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.

Preventing Rejection of Embryonic Stem Cell-Based Tissues


Embryonic stem cells (ESCs) are derived from human embryos. Because they are pluripotent, or have the capacity to make any adult cell type, ESCs are thought to hold great promise for cell therapy as a source of differentiated cell types.

One main drawback to the use of ESCs in regenerative medicine is the rejection of ESC-derived cells by the immune system of the patient. Transplantation of ESC-derived tissues would require the patient to take powerful anti-rejection drugs, which tend to have a boatload of severe side effects.

However, a paper reports a strategy to circumvent rejection of ESC-derived cells. If these strategies prove workable, then they might clear the way to the use of ESCs in regenerative medicine.

The first paper comes from the journal Cell Stem Cell, by Zhili Rong, and others (Volume 14, Issue 1, 121-130, 2 January 2014). In this paper, Rong and his colleagues from the laboratory of Yang Xu at UC San Diego and their Chinese collaborators used mice whose immune systems had been reconstituted with a functional human immune system. These humanized mice mount a robust immune response against ESCs and any cells derived from ESCs.

In their next few experiments, Xu and others genetically engineered human ESCs to routinely express two proteins called CTLA4-Ig and PD-L1. Now this gets a little complicated, but stay with me. The protein known as CTLA4-Ig monkeys with particular cells of the immune system called T cells, and prevents those T cells from mounting an immune response against the cells that display this protein on their surfaces. The second protein, PD-L1, also targets T cells and when T cells bind to cells that have this protein on their surfaces, they are completely prevented from acting.

CTLA-4 mechanism

Think of it this way: T cells are the “detectives” of the immune system. When they find something fishy in the body (immunologically speaking), they get on their “cell phones” and call in the cavalry. However, when these detectives come upon these cells, their cell phones are inactivated, and their memories are wiped. The detectives wander away and then do not remember that they ever came across these cells.

Further experiments showed that any derivatives of these engineered ESCs, (teratomas, fibroblasts, and heart muscle cells) were completely tolerated by the immune system of these humanized mice.

This is a remarkable paper. However, I have a few questions. Genetic engineering of these cells might be potentially dangerous, depending upon how it was done, where in the genome the introduced genes insert, and how they are expressed. Secondly, if cells experience any mutations during the expansion of these cells, these mutations might cause the cells to be detected by the immune system. Third, do these types of immune repression last long-term? Clearly more work will need to be done, but these questions are potentially addressable.

My final concern is that if this procedure is used widespread, it might lead to the wholesale destruction of human embryos. Human embryos, however, are the youngest, weakest, and most vulnerable among us. What does that say about us if we do not value the weakest among us and dismember them for their cells? Would we allow this with toddlers?

Thus my interest and admiration for this paper is tempered by my concerns for human embryos.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

“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.  

MSNBC Host Says That Life Begins Whenever You Feel Like It Does


I lived in Great Britain for three years for my first postdoctoral research fellowship at Sussex University. To be completely honest, I never got into the whole royal family thing, but the birth of George Alexander to Prince William and Kate Middleton is certainly an event to celebrate. George has little chance of ever ascending to the throne, but he is certainly a bundle of joy to his parents and to the British people.

Therefore, I find it rebarbative that media kill joys have used the joyous birth of William and Kate’s baby to be an opportunity to talk about abortion. In addition to this, one particular pro-choice news correspondent, Melissa Harris-Perry decided to wax philosophically about the nature of the unborn.

After noting the worldwide excitement that has surrounded Kate Middleton’s pregnancy and birth, MSNBC host Melissa Harris-Perry compared the buzz surrounding the British royal birth to Texas abortion politics, and then offered her own answer to the question “when does life begin:”

“When does life begin? I submit the answer depends an awful lot on the feeling of the parents. A powerful feeling – but not science,”

News correspondents say stupid things, but this has to rank as one of the most brain-dead things I have ever heard. Let’s not forget who said it, since Melissa Harris-Perry, is the news anchor who wore tampon earrings and received Planned Parenthood’s Maggie Award.

Once the egg in the fallopian tube of the mother fuses with a sperm cell from the father, the egg undergoes a complex sequences of biochemical and cellular events that culminate in the fusion of the genetic material of the mother with that of the father. This marks the end of the process known as fertilization and the beginning of the embryonic stages of development. The embryo has begun the journey of human development, growth, and maturation that will not stop until the individual dies. The embryo is genetically distinct from the mother and the father, and is a human being, albeit, a very young human being. The embryo is not a plant, an alligator, or some facsimile or something else, it is human, but a young human. That is not a feeling, but a scientific fact.

Can we kill the embryo just because it is very young? Reflection leads me to say no, no, a thousand times no. Do we value two-year old children more than one-year old children? Do we value six-year old children more than four-year old children? Age is irrelevant to the moral worth of an individual.

But, you say, the embryo is underdeveloped relative to a new-born baby. Does the extent of development determine moral worth? Again, a one-month old baby is more developed than a two-week old baby. Does that make the one-year old baby more valuable? No. Are teenagers who are more physically developed more morally valuable than eight-year old children? No. Therefore, the extent of development is not a good measure of a human being’s moral worth.

Ms. Harris-Perry seems to thing that feelings or perhaps she means how deeply a mother wants her baby is the factor that determines if he or she should continue to live. Again I say no. This would justify genocide. The dictators of North Korea can simply say that killing their own people is due to the fact that they did not want them anymore. They had those kind of feelings you know. How about Hitler and the Third Reich and their slaughter of six million Jews and many millions of  others? Hitler and his officers killed them because they did not feel that Jews and others were worthy of life. In fact, Harris-Perry’s ethic can justify any heinous, insidious acts simply on the basis of feelings.

This is, as I have said, brain-dead and she should be called out for it. The unborn human beings are still human beings regardless of how we feel about them. That is a fact of genetics and embryology regardless of your feelings about it. If MSNBC has news correspondents that say things that are this stupid, then maybe they deserve to have such low ratings.

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.