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.

New Liver Stem Cell Might Aid in Liver Regeneration

For patients with end-stage liver disease, a liver transplant is the only viable option to stave off death. Liver failure is the 12th leading cause of death in the United States, and finding a way to regenerate failing livers is one of the Holy Grails of liver research. New research suggests that one it will be feasible to use a patient’s own cells to regenerate their liver.

Researchers at the Icahn School of Medicine at Mount Sinai have discovered that a particular human embryonic stem cell line can be differentiated into a previously unknown liver progenitor cell that can differentiate into mature liver cells.

“The discovery of the novel progenitor represents a fundamental advance in this field and potentially to the liver regeneration field using cell therapy,” said Valerie Gouon-Evans, the senior author of this study and assistant professor of medicine at the Icahn School of Medicine. “Until now, liver transplantation has been the most successful treatment for people with liver failure, but we have a drastic shortage of organs. This discovery may help circumvent that problem.”

Gouon-Evans collaborated with the laboratory of Matthew J. Evans and showed that the liver cells that were made from the differentiating liver progenitor cells could be infected with hepatitis C virus. Since this is a property that is exclusive to liver cells, this result shows that these are bona fide liver cells that are formed from the progenitor cells.

One critical step in this study was the identification of a new cell surface protein called KDR, which is the vascular endothelial growth factor 2. KDR was thought to be restricted to blood vessels, blood vessels progenitor cells (EPCs), and blood cells.  However, the Evans / Gouon-Evans study showed that activation of KDR in liver progenitor cells caused them to differentiate into mature liver cells (hepatocytes).  KDR is one of the two receptors for VEGF or vascular endothelial growth factor.  Mutations of this gene are implicated in infantile capillary hemangiomas.

KDR Protein Crystal Structure
KDR Protein Crystal Structure

The next step in this work is to determine if liver cells formed from these embryonic stem cells could potentially facilitate the repair of injured livers in animal models of liver disease.

Misrepresentation of the Embryological Facts of Cloning by Reporters

Wesley Smith at National Review Online has been keeping tabs on the reporting of the Cell paper by Shoukhrat Miltalipov from the Oregon Health and Science University. The misrepresentation has been extensive but it’s not really all that surprising given the ignorance and lack of clear thinking on this issue. Nevertheless, Smith has kept up his yeoman’s work, cataloging the factual errors for reporters in multiple publications.

For his first example, see here, where Loren Grush on Fox wrote:

Through a common laboratory method known as somatic cell nuclear transfer (SCNT), ONPRC scientists, along with researchers at Oregon Health & Science University, essentially swapped the genetic codes of an unfertilized egg and a human skin cell to create their new embryonic stem cells…The combination of the egg’s cytoplasm and the skin cell’s nucleus eventually grows and develops into the embryonic stem cell.

Grush, as Smith points out, is quite wrong. Introducing a nucleus from a body cell into the unfertilized egg and inducing it does not turn the egg into embryonic stem cells, but turns it into a zygote. The zygote them undergoes cleavage (cell division) until it reaches the early/mid blastocyst stage 5-6 days later, then immunosurgery is used to isolated the inner cell mass cells, after which they are cultured. Somatic cell nuclear transfer is a stand-in for fertilization. It produces an embryo and all the redefinition in the world will not change that.

Next comes my favorite newspaper, the Wall Street Journal, which normally has decent to pretty good scientific reporting, but this one story from Gautam Naik contains a real howler:

Scientists have used cloning technology to transform human skin cells into embryonic stem cells, an experiment that may revive the controversy over human cloning. The researchers stopped well short of creating a human clone. But they showed, for the first time, that it is possible to create cloned embryonic stem cells that are genetically identical to the person from whom they are derived.

As Smith points out, Miltalipov and others did not stop short of creating a human clone, then explicitly made a cloned human embryo and therefore made a cloned young human being.

Then there is this humdinger from an online Australian news report:

US researchers have reported a breakthrough in stem cell research, describing how they have turned human skin cells into embryonic stem cells for the first time. The method described on Wednesday by Oregon State University scientists in the journal Cell, would not likely be able to create human clones, said Shoukhrat Mitalipov, senior scientist at the Oregon National Primate Research Center. But it is an important step in research because it doesn’t require the use of embryos in creating the type of stem cell capable of transforming into any other type of cell in the body.

Oh my gosh, folks the paper describes the production of cloned embryos expressly for the purpose of dismembering them and destroying them. This “doesn’t require the use of embryos” crap reveals a very basic ignorance of how the experiment was done. See Smith’s excellent post for more details.

Then there is this story from one of my least favorite papers, the LA Times:

Some critics continue to argue that it’s unethical to manipulate the genetic makeup of human eggs even if they’re unfertilized, and others warn about potential harm to egg donors. The biggest ethical issue for the OHSU team, though, is that it artificially created a human embryo, albeit one that was missing the components needed for implantation and development as a fetus.

Come on people! The cloned embryo does not have the components needed to implant because there is no womb into which it can be implanted. Dolly was made the same way. Surely Dolly had the components required to implant.  The problem here is one of will, since these embryos were made to be destroyed. Not capacity. What was done to those embryos was dismemberment. Would we object if they were toddlers?

Just to show that obfuscation is not wholly an American news feature, there is this story from the German newspaper Deutche Welle:

Scientists, for the first time, have cloned embryonic stem cells using reprogrammed adult skin cells, without using human embryos…The process used by Mitalipov is an important step in research because it does not require killing a human embryo–that is, a potential human being–to create transformative stem cells.

As Smith points out, this research made a human embryo that was then killed to make embryonic stem cells. Calling this research humane is to redefine humane to the point of absurdity.

Finally this jewel of blithering ignorance from bioethicist Jonathan Moreno in his column in the Huffington Post:

Despite some confused media reports, the Oregon scientists did not clone a human embryo but a blastocyst that lacks some of the cells needed to implant in a uterus.

And you wonder why people like me have lost all faith in American bioethics. As a developmental biologist, this one just grates on me.  A blastocyst has two cell populations; an outer trophectoderm composed of trophoblast cells that will form the placenta and the inner cell mass cells on the inside of the embryo, which will form the embryo proper and a few placental structures. To be a blastocyst is to have the equipment to implant.

To drive the nail into the coffin, Smith quotes the father of embryonic stem cells James Thomson from an MSNBC interview:

See, you are trying to redefine it away…If you create an embryo by nuclear transfer, if you gave it to somebody who didn’t know where it came from, there would be no test you could do on that embryo to say where it came from. It is what it is. By any reasonable definition, at least as some frequency, you are creating an embryo. If you try to redefine it away, you are being disingenuous.

Check out Smith’s posts. They are all worth reading. Maybe the press will learn some embryology, but I doubt it.

Postscript:  Brendan P. Foht writes at the Corner on National Review Online that in 2010 Shoukhrat Mitalipov, the leader of the Oregon cloning team, reported that he had achieved a single pregnancy using cloned monkey embryos that were made with exactly the same technology as was employed with human eggs in his 2013 Cell paper.  The fetus developed long enough to have a heartbeat detectable through ultrasound. Although the pregnancy failed after 81 days (about half the normal gestation period for that species), the fact that a pregnancy would develop so far indicates that reproductive cloning of primates is in principle possible.  This definitively shows that all this talk about the embryos made in Mitalipov’s lab not being able to implant is pure drek.

New Cell Type Derived from Embryonic Stem Cells for Possible Treatment of Brain Diseases

This story comes from my alma mater, UC Irvine. Go anteaters!! No really, UC Irvine’s mascot is the anteater, not to be confused with the aardvark.

Edwin Monuki at the Sue and Bill Gross Stem Cell Research Center with his graduate student Momoko Watanabe and other colleagues devised culture conditions to differentiate embryonic stem cells in “choroid plexus epithelial cells.” Monuki and he team were able to make choroid plexus epithelial cells (CPECs) from mouse and human embryonic stem cell (ESC) lines.

Now you are probably reading this and screaming, “what the heck are CPECs?” Calm down, we will explain:

The central nervous system is surrounded by a clear fluid known as cerebrospinal fluid or CSF. CSF flows all around the brain and spinal cord and also flows inside it. The CSF has several functions. These functions include buoyancy, protection, stability, and prevention of stroke (ischemia).

The CSF provides buoyancy to the brain, since the brain is large a potentially heavy. However, by filling the brain from within and around it with CSF decreases the density of the brain. CSF allows the brain to maintain a density that prevent it from collapsing under its own weight. If the brain were denser, then it would compact and cut off the blood supply of the cells in the lower part of the brain. This would kill off neurons.

Protection provided by the CSF comes during those times the head is struck. The CSF prevents the brain from coming into contact with the skull. The stability provided by the CSF

CSF flows throughout the inside of the brain through cavities known as “ventricles.” These ventricles provide reservoirs through which the CSF flows and is absorbed back into the bloodstream. This constant movement of the CSF through the CNS rinses it and removes metabolic wastes from the central nervous system. It also guarantees an even distribution of neural materials through the central nervous system.

CSF also helps prevent strokes, since the low pressure of the CSF in the skull translates into low intracranial pressure, which facilitates blood perfusion.

Now that have seen that CSF is very important in the life of the brain, where does it come from?  The answer is the CPECs make CSF as a fine filtrate from blood plasma. A minority of the CSF is also made by the walls of the brain ventricles. Nevertheless, CSF circulates from the lateral ventricles to the interventricular foramen, to the third ventricle, through the cerebral aqueduct, to the fourth ventricle, through the median aperture and lateral apertures to the subarachnoid space over the brain and spinal cord where arachnoid granulations return the CSF to the bloodstream.

Thus, CPECs make CSF, which helps remove metabolic wastes and other toxic compounds from the brain. In various neurodegenerative diseases, the choroid plexus and CPECs degenerate too and fail to efficiently remove debris and other rubbish from the central nervous system. Transplantation experiments in rodents have demonstrated that implanting a healthy set of CPECs can restore CSF function and slow down the damage done to the brain by neurodegenerative diseases (see Matsumoto et al., Neurosci Lett. 2010 Jan 29;469(3):283-8).  The problem is the lack of good cultures of CPECs.

Monuki and Watanabe and company seem to have fixed this problem. Monuki commented on his publication, “Our method is promising, because for the first time we can use stem cells to create large amounts of these epithelial cells (CPECs), which could be utilized in different ways to treat neurodegenerative diseases.” Monuki is an associate professor of pathology and laboratory medicine and cell and developmental biology at UC Irvine.

To make CPECs from ESCs, Monuki and his team differentiated cultured ESCs into neural stem cells. The neural stem cells were then differentiated into CPECs. This strategy makes a great deal of sense, since a neural stem cell population seems to exist in the CPECs (see Itokazu Y et al., Glia. 2006 Jan 1;53(1):32-42.

According to Monuki, there are three ways that these cells could be used to treat neurodegenerative diseases. First, the CPECs could be used to make more CSF and that would help flush out the proteins and other toxic compounds that kill off neurons. The down side of this is that it would also increase intracranial pressure, which is not optimal. Secondly,. the CPECs could be engineered into superpumps that transport high concentrations of therapeutic agents into the brain. Third, cultured CPECs could be used as a model system to screen drugs and other agents that shore up the endogenous CPECs in the patient’s brain.

Monuki’s next step is to develop an effective drug screening system in order conduct proof-of-concept studies to determine how CPECs afect the brain in mouse models of Huntington’s disease and other pediatric neurological diseases.

Nobel Prize Goes To Stem Cell Scientists

The Nobel Prize in physiology or medicine this year has been awarded to a British researcher named John Gurdon and a Japanese stem cell pioneer named Shinya Yamanaka. Both researchers showed that adult cells in our bodies have all the genes necessary to be reprogrammed into a more developmentally primitive state. Their discoveries, scientist hope, may be the basis for new treatments for diseases like Parkinson’s, diabetes and for studying the roots of diseases in the laboratory.

The Nobel prize committee at Stockholm’s Karolinska Institute said the discovery has “revolutionized our understanding of how cells and organisms develop.”

In 1962, John Gurdon showed that DNA from mature cells such an intestinal cells or skin cells could be used to generate new tadpoles. This revolutionary experiment definitely demonstrated that the genome of adult cells still possessed all the genes necessary to drive the formation of all cells of the body.

Ian Wilmut’s work at the Roslin Institute in Scotland that culminated in the cloning of Dolly the sheep utilized the same process that Gurdon had designed in frogs and showed that it could work in mammals.

Gurdon did his landmark experiment in 1962, the year the other Nobel prize winner, Shinya Yamanaka, was born. More than 40 years after Gurdon’s discovery (2006), Yamanaka used an incredibly simple yet elegant recipe to convert mature cells into primitive cells that could be differentiated into different kinds of mature cells.

These primitive cells, induced pluripotent stem cells or iPSCs were equivalent to embryonic stem cells. Embryonic stem cells, however, were embroiled in controversy, since derivation of embryonic stem cell lines required the destruction of human embryos. However, Yamanaka’s method provided a way to get such primitive cells without destroying embryos.

In the words of the Nobel committee: “The discoveries of Gurdon and Yamanaka have shown that specialized cells can turn back the developmental clock under certain circumstances. These discoveries have also provided new tools for scientists around the world and led to remarkable progress in many areas of medicine.”

As previously mentioned on this blog, Japanese scientists reported using Yamanaka’s approach to convert skin cells from mice into eggs that produced baby mice.

The 79-year-old Gurdon has served as a professor of cell biology at Cambridge University’s Magdalene College. Currently, he works at the Gurdon Institute in Cambridge, which he founded.

Yamanaka is 50 years old and previously worked at the Gladstone Institute in San Francisco and Nara Institute of Science and Technology in Japan. Currently, Yamanaka is at Kyoto University but retains his affiliation with the Gladstone Institute. Yamanaka is the first Japanese scientist to win the Nobel medicine award since 1987.

The work of Gurdon and Yamanaka earned them a Lasker award for basic research in 2009. Reprogramming cells has also been used in basic research in order to model certain genetic diseases in a culture dish. Cellular reprogramming allows scientists to create particular kinds of tissues with the exact abnormality they want to study. For example, scientists can make lung tissue afflicted with the mutations that cause cystic fibrosis, or brain tissue with Huntington’s disease. By reprogramming cells from patients that have a genetic disease, they can create new tissue with the same genetic flaws, and study it in the lab. Such a strategy can provide new insights into the roots of the problem.

In addition, that approach allows them to screen drugs in the lab for possible new medicines.

It is worth mentioning that Yamanaka’s motivation for developing iPSCs was a moral one. You see, he loved stem cell research, but did not want to destroy embryos. As told in a story published in the New York Times:

Inspiration can appear in unexpected places. Dr. Shinya Yamanaka found it while looking through a microscope at a friend’s fertility clinic. … [H]e looked down the microscope at one of the human embryos stored at the clinic. The glimpse changed his scientific career. “When I saw the embryo, I suddenly realized there was such a small difference between it and my daughters,” said Dr. Yamanaka. … “I thought, we can’t keep destroying embryos for our research. There must be another way.”

Dr. Yamanaka’s training taught him that there is no essential difference between a human embryo and a human adult. Differences in size, location, degree of dependence, and extent of development are not morally significant when it comes to the essential nature of an entity. The embryo is smaller than an adult, but so what? Are smaller people less human? Are the less developed less human? Or course not; they are less adult, but not less human. Does the degree of dependence determine your humanity? Then children and the aged are less human as are people after surgery. Surely this is absurd. Location? Now that’s even more ridiculous. There are the only differences between a human embryo and a human adult and they are purely accidental rather than essential differences.

It is amazing what a moral conviction can do. And note that Yamanaka is not a Christian. Therefore, you cannot say that he is a Christian fundamentalist pro-lifer. His is a purely scientific conclusion.

However, if I may opine on this – Why is human life valuable in the first place? It seems to me that people are valuable because they are a unique creation of God who loves them and values them. Therefore, they are inherently valuable and we should treat each life as precious. The conclusion that the embryo is not essentially different from a human adult is a scientific conclusion, but the conclusion that human life is valuable in the first place, is a religious conclusion.

A Return to 2004

My favorite bioethics blogger Wesley Smith has just been given a new gig at National Review called the Human Exceptionalism Blog.

This recent post examines an article in Fortune magazine that bemoans the lack of success with translating embryonic stem cell research into cures. It must be due to a lack of funding, right?

Smith puts the kibosh on that one pretty fast. Read it here.

Federal Court Rules in Favor of Federal Funding for Embryonic Stem Cell Research Legal

On August 24, 2012, a three-judge panel ruled that the government had properly interpreted a law that bans the use of federal funds research that destroys human embryos. Many legal observers, however, opined that this ruling will not end the controversy over this issue, and one of the judges on the three-judge panel importuned Congress to clarify what the government could and could not do with respect to human embryos.

This ruling upholds the dismissal of the case by the US Court of Appeals for the District of Columbia Circuit. The case, Sherley v. Sebellius, 11-5241, sought to prevent the US Department of Health and Human Services from using federal money to fund human embryonic stem cell research. The plaintiffs contended that funding human embryonic stem cell research would violate the Dickey-Wicker Amendment, which was passed as a rider to other legislation in 1996.

Predictably, biotechnology companies interested in embryonic stem cell-based treatments and other more academic embryonic stem cell researchers were quite pleased with the ruling. For example, Gary Rabin of Advanced Cell Technology said: “This court ruling should be of considerable benefit to our embryonic stem cell-based clinical programs. It effectively removes major speed bumps for the National Institutes of Health in terms of approving the several stem cells lines that we have submitted for their consideration for funding. We expect that a number of our embryonic stem cell lines will be approved for funding in coming months.”

The plaintiffs in this case were: a) James L. Sherley, who is a former a former member of the MIT faculty, but presently works as a senior scientist at the Boston Biomedical Research Institute; b) Theresa Deisher, who is the founder, managing member, and research and development director of AVM Biotechnology; and Nightlight Christian Adoptions, which is a non-profit, licensed adoption agency dedicated to protecting and finding adoptive parents for human embryos conceived through in vitro fertilization; c) the Christian Medical Association, a non-profit association of doctors dedicated to improving ethical standards of health care in the United States and abroad.

The main argument put forward by the plaintiffs in this lawsuit is that the present NIH Guidelines for the funding of embryonic stem cell projects violate existing federal law that bans the use of federal funds for the destruction of human embryos. Because the NIH created and sent its guidelines with a preconceived determination to fund human embryonic stem cell research and without considering scientifically and ethically superior alternatives, the guidelines are invalid regulations that violate the federal Administrative Procedure Act and, therefore, should be struck down.

With respect to the merits of the plaintiff’s case, it seems rather obvious, to me at least, that the NIH guidelines violate federal law. The Dickey-Wicker amendment was sponsored by former Congressman Jay Dickey (RAK) and Senator Roger Wicker (R-MS) who was then a member of the House of Representatives)], and applied to every Health and Human Services (“HHS”) appropriations bill governing the National Institutes of Health (NIH) since 1995. The bill states, “None of the funds made available by this Act may be used for . . . research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero under 45 C.F.R. § 46.204(b) and section 498(b) of the Public Health Service Act (42 U.S.C. § 289g(b).” Even for lawyer-speak, that seems pretty clear to me. Embryonic stem cell research includes the derivation of new embryonic stem cell lines from human embryos.  The derivation process destroys the embryo, which is precisely the thing that the Dickey-Wicker amendment prohibits the NIH from funding with federal money.

However, the federal judges did not see it that way for one reason:  funding for embryonic stem cell research also includes work on already established embryonic stem cell lines.  These lines are already in existence that the decision to kill the embryos has already occurred.  Therefore, the judges thought that the Dickey-Wicker amendment was simply not specific enough on the matter to merit stopping all funding for embryonic stem cell work.

While this argument has merit, it dodges the fact that embryo-destroying research will be funded by the NIH because of this ruling and some of this research will include laboratories that destroy embryos to make embryonic stem cells.  This is clear, since several sources have noted the increase in embryonic stem cell lines approved for NIH funding.  For example the Nature Medicine blog had this to say:  “Since the beginning of the month, the NIH has quietly kept adding ES cell lines to its registry, bringing the total tally up to 128.”  Thus the NIH funding policy has definitely led to the destruction of more embryos.

Where did this legal battle originate?  It began in 1994, during the presidency of Bill Clinton.  Before 1994, there was a ban on human embryo experimentation.  The Clinton Administration took steps to reverse this ban, and allow embryo-destructive research on donated embryos left over from fertility clinics while still prohibiting research that created its own embryos for research purposes.;  This policy shift was in response to the recommendation of an advisory committee known as the Human Embryo Research Panel (“HERP”).  This was an ad hoc committee formed to address the question of embryo research.  The phrase “ad hoc” is a Latin phae that means “for this.”  It simply means that this committee was formed for to address this particular question.  In testimony before the House Appropriations Committee, NIH Director Varmus stated that NIH would have funded six out of nine applications for grants involving embryo-related research “if the NIH had been able to proceed according to the recommendations and the President’s directive.”  Varmus also stated that he “firmly agree[d]” with particular sections of the HERP report, and further told the Committee that NIH was currently deciding whether to go forward with funding embryo-destructive research on donated human embryos.

However, before NIH was able to approve any grants that funded embryo-destructive research, Congress passed the Dickey-Wicker Amendment for the first time.  Opponents of the amendment argued that prohibiting federal funding of embryos research would push such research into the dark recesses of private industry where it would not be properly regulated.  Also, the understanding of the amendment was universal throughout Congress: it would prohibit ALL federal funding of embryo-destructive research.  Democratic senator from California, Barbara Boxer, understood the Dickey-Wicker Amendment as creating “a total prohibition of Federal funding for human embryo research,” and Republican Congressman John Porter understood the amendment in this way as well.  In fact Porter tried to pass an alternative rider to the Dickey-Wicker Amendment that would have prohibited federal funding for only creation of new embryos, but not other types of embryo-destructive research with donated embryos, but his rider was defeated.

Even more significantly, the NIH understood the amendment that way from 1996-1999 when they enforced it.  DNA research with DNA from human embryos that did not necessarily kill the embryos was prohibited from receiving federal funding under the NIH’s understanding of the Dickey-Wicker Amendment.  In a 1996 letter (October 10)  to Georgetown University Medical School researcher Mark Hughes who was using federally funded equipment to conduct tests on DNA derived from embryos, NIH “clarif[ied] . . .the NIH position on embryo research.” The agency explained that “analysis from DNA derived from a human embryo” violated the federal prohibition on research involving embryos and that NIH equipment “may not be used for embryo work of any kind.”

However, four years later, the NIH altered its position and issue Guidelines authorizing the funding of human embryonic stem cell research (65 Fed. Reg. 51976, Aug. 25, 2000).  Before the 2000 Guidelines were published, then-HHS General Counsel Harriet S. Rabb issued a memorandum on January 15, 1999, that supported the National Institutes of Health (NIH) claim that the Dickey-Wicker Amendment ought to be re-interpreted to ban federal funding of the derivation of embryonic stem cells – the procedure by which human embryos are destroyed to harvest their embryonic stem cells – but not research utilizing the derived embryonic stem cells.  This is in direct contradiction to the clear understanding of the Dickey-Wicker amendment put forward by Congress.

To remedy this reinterpretation of the Dickey-Wicker Amendment (DWA), seven senators sent a letter to then Secretary of Human Health and Services, Donna Shalala.  In that letter they stated that “Congress never intended for the National Institutes of Health to give incentives for the killing of human embryos for the purpose of stem cell research.”  The warnings of the senators and the comments from many dissenting parties were ignored by the NIH.

When President Bush took office, he rescinded the Clinton rules and upheld the original interpretation of the DWA.  This ended when Barak Obama became president in 2008.  President Obama revoked President Bush’s Executive Order 13435 (June 22, 2007) and ordered NIH “support and conduct responsible, scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent permitted by law.”

This was the reason for the lawsuit filed by Sherley and his co-plaintiffs.  They wanted the Federal government to simply recognize the law as it was originally written and interpreted.  Unfortunately, the judges ignored all that and went with their own shtick.  This is sad, and it should be remedied, but for now the situation seems to be that Congress’ laws do not mean what they originally say.

Embryonic Stem Cell-Derived Nerve Cells Restore Hearing to Deaf Animals

In a remarkable study, a research team from the University of Sheffield in England has improved the hearing of deaf animals by using embryonic stem cells. This result should certainly give new hope to those who suffer from hearing disorders.

In this study, an uncommon for of deafness that affects perhaps less than 1% and no more than 15% of all hearing-impaired patients, was treated. Even though this treatment would not benefit all cases of hearing impairment, the strategy developed in this study could be expanded to apply to other cases of deafness. Since these results are strictly pre-clinical in nature, it will be years before human patients might benefit from them.

This work used gerbils are a model system and the results were reported in the international journal Nature. The research team was led by Dr. Marcelo Rivolta and the scientists in his laboratory.

To induce deafness in the gerbils, the scientists ablated (killed off) those nerve cells that transmit auditory information from the ear to the brain. The nerve cells are called “spiral ganglia neurons” or SGNs, and if a patient suffers damage to the SGNs, they will not be able to receive a cochlear implant to restore their hearing. Therefore, this experiment attempted to replace these SGN cells in order the restore hearing.

Rivolta’s group used human embryonic stem cell (hESC) lines H7, H14, and Shef1 and treated them with two growth factors, FGF3 and FGF10. The combination of these two growth factors induced the expression of a whole host of genes normally found in SGN cells (for example, Pax8 & Sox2). These treatments converted the hESCs into otic neural progenitors (ONPs).

In order to destroy the SGN cells in the ears of gerbils, Rivolta and others used a drug called ouabain. This drug, when injected into the inner ear, will destroy the SGN cells and make the animals completely deaf. In the next experiment, Rivolta et al. transplanted the immature nerve cells into the ears of 18 gerbils. One ear received the transplantation, while the other ear was kept as is as a control.

10 weeks later, they used electrophysiology tests to measure the response of the brain to sound. Of the 18 gerbils transplanted with the hESC-derived ONPs, the animals had recovered their hearing by an average of 46%. The recovery differed from animal to animal, but it ranged from modest recovery to almost complete in others.

All animal subjects were kept on anti-rejection medications to prevent rejecting the implanted human cells. In order to prevent tissue rejection in human patients, either induced pluripotent stem cells should be used, or hESCs that match the tissue types in the patient.

Rivolta’s team is also in the process of making immature versions of a second kind of inner-ear cell, that is, the “hair cell” that detects the auditory vibrations in the cochlea. The induction of ONPs from hESCs tends to produce two types cells: ONPs and otic epithelial progenitors (OEPs), which are the precursors of cochlear “hair cells.” Since damage to hair cells is far more common in cases of hearing loss, implantation of such cells should be able to treat far more cases of hearing loss. Unfortunately, this has not yet been tested in animals, according to Rivolta.

Yehoash Raphael of the University of Michigan, who didn’t participate in the work, said it’s possible the stem cell transplants worked by stimulating the gerbils’ own few remaining nerve cells, rather than creating new ones. But either way, “this is a big step forward in use of stem cells for treating deafness.”

Likewise, Lawrence Lustig of the University of California, San Francisco, said, “It’s a dynamite study (and) a significant leap forward.”

Scientists Create Germ Cell-Supporting Embryonic Sertoli-Like Cells From Skin Cells

Stem cell researchers from the Whitehead Institute in Cambridge, Massachusetts have used a novel, stepwise cell reprogramming protocol to convert skin cells into embryonic Sertoli-like cells.

Sertoli cells are found in the testes of men and they provide vital support, protection, and nutrition to developing sperm cells. Sertoli cells also possess “trophic” properties, which simply mean that they secrete factors that help cells grow and survive. In fact, Sertoli cells have been used to protect and promote the growth, and survival of non-testicular cellular grafts in transplantations. Mature Sertoli cells, however, do not divide, and primary immature Sertoli cells have the unfortunate tendency to degenerate during prolonged culture in the laboratory. Therefore, it is desirable to find some kind of alternative source of Sertoli cells independent of the donor testis cells, but for basic research and clinical applications.

Whitehead Institute Founding Member Rudolf Jaenisch said, “The idea is if you could make Sertoli cells from a skin cell, they’d be accessible for supporting the spermatogenesis process when conducting in vitro fertilization assays or protecting other cell types such as neurons when co-transplanted in vivo. Otherwise, you could get proliferating cells only from fetal testis.”

Researchers in the Jaenisch lab seem to have overcome the supply and lifespan challenges of cultured Sertoli cells by means of using cellular reprogramming to direct one mature cell type, in this case a skin cell, into immature Sertoli cells. The process of cellular reprogramming, otherwise known as trans-differentiation, reprograms a cell directly from one mature cell type to another without first de-differentiating the cell back to an embryonic stem-cell stage. Unlike other reprogramming methods that generate induced pluripotent stem cells (iPSCs), trans-differentiation does not rely on the use of genes that can cause cancer.

The Whitehead Institute scientists trans-differentiated mouse skin cells into embryonic Sertoli-like cells by dividing the trans-differentiation process into two main steps that mimic Sertoli cell development inside the testes. This first step involves the progression transformed skin fibroblasts from their free-moving, unorganized mesenchymal state into an organized, sheet-like epithelial state. For the second step, the cells were induced so that they acquired the ability to attract each so that they formed aggregates that are very similar to those observed in co-cultures of embryonic Sertoli cells and germ cells.

Next, Jaenisch’s lab workers invented a cocktail that consisted of five different transcription factors that specifically activate the developmental program for embryonic Sertoli cells. The cells that resulted from this induction behaved in ways that were reminiscent of embryonic Sertoli cells. They aggregated, formed tubular structures similar to seminiferous tubules found in the testes, and secreted a host of Sertoli-specific factors. When these reprogrammed cells were injected into the testis of fetal mice, the trans-differentiated cells properly migrated to the right location and integrated into the seminiferous tubules. The injected cells behaved exactly like endogenous embryonic Sertoli cells, even though they expressed a few genes differently.

Yossi Buganim, a postdoctoral researcher in the Jaenisch lab and first author of the Cell Stem Cell paper said: “The injected trans-differentiated cells were closely interacting with the native germ cells, which shows [sic] that they definitely do not have any bad effect on the germ cells. Instead, they enable those germ cells to survive.”

Buganim also showed that when their embryonic Sertoli-like cells made from trans-differentiated skin cells were used to sustain other cultured cells in a Petri dish, the cells thrived and lived longer than cells sustained by actual native Sertoli cells. The reprogrammed Sertoli cells supported and nourished the cultured cells and acted like tried and true Sertoli cells.

These encouraging results from their cell culture work have inspired Buganim to investigate if the embryonic Sertoli-like cells retain their enhanced supportive capacity after transplantation into the brain. Once in the brain, the cells could potentially sustain ailing neurons. If these cells truly have this ability, they could have clinical applications. Such applications would include the support and protection of implanted neurons in regenerative therapies for neurodegenerative disorders such as ALS and Parkinson’s disease.

Embryonic Stem Cell Lines Derived from Embryos Frozen for 18 Years

How long do human embryos survive in cryostorage? To be completely honest, no one truly knows. According to the Planer PLC Group, a cryopreservation company, a baby was born from an embryo that had frozen for 16 years at their institution. However, it is possible that embryos might live even longer in cryostorage. Furthermore, one study that examined more than 11,000 cryopreserved human embryos determined that the length of time for which the embryo was frozen had no significant effect on post-thaw survival for in vitro fertilisation (IVF) or oocyte donation cycles, or for embryos frozen at the pronuclear or cleavage stages. This study also showed that the duration of storage did not have any significant effect on clinical pregnancy, miscarriage, implantation, or live birth rate, whether from IVF or oocyte donation cycles (Riggs R, Mayer J, Dowling-Lacey D, Chi TF, Jones E, Oehninger S (November 2008). “Does storage time influence postthaw survival and pregnancy outcome? An analysis of 11,768 cryopreserved human embryos”. Fertil. Steril. 93 (1): 109–15).

However, some embryos do not survive the freezing process. Also, some embryos that are frozen are very low-quality embryos that have an exceedingly low probability of ever making a baby. Since these embryos are of very little value from a reproductive standpoint, they might be of use to stem cell biologists who want to make embryonic stem cells from them. Several studies have shown that low quality embryos are excellent sources of material for embryonic stem cells. For example, Lerou PH, et al., Human embryonic stem cell derivation from poor-quality embryos.Nat Biotechnol. 2008 Feb;26(2):212-4. In this paper Daley’s lab derived embryonic stem cell lines at rates comparable to the rates of embryonic stem cell derivation with high-quality embryos. Another paper by Shetty R and Inamdar MS, “Derivation of human embryonic stem cell lines from poor quality embryos,” in Methods Mol Biol. 2012;873:151-61, Indian researchers derived embryonic stem cell lines from low-quality embryos.

A Chinese laboratory has also used low quality embryos that were discarded by fertility clinics. 166 poor quality embryos gave rise to 4 new embryonic stem cell lines in this paper (see Lui W, et al., J Genet Genomics. 2009 Apr;36(4):229-39). Therefore, this practice is well established.

What is questionable is whether or not the embryo is actually dead. Remember, even though grade III embryos are not desirable because they show lower rates of implantation, they still give rise to live births occasionally. For example, one study showed that grade iV embryos (worse than grade III) gave pregnancy rates of 18.2%. Therefore, these studies are being done with low-grade embryos – not embryos that are clinically dead.

As I have noted before in a previous post, defining death in an embryo is difficult to do, but when there are far more dead cells in the embryo than live ones, the chance of the embryo giving rise to a baby becomes so low as to be impossible. If 60% of the cells in the embryos are dead, then the embryo is usually defined as clinically dead. Such an embryo, if it has not experienced early developmental arrest, can be a reasonable source of embryonic stem cells, according to work from the Daley lab

With this in mid, there is a paper from a research group at Chulalongkorn University and Chilalongkorn Memorial Hospital, Bangkok, Thailand, that shows that embryonic stem cells can be successfully made from embryos that had been frozen for 18 year. This paper shows that even embryos that have been frozen for almost two decades can still yield embryonic stem cells.

Evaluations of these embryonic stem cell lines revealed that they were as pluripotent as similar lines derived from embryos that had only been frozen for a few years.

Jane Taylor, a collaborator in this paper from the MRC Centre for Regenerative Medicine at the University of Edinburgh, Scotland, said, “The importance of this study is that is it identifies an alternative source for generating new embryonic stem lines, using embryos that have been in long-term storage.”

These frozen embryos, if they were not clinically dead, were still human beings. They merely needed to be implanted into a mother’s womb in order to execute their intrinsic developmental program that implants itself into the mother’s womb. By using these embryos to derive embryonic stem cell lines, their lives were ended. All other arguments that try to downgrade the essential status of these embryos because they are too young, too small, in the wrong place or too different from an adult rely upon accidental qualities of the embryo. That is, qualities that are temporary and not integral to the essence of the embryo. Its essence is that of a human being. When it grows larger, it is still a human being and the fact that it executes its intrinsic developmental program to do so merely demonstrates its human essence. The same can be said about it appearance, and age.

Location is an even more problematic criterion by which to disqualify the embryo from the human race.

Therefore, these embryos were destroyed and their human lives, killed. Surely there is a better way to do regenerative science that both respects the value of human life and creates technologies to heal us. Interested? Read the other posts on this blog.

Embryonic Stem Cells – Not all Genes are On

Early thinking about embryonic development and differentiation tended to view development as a matter of going from a cell with all kinds of genes on to progeny cells that have a host of these genes turned off and only a small subset of the original cache of genes turned on. If those genes were muscle-specific genes, then the cell became a muscle cell, and if they were nervous system-specific genes, then the cell became a neuron or glial cell.

Several different experiments questioned this conventional wisdom, and in particular, microarray experiments that allowed researchers to examine the gene expression pattern of the entire genome at a time showed that this was not the case. Instead of a host of genes being on in embryonic cells, a particular subset of genes were on, and as the embryo grew and aged, some cells shut one set of genes and turned on others, while a different group of cells turn off yet another set of genes and turned on a completely distinct set of genes.

With embryonic stem (ES) cells, the gene expression pattern depended on the culture system. Therefore, it was always difficult to interpret the results of such experiments.

This problem has now been largely solved, since Austin Smith at the Welcome Trust Stem Cell Institute in Cambridge (UK) has developed a culture system to standardize these conditions for embryonic stem cells. By employing this new methods, Hendrik Marks at the Nijmegen Centre for Molecular Sciences of the Radboud University Nijmegen, the Netherlands, showed that the ground state genes expression of embryonic stem cells is surprising.

There are only a few genes that are activated in embryonic stem cells. However, other genes that are not activated are not actively repressed. Instead that are ready to go and are in a kind of “on hold” status. The protooncogene (a gene that drives cells to divide and grow) c-myc, was thought to be essential for embryonic stem cell growth and division is hardly detectable.

This provides added clues as to how to keep ES cells as ES cells or how to drive them to differentiate into one cell type or another.

According to Marks, formerly researchers thought that “ES cells would subsequently differentiate by turning genes off that are not relevant for a specific specialization, to finally reach the correct combination of active genes for a particular specialization. We now see the opposite: genes are selectively turned on.”

The proteins that bind to DNA and direct gene expression, however, the so-called “epigenome,” are already prepared for action. Thus ES cells are poised to become one thing or another, and the environmental cues that they receive coaxe them into one differentiation pathway or another.

This finding also calls into question the work of Ronald Bailey who thinks that ES cell research is not immoral for the following reason: “So what about the claims that incipient therapies based on human embryonic stem cell research are immoral? That brings us to the question of whether the embryos from which stem cells are derived are persons. The answer: Only if every cell in your body is also a person.” Bailey continues: “Each skin cell, each neuron, each liver cell is potentially a person. All that’s lacking is the will and the application of the appropriate technology. Cloning technology like that which famously produced the Scottish sheep Dolly in 1997 could be applied to each of your cells to potentially produce babies.”

To support his claim, he quotes the Australian bioethicist Julian Savulescu from the 1999 Journal of Medical Ethics: “What happens when a skin cell turns into a totipotent stem cell [a cell capable of developing into a complete organism] is that a few of its genetic switches are turned on and others turned off. To say it doesn’t have the potential to be a human being until its nucleus is placed in the egg cytoplasm [i.e., cloning] is like saying my car does not have the potential to get me from Melbourne to Sydney unless the key is turned in the ignition.”

Savulescu is simply wrong. Many experiments have called this account of development into question, and now Marks’ experiments have placed the nail in the coffin. Furthermore, his analogy that Ta body cell does not have the “potential to be a human being until its nucleus is placed in the egg cytoplasm [i.e., cloning] is like saying my car does not have the potential to get me from Melbourne to Sydney unless the key is turned in the ignition,” is also flawed. The cell of our body are not undergoing development. Development is a process we know a great deal about, and our cells are not undergoing development. Embryos are undergoing development and they are unique human persons. Embryos give rise to our bodies. We are human persons and we began to assume our adult form when the embryo initiated development (i.e., at the termination of fertilization). Development also involves the hierarchical activation and inactivation of various genes. This is not a process that occurs in adult human bodies. Embryos are the beginning of a human person and they are human persons. Savulescu’s analogy would be more accurate if we say that the engine without the car would be unable to get him to Sydney, Australia: It needs a frame, tires and so on. They also all need to be properly connected and integrated with each other to work. His analogy is simply inaccurate and bogus.

Likewise, what Bailey calls “the application of the appropriate technology,” during a cloning experiment is the wholesale creation of a new human being. To say that this new human being is one of your cells is to woefully misunderstand the biological nature has happened during cloning. An egg from a female has its nucleus removed and is fused with a cell from another part of your body. After appropriate manipulation, the egg starts to divide and undergo embryonic development. Even this cell has the same genetic information as the cell from your body, it will not development into an exact duplicate of yourself. There are too many random events that occur during development that cause the individual to become a unique person who may have some similarities with their genetic parent, but will not resemble them completely. Cloning is not a minor manipulation – it is the creation of a new life, and this is a process that our cells are not going through; they are not developing. Therefore, they are not “potential persons.”

Secondly, the embryo is not a potential person, it is a very young human person.  It is a potential adult person, but it is a person nonetheless.

Michael J Fox Changes Tune on Embryonic Stem Cells

Actor Michael J. Fox, whose acting career has included such greats as the “Back to the Future.” series, and the television series “Spin City,” and others has been diagnosed with early onset Parkinson’s disease (PD). He has also been a stalwart proponent of embryonic stem cell research. Apparently, he believes that embryonic stem cell research will provide a potential treatment for his PD and many other PD patients as well. The Michael J Fox Foundation has been a supporter of PD research, which includes embryonic stem cell research into PD treatments.

Michael J. Fox was the subject of some controversy a few years ago when he appeared in some political ads for Missouri 2006, Michael J. Fox endorsed Claire McCaskill, Democratic candidate for the senate from the state of Missouri, who is also an ardent supporter of embryonic stem cell research. In those ads, Fox told viewers in the ad that Ms. McCaskill supported stem cell research that could provide a cure for his Parkinson’s disease. There were also accusations that Fox had gone off his PD-controlling medications during the period of time the ad was shot in order to increase his symptoms and elicit sympathy. The radio talk show host Rush Limbaugh suggested that Fox could have been acting, but many people emailed Limbaugh saying that Fox typically went off his medication before testifying before Congress.

Nevertheless, Fox no longer believes that embryonic stem cell research is the sina qua non of PD treatment. In an article at the New Scientist web site, Fox stated that the problems with stem cell-based treatments made him less sanguine about the possibilities of a stem cell-based treatment for PD.  This does NOT mean that Fox is no longer a supporter of embryonic stem cell research.  It simply means that one of the most vociferous advocates of embryonic stem cell research is unwilling to place all his hope in it as a viable cure for PD.  This is truly a remarkable development.

PD has been experimentally treated with cells from aborted fetuses.  These experiments are nothing short of gruesome, and they did not provide any evidence of lasting viable cures.  Furthermore, when the brains of individuals who had received the transplants were examine postmortem, the implanted cells showed the same pathologies as the surrounding tissue.  Therefore the implants were a rousing flop.  Some successes have been seen with transplantation of animal tissue, but these experiments were few and far between, and have risks of infecting patients with animal viruses.

With respect to stem cell treatments or PD, a highly-publicized Nature paper implanted dopamine-making neurons that were made from embryonic stem cells into the brains of PD mice.  While many of the symptoms improved, the implanted cells generated lots of tumors (see Roy N et al., Functional engraftment of human ES cell–derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes, Nature Medicine 12, 1259-68; November
2006).  Wesley J Smith has noted that Fox called these tumors “tissue residue.”  This is either ignorance or dishonesty.  100% of the rats in these experiments that received that implants developed tumors.  This is not tissue residue, they are tumors.

On the other hand, adult and umbilical cord stem cells have shown some remarkable successes, as have experiments with specific proteins called “neurotrophic factors,” which stimulate endogenous brain cells wot divide and make new connections with other cells.  For example, PD rats that were treated with umbilical cord stem cells showed significant recovery in motion and behavior (Weiss ML, et al., Stem Cells 24, 781-792, March 2006).  Additionally, researchers from Kyoto University treated PD mice by transplanting nerve cells developed from their own bone marrow stromal cells (Mari Dezawa et al., Journal of Clinical Investigation 113:1701-1710, 2004).

When it comes to neurotrophic factors,  University of Kentucky scientists treated ten Parkinson’s patients with a protein called glial cell line derived neurotrophic factor to stimulate the patients’ own brain stem cells and showed significant improvement in symptoms (Slevin JT, et al., Journal of Neurosurgery 102, 216-222, February 2005).  Also British researchers injected a protein known as a “neurotrophic factor” into the brains of 5 Parkinson’s patients and found that it stimulated the patients’ own adult neural stem cells. This treatment provided an average 61% improvement in motor function (Gill SS et al., Nature Medicine 9, 589-595; May 2003).  Later autopsies of these treated patients demonstrated that the neurotrophic factors stimulated sprouting of new neurons in the brain (Love S. et al., Nature Medicine 11, 703-704, July 2005).

Likewise, all present clinical trials for PD are all adult stem cell- or induced pluripotent stem cell-based.

Another treatment for PD that is not stem cell-based is Deep Brain Stimulation (DBS).  DBS uses a surgically implanted medical device called a brain pacemaker that sends electrical impulses to specific parts of the brain.  DBS in select brain regions has provided remarkable therapeutic benefits for otherwise treatment-resistant movement disorders like PD (see Kringelbach ML, et al., Nature Reviews Neuroscience. 2007;8:623–35).

Therefore Fox was certainly right to change his perspective on embryonic stem cells. If only he would see that destroying the youngest and most vulnerable members of humanity is too high a price to pay for the cures of others.  There are better and more humane and ethically-sound ways to treat PD, and those ways are being pursued.

Bone Marrow Stem Cells Make the Blind (Lab Animals) See

There has been a great deal of discussion of embryonic stem cell-derived retinal pigment cells and the transplantation of these cells into the retinas of two human patients who subsequently showed improvements in their vision. One of these patients had a degenerative eye disease called “Stargardt’s macular dystrophy,” and the other had dry, age-related macular degeneration.

Stargardt Macular Dystrophy (SMD) is one of the main causes of eyesight loss in younger patients (affects 1/10,000 children), and retinal damage begins somewhere between the ages of 6 – 20. Visual impairment is usually not obvious to the patient until ages 30 – 40. Children with SMD usually notice that they have difficulty reading. They may also complain that they see gray, black or hazy spots in the center of their vision. Additionally, SDM patients take a longer time to adjust between light and dark environments.

Mutations in the ABCA4 gene seem to be responsible for most cases of SDM.  Defects in ABCA4 prevent the photoreceptors from disposing of toxic waste products that accumulate within build up in the disc space of the photoreceptors.  These toxic waste products are a consequence of housing light-absorbing pigments, and intense light exposure.  The pigment, all-trans retinal, binds to membrane lipids, and this forms a compound called NRPE (short for N retinylidene-phosphatidylethanoliamne, which is a mouth-full).  The protein encoded by ABCA4 moves NRPE into the cytoplasm of the photoreceptor cells, but if ABCA4 is not functional, NRPE accumulates in the disc space and binds more all-trans retinal to form a toxic sludge called “lipofuscin.”  Lipofuscin is taken up from the photoreceptors by the RPE cells and it kills them (see Koenekoop RK. The gene for Stargardt disease, ABCA4, is a major retinal gene: a mini-review. Ophthal Genet. 2003;24(2):75–80).  Mutations in other genes (ELOVl4, PROM1, and CNGB3) also cause SDM.

Dry, age-related macular degeneration is associated with the formation of small yellow deposits in the retina known as “drusen.”  Drusen formation leads to a thinning and drying of the macula that eventually causes the macula to lose its function.  There is loss of central vision and the amount of vision loss is directly related to the amount of drusen that forms.  Early stages of age-related macular degeneration is associated with minimal visual impairment, but is characterized by large drusen and abnormalities in the macula.  Drusen accumulates near the basement membrane of the retinal pigment epithelium.  Almost everyone over the age of 50 has at least one small druse deposit in one or both eyes.  Only those eyes with large drusen deposits are at risk for late age-related macular degeneration.

All of this is to say that these diseases are progressive.  They have no cure and little can be done for treatment.  Secondly, people rarely get better.  However, both patients in this study showed quantifiable improvements.  The patient with age-related macular degeneration went from being able to see 21 letters in the visual acuity chart (20/500 vision for the patient, with 20/20 being perfect vision) to 28 letters (20/320).  This improvement remained stable after 6 weeks.  The patient with SMD was able to detect hand motions only, but after the stem cell injection, she could count fingers and see one letter in the eye chart by week 2, and was able to see five letters (20/800) after 4 weeks.  She also was able to see colors and contrast better and had better dark adaptation in the treated eye.

Now there are some caveats for this report.  First of all, the patient with SMD showed distinct structural improvements in the retina of the injected eye.  This patient also had distinct improvements in visual acuity.  However, the patient with dry, age-related macular dystrophy had no detectable structural improvements in the injected eye. The paper states, “Despite the lack of anatomical evidence, the patient with macular degeneration had functional improvements.”  Additionally, the non-injected eye also showed some visual improvements.  Note the words of the paper:  “Confounding these apparent functional gains in the study eye, we also detected mild visual function increases in the fellow eye of the patient with age-related macular degeneration during the postoperative period.”  Therefore, this experiment is highly preliminary and has equivocal results.  The SMD patient does show recognizable improvements, but this is only one patient.

While we are considering the efficacy of embryonic stem cells in the treatment of retinal degenerative diseases, a paper that was published in 2009 shows that bone marrow stem cells that have a cell surface marker celled “CD133” can become retinal pigment (RPE) cells.  This paper was published in the journal “Stem Cells,” and the principal author was Jeffrey Harris who did his work in the laboratory of Edward W. Scott at the University of Florida.  These cells were extracted from the bone marrow of mice and implanted into the retinas of albino mice.  Since the donor mice had pigmented skin and fur coats, the bone marrow cells were capable of making pigmented cells.  Once the CD133 cells were implanted, they survived and became pigmented.  When examined in postmortem sections, it was exceedingly clear that the transplanted CD133 cells expressed RPE-specific genes and assumed a RPE-like morphology.  Additionally, the implanted bone marrow cells also contributed functional recovery of retina.  A second set of experiments showed that human CD133 cells from umbilical cord could also integrate into mouse retinas and differentiate into RPEs.

This paper shows that embryonic stem cells are probably not necessary for retinal repair of RPE-based retinal degeneration.  Umbilical cord CD133 stem cells or bone marrow stem cells can differentiate into RPEs when transplanted into the retina.  While this paper does not address whether or not such differentiation occurs in human patients, such results definitely warrant Phase I studies. Thus once again, embryonic stem cells seem not be necessary.

California Stem Cell Report Includes No Critics

Wesley Smith at his blog notes that the California Stem Cell Report, which will include public testimony to the Institute of Medicine (IOM), an arm of the National Institutes of Health (NIH), will include scientists who were awarded lucrative grants by the California Institute for Regenerative Medicine (CIRM), but no critics of the program.  His source is a very critical Los Angeles Times article.

The critics of CIRM are not pro-life advocates who oppose embryonic stem cell research on principle.  Instead critics include the Little Hoover Commission, which issued this blistering report of CIRM, and the Oakland-based Center for Genetics and Society.  These organizations were afraid that there were too many conflicts of interest on the grant-awarding panel.  In the words of the Little Hoover Commission:

CIRM’s 29-member oversight committee includes representatives from institutions that have benefitted from grants the committee approved. This structure, along with overly long terms and the inability to nominate its own leaders or hold them accountable, fuels concerns that the committee never can be entirely free of conflict of interest or self-dealing, notwithstanding a court ruling that established the legality of such a structure. Legal is not necessarily optimal, however, and litigation over this issue delayed CIRM from beginning its work. As long as the board remains in its present form, its structure will draw scrutiny, diverting CIRM resources.

No representatives from either of these critical institutions are on the witness list.  Why aren’t members of the public allowed to address the IOM?  According to the LA Times,  the proprietor of the California Stem Cell Report, David Jensen, says he asked the IOM why no objective witnesses were on the hearing list, and an IOM public relations person directed him to a survey form members of the public could fill out (though the link for the form on the IOM’s website was dead when I checked it).  Apparently, members of the public will also be permitted to address the IOM panel at Tuesday’s hearing. They’ll each get up to five minutes.

CIRM is selling the people of California a bill of goods.  In 2014, CIRM will be back to the people of California with their hand out for more money.  If the process is so objective, then what do they have to hide?  3 billion dollars later and little to show for it except for lots of dead human embryos.  People will be more than a little miffed; and they should be.