cells can survive in conditions in which usually cancer cells can survive. Upon further investigation and clinical trials, these cells could prove a revolutionary treatment option for numerous diseases, including heart disease, stroke and for tissue damage and neural regeneration,” said Chazenbalk.

Purifying and isolating Muse-AT stem cells does not require the use of a cell sorter or other specialized, high-tech machinery. Muse-AT stem cell can grow in liquid suspension, where they grow as small spheres or as adherent cells that pile on top of each other to form aggregates, which is rather similar to embryonic stem cells and the embryoid bodies that they form.

Isolation and morphologic characterization of Muse-ATs. (A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity. doi:10.1371/journal.pone.0064752.g001
Isolation and morphologic characterization of Muse-ATs.
(A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity.

We have been able to isolate these cells using a simple and efficient method that takes about six hours from the time the fat tissue is harvested,” said Chazenbalk. “This research offers a new and exciting source of fat stem cells with pluripotent characteristics, as well as a new method for quickly isolating them. These cells also appear to be more primitive than the average fat stem cells, making them potentially superior sources for regenerative medicine.”

Embryonic stem cells and induced pluripotent stem cells are the two main sources of pluripotent stem cells. However, both of these stem cells have an uncontrolled capacity for differentiation and proliferation, which leads to the formation of undesirable teratomas, which are benign tumors that can become teratocarcinomas, which are malignant tumors. According to Chazenbalk, little progress has been made in resolving this defect (I think he overstates this).

Muse-AT stem cells were discovered by a research group at Tokohu University in Japan and were isolated from skin and bone marrow rather than fat (see Tsuchiyama K, et al., J Invest Dermatol. 2013 Apr 5. doi: 10.1038/jid.2013.172). The Japanese group showed that Muse-AT stem cells do not form tumors in laboratory animals. The UCLA group was also unable to get Muse-AT stem cells to form tumors in laboratory animals, but more work is necessary to firmly establish that these neither form tumors nor enhance the formation of other tumors already present in the body.

Chazenbalk also thought that Muse-AT stem cells could provide an excellent model system for studying the effects of cellular stress and how cancer cells survive and withstand high levels of cellular stress.

Chazenbalk is understandable excited about his work, but other stem cells scientists remain skeptical that this stem cells population has the plasticity reported or that these cells are as easily isolated as Chazenbalk says.  For a more skeptical take on this paper, see here.

Developmental Regression: Making Placental Cells from Embryonic Stem Cells

A research group from Copenhagen, Denmark has discovered a way to make placental cells from embryonic stem cells. In order to do this, the embryonic stem cells must be developmentally regressed so that they can become wither placenta-making cells rather than inner cell mass cells.

This study is significant for two reasons. First of all, it was thought to be impossible to make placental cells from embryonic stem cells because embryonic stem cells (ESCs) are derived from the inner cell mass cells of 4-5-day old human blastocysts. These early embryos begin as single-celled embryos that divide to form 12-16-cell embryos that undergo compaction. At this time, the cells on the outside become trophoblast cells, which will form the trophectoderm and form the placenta and the cells on the inside will form the inner cell mass, which will form the embryo proper and a few extraembryonic structures. Since ESCs are derived from inner cell mass cells that have been isolated and successfully cultured, they have already committed to a cell fate that is not placental. Therefore, to differentiate ESCs into placental cells would require that ESCs developmentally regress, which is very difficult to do in culture.

Secondly, if this could be achieved, several placental abnormalities could be more easily investigated, For example, pre-eclampsia is a very serious prenatal condition that is potentially fatal to the mother, and is linked to abnormalities of the placenta. Studying a condition such as pre-eclampsia in a culture system would definitely be a boon to gynecological research.

Because human ESCs can express genes that are characteristic of trophoblast cells if they are treated with a growth factor called Bone Morphogen Protein 4 (BMP4), it seems possible to make placental cells from them (see Xu R.H., Chen X., Li D.S., Li R., Addicks G.C., Glennon C., Zwaka T.P., Thomson J.A. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 2002;20:1261–1264, and Xu RH. Methods Mol Med. 2006;121:189-202). However, a study by Andreia S. Bernardo and others from the laboratory of Roger Pedersen at the Cambridge Stem Cell Institute strongly suggested that BMP4 treatment, even in the absence of FGF signaling (another growth factor that has to be absent for BMP4 to induce trophoblast-like gene expression from ESCs) the particular genes induced by BMP4 are not exclusive to trophoblast cells and more closely resemble mesodermal gene profiles (see AS Bernardo, et al., Cell Stem Cell. 2011 Aug 5;9(2):144-55).

Into the fray of this debate comes a paper by stem cells scientists at the Danish Stem Cell Center at the University of Copenhagen that shows that it is possible to rewind the developmental state of ESCs.

In this paper, Josh Brickman and his team discovered that if they maintained mouse ESCs under specific conditions, they could cause the cells to regress into very early pre-blastocyst embryonic cells that can form trophoblast cells or ICM cells.

“It was a very exciting moment when we tested the theory, said Brinkman. “We found that not only can we make adult cells but also placenta, in fact we got precursors of placenta, yolk sac as well as embryo from just one cell.”

“This new discovery is crucial for the basic understanding of the nature of embryonic stem cells and could provide a way to model the development of the organism as a whole, rather than just the embryonic portion,” said Sophie Morgani, graduate student and first author of this paper. “In this way we may gain greater insight into conditions where extraembryonic development is impaired, as in the case of miscarriages.”

To de-differentiate the ESCs, Brinkman and his colleagues grew them in a solution called “2i.”  This 2i culture medium contained inhibitors of MEK and GSK3.  MEK is a protein kinase that is a central participant in the “MAP kinase signaling pathway, which is a signaling pathway that is central to cell growth and survival.  This particular signaling pathway is the target of the anthrax toxin, which illustrates its importance,  GSK3 stands for “glycogen synthase kinase 3,” which is a signaling protein in the Wnt pathway.

When the mouse ESCs were grown in 2i medium they expressed genes normally found only in pre-blastocyst embryos (Hex, for example).  Therefore, the 2i medium directs mouse ESCs to de-differentiate.  When ESCs grown in 2i were implanted into mouse embryos, they divided and differentiated into cells that were found in placental and embryonic fates.  This strongly argues that the ESCs grown in 2i became pre-blastocyst embryonic cells.  When the ESCs grown in 2i were also grown with LIF, which stands for “leukemia inhibitory factor” (LIF is a protein required for the maintenance of mouse ESCs in culture), the 2i cells were maintained in culture and grew while maintaining their pre-blastocyst status.  These cells differentiated into placental cells, embryonic or fetal cells.  Essentially, the 2i-cultured cells when from being pluripotent to being “totipotent,” or able to form ALL cell types in the embryo, fetus, or the adult.

ESC de-differentiation in totipotence

“In our study we have been able to see the full picture unifying LIF’s functions: what LIF really does, is to support the very early embryo state, where the cells can make both embryonic cells and placenta. This fits with LIFs’ role in supporting implantation,” said Brinkman.

This study definitively shows that ESCs are NOT embryos.  ESCs can regress in their development but embryos develop forward, becoming more committed as they develop and more restricted in the cell fates they can form.  This should effectively put the nail in the coffin of Lee Silver’s argument against Robert P. George that embryonic stem cells are embryos.  They are definitely and unequivocally, since embryos do NOT develop in reverse, but ESCs can and do.

Robert P. George argues that early human embryos, like the kind used to make ESCs are very young  members of the human race and deserve, at the minimum, the right not to be harmed.  Silver counters that George’s argument is inconsistent because George would not extend the same right to an ESC cell line, which is the same as an embryo.  His reasoning is that mouse ESCs can be transplanted into other mouse embryos that have four copies of each chromosome.  The messed up mouse embryo will make the placenta and the ESCs will make the inner cell mass and the mouse will develop and even come to term.  This is called tetraploid rescue, and Silver thinks that this procedure is a minor manipulation, but that it shows that ESCs are functionally the same as embryos.

I find Silver’s argument wanting on just about all fronts.  This is not a minor manipulation.  The tetraploid embryo is bound for certain death, but the implanted ESCs use the developmental context of the tetraploid embryo to find their place in it and make the inner cell mass.  The ESCs do not do it all on their own, but instead work with the tetraploid embryo in a complex developmental give-and-take to make an embryo with the placenta from one animal and the embryo proper from another.

Thus Silver’s first argument does not demonstrate what he says it does.  All it demonstrates is that ESCs can contribute to an embryo, which is something we already knew and expected.  This new data completes blows Silver’s assertion out of the water, since ESCs can take developmental steps backward and embryos by their very nature and programming, do not.  Thus these two entities are distinct entities and are not identical.  The early embryo is a very young human person, full stop.  We should stop dismembering them in laboratories just to stem our scientific curiosity.