Studying Tough-to-Examine Disease by Using Brain Cells Made from Stem Cells


Diseases that are hard to study, such as Alzheimer’s, schizophrenia, and autism can be examined more safely and effectively thanks to an innovative new method for making mature brain cells from reprogrammed skin cells. Gong Chen, the Verne M. William Chair in Life Sciences and professor of biology at Penn State University and the leader of the research team that designed this method said this: “The most exciting part of this research is that it offers the promise of direct disease modeling, allowing for the creation, in a Petri dish, of mature human neurons that behave a lot like neurons that grow naturally in the human brain.”

Chen’s method could lead to customized treatment for individual patients that are based on their own genetic and cellular profile. Chen explained it this way: “Obviously we do not want to remove someone’s brain to experiment on, so recreating the patient’s brain cells in a Petri dish is the next best thing for research purposes and drug screening.”

In previous work, scientists at the University of Wisconsin in James Thomson’s laboratory and in Shinya Yamanaka’s laboratory at Kyoto University in Kyoto, Japan discovered a way to reprogram adult cells into pluripotent stem cells. Such stem cells are called induced pluripotent stem cells or iPSCs. To make iPSCs, scientists infect adult cells with genetically engineered viruses that introduce four specific genes (OCT4, SOX2, KLF4 and cMYC for those who are interested). These genes encode transcription factors, which are proteins that bind to DNA or to the machinery that directly regulates gene expression.  These transcription factors turn on those genes (e.g., OCT4, NANOG, REX1, DNMT3β and SALL4, and OCT4) that induce pluripotency, which means the ability to form any adult cell type.  Once in the pluripotent state, iPSCs can be cultured and grown life embryonic stem cells and can differentiate into adult cell types and tissues.

As Chen explained, “A pluripotent stem cell is a kind of blank slate.”  Chen continued, “During development, such stem cells differentiate into many diverse specialized cell types, such as a muscle cell, a brain cell, or a blood cell.  So, after generating iPSCs from skin cells, researchers then can culture them to become brain cells, or neurons, which can be studies safely in a Petri dish.”

Chen’s team invented a protocol to differentiate iPSCs into mature human neurons much more effectively than previous protocols.  This generates cells that behave neurons in our own brains and can be used to model the individualized disease of a single patient.

In the brain, neurons rarely work alone, but instead are usually in close proximity to star-shaped cells called astrocytes.  Astrocytes are very abundant cells and they assist neuron function and mediate neuronal survival.  “Because neurons are adjacent to astrocytes in the brain, we predicted that this direct physical contact might be an integral part of neuronal growth and health,” said Chen.  To test this hypothesis, Chen and his colleagues began by culturing iPSCs-derived neural stem cells, which are stem cells that have the potential to become neurons.  These cells were cultured on top of a one-cell-thick layer of astrocytes sop that the two cell types were physically touching each other.

Astrocytes
Astrocytes

“We found that these neural stem cells cultured on astrocytes differentiated into mature neurons much more effectively,” Chen said.  This contrasts Chen’s method with other neural stem cells that were cultured alone in a Petri dish.  As Chen put it, the astrocytes seems to be “cheering the stem cells on, telling them what to do, and helping them to fulfill their destiny to become neurons.”

While this sounds a little cheesy, it is undeniable that the astrocyte layer increases the efficiency of neuronal differentiation of iPSCs.  Personalized medicine is moving beyond the gene level, to the level of cellular organization and tissue physiology, and iPSCs are showing the way.

Neurons Made from the Skin Cells of Down Syndrome Patients Show Reduced Connectivity


The most common form of intellectual disability in the United States is caused by Down syndrome (DS). DS results when babies are born with an extra copy of an extra piece of chromosome 21. Individuals with DS show various types of intellectual deficits and other health problems as well, such as heart problems, poor muscle tone, an under-active thyroid, respiratory infections, hearing problems, celiac disease, eye conditions, depression or behavior problems associated with attention-deficit hyperactivity disorder or autism.

Even though Down syndrome patients have symptoms and health problems that are well described, how the extra chromosome causes such widespread effects is still largely mysterious.

In recently published research, Anita Bhattacharyya, who is a neuroscientist at the Waisman Center at the University of Wisconsin-Madison, reported that brain cells that were grown from skin cells taken from individuals with Down syndrome.

“Even though Down syndrome is very common, it’s surprising how little we know about what goes wrong in the brain,” says Bhattacharyya. “These new cells provide a way to look at early brain development.”

The skin cells taken from DS patients were grown in culture and genetically engineered to so that a fraction of them were transformed into induced pluripotent stem cells (iPSCs). Since iPSCs can be differentiated into any adult cell type, Bhattacharyya’s lab, working with collaboration with Su-Chun Zhang and Jason Weick, grew those iPSCs in culture and differentiated them into dorsal forebrain neurons, which they could test in the laboratory.

Neurophysiological tests of the DS neurons revealed that these neurons formed a reduced number of connections between them each other. Bhattacharyya says. “They communicate less, are quieter. This is new, but it fits with what little we know about the Down syndrome brain.” Brain cells communicate through connections called synapses, and the Down neurons had only about 60 percent of the usual number of synapses and synaptic activity. “This is enough to make a difference,” says Bhattacharyya. “Even if they recovered these synapses later on, you have missed this critical window of time during early development.”

Bhattacharyya and colleagues also examined the genes that were affected in the Down syndrome stem cells and neurons. They discovered that those genes on the extra chromosome were increased 150 percent, which is consistent with the contribution of the extra chromosome.

However, the output of about 1,500 genes elsewhere in the genome was strongly affected. “It’s not surprising to see changes, but the genes that changed were surprising,” says Bhattacharyya. The predominant increase was seen in genes that respond to oxidative stress, which occurs when molecules with unpaired electrons called free radicals damage a wide variety of tissues.

“We definitely found a high level of oxidative stress in the Down syndrome neurons,” says Bhattacharyya. “This has been suggested before from other studies, but we were pleased to find more evidence for that. We now have a system we can manipulate to study the effects of oxidative stress and possibly prevent them.”

DS includes a range of symptoms that might result from oxidative stress, Bhattacharyya says, including accelerated aging. “In their 40s, Down syndrome individuals age very quickly. They suddenly get gray hair; their skin wrinkles, there is rapid aging in many organs, and a quick appearance of Alzheimer’s disease. Many of these processes may be due to increased oxidative stress, but it remains to be directly tested.”

Oxidative stress could be especially significant, because it appears right from the start in the stem cells. “This suggests that these cells go through their whole life with oxidative stress,” Bhattacharyya adds, “and that might contribute to the death of neurons later on, or increase susceptibility to Alzheimer’s.”

Other researchers have created neurons with DS from induced pluripotent stem cells, Bhattacharyya notes. “However, we are the first to report this synaptic deficit, and to report the effects on genes on other chromosomes in neurons. We are also the first to use stem cells from the same person that either had or lacked the extra chromosome. This allowed us to look at the difference just caused by extra chromosome, not due to the genetic difference among people.”

The research, published the week of May 27 in the Proceedings of the National Academy of Sciences, was a basic exploration of the roots of Down syndrome. Bhattacharyya says that while she did not intend to explore treatments in this work, she did note that “we could potentially use these cells to test or intelligently design drugs to target symptoms of Down syndrome.”

Skin Cells Used to Make Personalized Bone Substitutes


Patient-specific bone substitutes have been produced by a team of scientists from the New York Stem Cell Foundation. Darja Marolt and Giuseppe Maria de Peppo from the New York Stem Cell Foundation (NYSCF) led the study that demonstrated that customizable, three-dimensional bone grafts that can be produced on-demand for patients from their own cells.

Marolt and de Peppo and their co-workers used skin grafts from their patients to isolate skin fibroblasts that were reprogrammed to induced pluripotent stem cells (iPSCs). Because iPSCs are made from the patient’s own cells, they have the same profile of cell surface proteins as the patient’s own tissues. Therefore, they are very unlikely to be rejected by the patient’s immune system. Also, iPSCs have the ability to differentiate into any cell type found in the adult body, and therefore, can be used to form bone cells.

iPS cells

After deriving iPSCs from patient skin cells, de Peppo, Marolt, and colleagues coaxed the cells to form osteoblasts (the cells that form bone), and seeded them onto a scaffold that mimicked three-dimensional bone structure. These structures were grown in a bioreactor that fed the cells oxygen and nutrients.

According to Marolt, “Bone is more than a hard mineral composite, it is an active organ that constantly remodels. Blood vessels shuttle important nutrients to healthy cells and remove waste; nerves provide connection to the brain; and, bone marrow cells form new blood and immune cells.”

Previous studies have demonstrated that cells from other sources also possess bone-forming potential. However, these same studies have revealed serious shortcomings of the clinical potential of such cells. A patient’s own bone marrow stem cells can form bone and cartilaginous tissue, but not the accompanying underlying vasculature and nerve compartments. Also, embryonic stem cell derived bone may prompt an immune rejection. Therefore, the use of iPSCs can overcome many of these limitations.

As de Peppo noted: “No other research group has published work on creating fully viable functional three-dimensional bone substitutes from humans iPS cells. These results bring us closer to achieving our ultimate goal, to develop the most promising treatments.”

Since bone injuries and defects are often treated with bone grafts that are taken from other parts of the body or a tissue bank. Alternatively, synthetic alternatives can also be used, but none of these possibilities provide the means for complex reconstruction and they may also be rejected by the immune system, or fail to integrate with surrounding connective tissue. n the case of trauma patients who suffer from shrapnel wounds or vehicular injuries , the traditional treatments provide only limited functional and cosmetic improvements.

To access the integrity of the bioreactor-made bone, the NYSCF team implanted them into animals. Implantation of undifferentiated iPSCs formed tumors, but transplantation of the iPSC-derived bone produced no tumors, but also produced grafts that effectively integrated into the bones, connective tissues and blood vessels of the animals.

Susan Solomon, CEO of NYSCF, said of this work, “Following from these findings, we will be able to create tailored bone grafts, on demand, for patients without any immune rejection issues. She continued: “it is the best approach to repair devastating damage or defects.”

The therapeutic relevance of this work aside, these adaptive bone substitutes can also serve as models for bone development and various bone pathologies. Such bone exemplars could serve as models for drug testing and drug development.

Decoding the Mechanisms Behind Stem Cell Reprogramming


Kenneth Zaret is the associate director of the Penn Institute for Regenerative Medicine and is also Professor of Cell and Developmental Biology at the University of Pennsylvania. Zaret’s laboratory has examined the process by which adult cells are reprogrammed to make induced pluripotent stem cells (IPSCs). Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs, and iPSCs seem to offer many of the advantages of embryonic stem without the moral messiness of destroying human embryos to make them.

The production of iPSCs required genetic engineering techniques that introduce genes into cells. Introducing four genes – Oct4, Sox2, Klf4, and c-Myc – into adult cells drives them to de-differentiate at become iPSCs, but this process is very slow – it can take up to a month – and is quite inefficient – one in 1,000 cells becomes an iPSC. Also, even though iPSCs share many characteristics with embryonic stem cells, they are not exactly the same and differ in some ways.

Oct4 in Mammalian ESC Pluripotency

See this site for this figure.

This study, which was published in the journal Cell, attempted to define the reasons why iPSC production takes so long and is so inefficient. Zaret and his group examined the genomes of adult cells, 48 hours after the introduction of the four genes, and compared them to the genomes of the starting adult cells, fully reprogrammed iPSCs, cells near the end of the re-programming process (pre-iPSCs), and embryonic stem cells.

At 48 hours, Zaret and others found that of the transcription factors that had been introduced and overexpressed in the adult cells, three of them, Oct4, Klf4, and Sox2, tended to bind to the “distal enhancer elements” of genes. The phrase “distal enhancer elements” refers to regions of genes that help control when the gene is turned on. Most genes consist of a sequence of DNA known as the “coding region” that contains the sequences that are used to make the messenger RNA that will be translated into protein. However, genes also have other sequences that tell the cell when and where to make the messenger RNA. These control sequences are called “enhancers.” The Oct4, Klf4, and Sox2 proteins bind to enhancers in target genes and influence their expression. Because these proteins do this early heavy lifting, Zaret called them “pioneer factors.”

Now, there is a problem. The DNA of the cell is not an open book, but is bound up into a compact structure called chromatin. DNA tightly wound into chromatin does not allow access to proteins. Therefore, the pioneer factors for cell reprogramming are ready to bind to enhancers, but the DNA of the genome is too tightly wound to permit their binding. What is the cell to do? This is the job of the c-Myc protein, which enhances the binding of the other pluripotency factors to chromatin.

chromatin

There is another problem, however, and this is the genuinely remarkable finding of Zaret’s lab: 48 hours after the initiation of reprogramming, large sections of the genome of the cell are refractory to the binding of the pioneering factors. In Zaret’s own words: “Basically, large chunks of the human genome were physically resisting these factors from entering. That provided some understanding that you’ve got to overcome the binding requirement to get these factors to their final destination.”

What caused these chunks of the genome to be off-limits to the pioneer factors? Chromatin results from the assembly of DNA with very positively-charged proteins called histones. Histones act as miniature spools around which the DNA is wound and packaged. Chemical modification of the histones can influence the tightness of the chromatin. For example, the attachment of acetate groups tends to make chromating rather loose, and gene expression can readily occur, but the attachment of methyl (CH3-) groups tends to cinch the chromatin down so tightly that little gene expression can occur.

Histone modification

In the case of the off-limits portions of the genome in adult cells undergoing reprogramming, a histone modification called “H3K9me3,” which is a short hand for saying lysine residue number 9 on histone #3 had three methyl groups attached to it, blocks the pioneer factors from accessing the DNA under its structural compaction. However, if Zaret and his workers treated cells with an inhibitor that prevents the enzymes from modifying histones in this manner, they found that the reprogramming process was significantly accelerated.

Zaret thinks that these findings might not only tell us about the roadblocks to reprogramming, but also give us clues as to a way to work around the difficulties to reprogramming. His lab has uncovered a normal mechanism by which cells protect themselves from being reprogrammed under normal circumstances. In his own words: “We went into this thinking that we were going to learn something about the mechanism of conversion to pluripotency, but at the end of the day we ended up discovering new ways that cells control gene expression by shutting down parts of their genome.”

The importance of this work is difficult to overstate. In the words of Susan Haynes, from the National Institutes of Health General Medical Sciences division, which funded Zaret’s work, “These studies provide detailed insights into how reprogramming factors interact with the chromatin of differentiated cells and start them down the path toward becoming stem cells. Dr. Zaret’s work also identified a major structural roadblock in the chromatin that the factors must overcome in order to bind DNA. This knowledge will help improve the efficiency of reprogramming, which is important for any future therapeutic applications.”