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.