Genes and Growth Factors that Control Neural Stem Cells

Neuron-producing stem cells in the brain are controlled by a host of mechanisms, and two of these have been more precisely enumerated thanks to work by Steven Levison and Teresa Wood at the University of Medicine and Dentistry of New Jersey and Anna Lasorella at Columbia University Medical Center.

The first study by Levison and Wood examined proteins that are soluble in the cerebrospinal fluid. Neural stem cells are in constant contact with the cerebrospinal fluid, and therefore, any signaling molecules that are secreted into the cerebrospinal fluid (CSF), can potentially influence the activity of neural stem cells.

Insulin-like growth factors are typically made in response to growth hormone. Because these insulin-like growth factors mediate the response of growth hormone, they are called somatomedins. Insulin-like growth factors (IGFs) also play roles in the development of the brain. There are two main IGFs, IGF-1 and IGF-2. IGF-I and its receptor (IGF-IR) are widely expressed in the central nervous system, and IGF-2 is expressed in a more restricted pattern. IGF-binding proteins are similarly expressed during varying phases of brain development. IGF-I regulates both neuronal and glial cell proliferation and differentiation, apparently by an initial increase in neural progenitor proliferation. Loss of function mutations in the genes that encode either IGF-I or IGF-IR result in brain retardation, and overexpression leads to brain overgrowth. A few cases of IGF-I or IGF-IR mutations have been described in humans, and both of them result in some form of mental retardation and even microcephaly (small head). Later in development, circulating IGF-I levels are elevated and brain levels-specific are reduced, but circulating IGF-I can cross the blood-brain barrier and influence brain biology. It seems to prevent programmed cell death of neurons (see D’Ercole AJ, Ye P 2008 Minireview: expanding the mind: insulin-like growth factor I and brain development. Endocrinology 149:5958–5962).

This study by Levison and Wood established that IGF-1 & 2 are essential for neural stem cell renewal and cell proliferation. IGF-1 maintains neural stem cell numbers by promoting cell division. However, IGF-2 drives the expression of those proteins necessary to main the undifferentiated state of the neural stem cells.

Since the concentration of both these proteins declines with age, it might explain the cognitive decline associated with aging.

The second study identified a molecular pathway that controls the retention and release of the brain-specific stem cells. Antonio Iavarone and Anna Lasorella at Columbia University Medical Center were able to establish that neural stem cells reside in small areas called “niches.” This molecular pathway also works to maintain the neural stem cell population.

According to Iavarone, “From this research, we knew that when stem cells detach from their niche, they lose their identity as stem cells and begin to differentiate into specific cell types.”

Stem cell niches in the brain are located right next to the “ventricles.” Ventricles are fluid-filled spaces within the central nervous system. These fluid-filled spaces are loaded with cerebrospinal fluid. the number of neural stem cells within these neural stem cell niches is carefully regulated so that enough cells are present for cell division, but enough are released into the brain to replenish dead or heavily-needed neurons. However, as explained by Anna Lasorella, associate professor of pathology and pediatrics, “the pathways that regulate the interaction of stem cells with their niche were obscured.”

In previous work, Iavarone and Lasorella showed that molecules called ID or inhibitor of differentiation proteins, regulate stem cell properties (Iavarone A, Lasorella A. Trends Mol Med. 2006 Dec;12(12):588-94). This present study determined how Id proteins regulate stem cell identity.

In this study, mice with loss-of-function mutations in the gene that encodes the Id protein. They also made strains in which the amount of the Id protein was not eliminated, but decreased. In the mice with no ID protein, the mice died within 24 hours of birth. The brains of these mice showed very low levels of neural stem cell proliferation and the entire neural stem cell population was greatly reduced.

When Iavarone and Lasorella and their co-workers examined what genes were reduced in the absence of Id proteins, they discovered some of these genes encoded proteins involved in cell adhesion. Therefore the Id proteins brings on-line a whole host of proteins that cause the neural stem cells to stick to their stem cell niche., This adhesion allows the neural stem cells to divide and increase in numbers. However, the Id protein is not completely segregated to the sister cell and this cell does not express the cell adhesion genes and detaches from the stem cell niche. The detachment from the stem cell niche induces differentiation in the neural stem cell, and the specific cell type it forms depends upon microenvironmental cues.

Therapeutic application of these finds will require a good deal more research.  Dr. Iavarone said. “Multiple studies show that NSCs respond to insults such as ischemic stroke or neurodegenerative diseases. If we can understand how to manipulate the pathways that determine stem cell fate, in the future we may be able to control NSC properties for therapeutic purposes.”

“Another aspect,” added Dr. Lasorella, “is to determine whether Id proteins also maintain stem cell properties in cancer stem cells in the brain. In fact, normal stem cells and cancer stem cells share properties and functions. Since cancer stem cells are difficult to treat, identifying these pathways may lead to more effective therapies for malignant brain tumors.”

Stephen G. Emerson, MD, PhD, director of the Herbert Irving Comprehensive Cancer Center at NewYork-Presbyterian Hospital & Columbia University Medical Center, added that, “Understanding the pathway that allows stem cells to develop into mature cells could eventually lead to more effective, less toxic cancer treatments. This beautiful study opens up a wholly unanticipated way to think about treating brain tumors.”

Gallbaldder Contains Stem Cell Source for Liver Regeneration

The research group of Guido Carpino at the University of Rome has announced at the 2012 International Liver Congress the existence of a stem cell population in the gallbladder. This is significant because the gall bladder is an organ that is often discarded during organ donations and surgical procedures, but this organ contains a multipotential stem cell population.

Biliary tree stem/progenitor cells (BTSCs) have been previously identified in human extra hepatic bile ducts. BTSCs can form liver, gall-bladder and pancreas-specific cell types in culture and when injected into a laboratory animal (See Vincenzo Cardinale, et al., Hepatology 2011;54(6):2159-72).

In the present study, Carpino and his co-workers discovered that in the gallbladders of normal and sick mice, a stem cell population was available that could be easily isolated and were able to repopulate the liver and improve liver function (see Vincenzo Cardinale, et al., Nature Reviews Gastroenterology and Hepatology 2012; 9: 231-240).

Stem Cell Differentiation Requires Proper Compaction of DNA

Human cells have a compartment that houses the chromosomes known as the nucleus. The nucleus in human cells contains so much DNA that if the DNA was laid out end to end, it would stretch out for about one meter. Think of that – almost every cell in your body has one meter’s length of DNA in it. In order to properly package all that DNA into the nucleus of the cell, it must be assembled into a structure called “chromatin.”

Chromatin involves winding the DNA around small spools, and the spools are coiled into a fiber and then the fiber is looped into rosettes. The spools are composed of proteins called “histones.” Histone proteins are extremely conserved from one organism to another. In fact, histone proteins from cow peas and extremely similar to histones from cows. This tells scientists that the function of histones is exactly the same from one organism to another. The tiny spools are composed of four “core histones” known as H2A, H2B, H3 and H4. Each tiny spool has two copies of each core histone protein. The DNA is then wound around each of these histone complexes 1.8 times. The core histone complex with its DNA wound about it is either called a “core particle,” or a “nucleosome.”

To form chromatin, the nucleosomes are bound together by another histone proteins called H1. H1 winds the nucleosomes together into a chromatin fiber that is about 30 nanometers in diameter. These chromatin fibers are looped into a 300 nanometer coiled chromatin fiber, and these coiled chromatin fibers are then wound into 700 nanometer condensed chromatin. A chromosome that has been observed during the height of cell division (a phase called metaphase for those who are interested) has a diameter of 1400 nanometers, and since there are two chromatids present during cell division that compose each chromosome, these measurements agree completely with other work.

All of this might seem rather dry and uninteresting, except that researchers at the Georgia Institute of Technology and Emory University have demonstrated that chromatin compaction is essential for embryonic stem cell differentiation. Embryonic stem cells (ESCs) express several different types of H1 subtypes, and ESCs that fail to express these H1 subtypes show reduced chromatin compaction and impaired differentiation. The diminished differentiation capacity of these genes seems to derived from the inability of these cells to properly silence particular genes.

Yuhong Fan, assistant professor in the Georgia Tech School of Biology said, “While researchers have observed that embryonic stem cells exhibit a relaxed, open chromatin structure and differentiated cells exhibit a compact chromatin structure, our study is the first to show that this compaction is not a mere consequence of the differentiation process but is instead a necessity for differentiation to proceed normally,”

In this study, which was led by Fan and Todd McDevitt, who is an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, a ESC strain was used that lack three H1 subtypes; H1c, H1d, and H1e. These three H1 subtypes show increased expression levels during ESC differentiation, and the ESC strains triply deficient for these three H1 subtypes were unable to differentiate, even under standard culture conditions that normally induce differentiation. They also failed to differentiate in embryoid bodies, and could not form neural cells.

Anthony Carter, who oversees grants at National Institutes of Health’s National Institute of General Medical Sciences that deal with the effects of chromatin structure on gene expression, made these comments about this work: “This study has uncovered a new, regulatory function for histone H1, a protein known mostly for its role as a structural component of chromosomes. By showing that H1 plays a part in controlling genes that direct embryonic stem cell differentiation, the study expands our understanding of H1’s function and offers valuable new insights into the cellular processes that induce stem cells to change into specific cell types.”

When the triply-deficient ESCs were subjected to differentiation protocols, they remained tightly packed together, much like the early embryo. The cells also expressed genes that are found in the inner cell mass of the early embryo, such as Oct4. In order for the cells of the early embryo to properly differentiate, genes like Oct4 must undergo programmed down-regulation.

Accord to Fan, ““H1 depletion impaired the suppression of the Oct4 and Nanog pluripotency genes, suggesting a novel mechanistic link by which H1 and chromatin compaction may mediate pluripotent stem cell differentiation by contributing to the epigenetic silencing of pluripotency genes. While a significant reduction in H1 levels does not interfere with embryonic stem cell self-renewal, it appears to impair differentiation.”

In order to make embryoid bodies, this study utilized a rotary suspension technique that was developed by McDevitt and his co-workers. Normally, scientists use the so-called “hanging drop” method in which cells are placed in a drop of medium with few growth factors (20% serum usually), and suspended upside down on a microscope slide in a sealed chamber that prevents desiccation. under these conditions, the ESCs will form roundish little balls of cells that differentiate on the inside. These are known as embryoid bodies, and McDevitt’s technique forms three-dimensional embryoid bodies at very high-efficiency.

Embryoid bodies contain cell types of all three primary embryonic germ layers (endoderm, mesoderm and ectoderm). However, when the triply-deficient ESC line was subjected the McDevit’s embryoid body-making protocol, that lacked differentiated cell types and largely resisted differentiation.

As noted by McDevitt, “H1 triple-knockout embryoid bodies displayed a reduced level of activation of many developmental genes and markers in rotary culture, suggesting that differentiation to all three germ layers was affected.”

Fan and MCDevitt’s groups tried to add back H1 subtypes to the triply-deficient ESC strain. According to Fan, “When we added one of the deleted H1 subtypes to the embryoid bodies, Oct4 was suppressed normally and embryoid body differentiation continued. The epigenetic regulation of Oct4 expression by H1 was also evident in mouse embryos.”

This work also examined the ability of the triply-deficient ESC strain to differentiate into neural cells. However, the H1 triple-knockout ESCs could form neither neuronal nor glial cells, and unable to contribute to the formation of a neural network. Only 10% of the H1 triple-knockout embryoid bodies formed neurites and they produced on average eight neurites each in comparison to normal embryoid bodies, which produced, on average, 18 neurites.

In he future, Fan and McDevitt would like to investigate if controlling H1 histone levels can be used to influence the reprogramming of adult cells to form induced pluripotent stem cells, which have the capacity to differentiate into tissues in a way similar to embryonic stem cells.

Half of all Americans are Pro-Life

The Gallup Pole’s new numbers have shown that at least half of all Americans, which includes, Republicans, Democrats, and Independents, are pro-life. This does not mean that 50% of all Americans think that abortion should be illegal, but that enough of them think that abortion is immoral enough to call themselves as pro-life.  This suggests that the pro-life movement has gone from fringe to mainstream.  It may not be too long before enough Americans view abortion as immoral so that it becomes a restricted practice, and is permissible only under particular circumstances.

See Wesley J Smith’s article about it here.

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.

Regenerated Hair from Adult Stem Cells

Japanese researchers led by Takashi Tsuji from the Research Institute for Science and Technology at Tokyo University of Science have made bioengineered hair follicle germ cells from adult epithelial stem cells and dermal papillae cells. These hair follicle germ cells form functional hair follicles and grow hair. This is a proof-of-concept experiment for bioengineered organ replacement that may then proceed to human clinical trials.

These bioengineered follicle germs were made with epithelial and mesenchymal stem cells from skin found on the backs of mouse embryos (stage E18 for those who are interested). Once these cells were dissociated, they were combined with stem cells from adult hair follicles (the bulge region).

In a previous paper, Tsuji’s lab showed that a bioengineered hair follicle germ that was reconstituted from embryonic follicle germ-derived epithelial and mesenchymal cells could generate a bioengineered hair follicle and shaft if they used their new technique (Nakao, K. et al. The development of a bioengineered organ germ method. Nat. Methods 4, 227–230 (2007)). However, the Nature Methods paper did not transplant these bioengineered hair follicles into the skin of laboratory mice to determine if they could produce fully functional hair regeneration that includes hair shaft elongation, hair cycles, connections with surrounding tissues, and the regeneration of stem cells and their niches.

In this recent publication, Tsuji’s co-workers in his lab rigorously established that these bioengineered hair follicles could do everything a naturally produced hair follicle could do. In order to direct the growth of the hair toward the surface of the skin, Tsuji and others used a tiny plastic container with a fine nylon thread in the middle to direct the growth of the hair shaft. Previous experience had shown that implanting the bioengineered hair follicles into the skin caused them to form “epithelial cysts,” or fluid-filled vesicles that did not form hairs. The reason for this abnormal behavior is that the implanted follicles are connected with the surface of the skin, and therefore, lack polarity. The small, plastic containers provides a surface upon which the cells can grow toward the skin surface, and the nylon thread directs the extension of the hair shaft toward the skin surface.

These hair follicles expressed all the right genes and also cycled the way normal hair follicles cycle (growth of the hair, cessation of growth, dumping the hair shaft, and then regrowth of the hair shaft). This study definitely demonstrates the ability of adult tissue-derived follicular stem cells to serve as bioengineered organ replacements therapies.