Defects in in vitro generated stem cells

There are two methods for reprogramming mature cells to pluripotent stem cells, which can give rise to all cells of the body. The first direct comparison of the methods reveals that both can cause subtle molecular defects.

Pluripotent stem cells hold promise for disease modelling and therapeutics, because they have the potential to differentiate into almost all cell lineages. In particular, there is much interest in patient-derived pluripotent stem cells, which are genetically matched to the patient’s own cells, minimizing the risk of rejection by the immune system.

In the past decade, two cell-reprogramming methods have been successfully used to generate patient-derived pluripotent stem cells: (1) cloning and (2) direct reprogramming of differentiated cells to induced pluripotent stem cells, through the addition of a defined transcription-factor cocktail.

However, the molecular differences between cells derived using each method remain unclear.

Derivation of induced pluripotent stem (iPS) cells is an appealing technology, because iPS cells can be reproducibly derived from patient samples. But comparison of iPS cells with the pluripotent embryonic stem (ES) cells generated during normal embryogenesis shows that human iPS cells are not completely reprogrammed, and reveals epigenetic differences between the two cell types (epigenetic marks are lingering genomic modifications that affect gene expression without changing DNA sequence).

Cloning — also called somatic cell nuclear transfer (SCNT) — involves transfer of the nuclear material from a mature donor cell into an egg from which the nucleus has been removed. Pluripotent cells, called nuclear transfer ES (NT ES) cells, which are genetically matched to the donor, then arise as the egg begins to develop into an embryo. Generation of patient-specific NT ES cells from adult human cells is now feasible. Although SCNT does not involve introducing transcription factors that have the potential to cause cancer (which is a problem with iPS cell generation), the protocol is technically difficult.

Figure 1: Comparing techniques for generating stem cells.

Comparing techniques for generating stem cells.

Three techniques can be used to generate pluripotent stem cells in vitro:

a) Induced pluripotent stem (iPS) cells are generated from mature cells, which can be directly converted by the addition of a transcription-factor cocktail.

b) In somatic cell nuclear transfer (SCNT), the nucleus is removed from an egg and replaced with the nucleus from a mature donor cell. As this hybrid cell develops into an embryo, pluripotent stem cells called nuclear transfer embryonic stem (NT ES) cells can be extracted from a region called the inner cell mass (ICM).

c) Embryos derived from in vitro fertilization (IVF) give rise to IVF ES cells that can be extracted from the ICM.

Incomplete demethylation patterns correlated with abnormal gene transcription were observed in iPS cells. NT ES cells were more similar to IVF ES cells, although some transcriptional alterations were apparent in both reprogrammed cell types.

There is an abundance of factors that can be used to reprogram cells and expand them in vitro, and each can influence the epigenetic and functional properties of reprogrammed cells in distinct ways. This complexity disrupts simplistic attempts to define and obtain ‘perfect’ stem cells. (Vladislav KrupalnikJacob H. HannaNature, 511,160 – 162 (10 July 2014)) The Research article subject to this review: Ma et al., Abnormalities in human pluripotent cells due to reprogramming mechanisms, Nature, 511,177–183(10 July 2014)doi:10.1038/nature13551


Cancer’s madness

by Richard Saltus (HHMI Bulletin, Spring 2014)

Computational approaches reveal that massive chromosome alterations give cancer an edge.

Cancer cells are known for the rampant disorder in their genomes: extra or absent chromosomes or parts of chromosomes, long stretches of DNA gone missing or present in too many copies. “It looks like someone threw a stick of dynamite into the nucleus,” says HHMI Investigator Stephen Elledge of Harvard Medical School and Brigham and Women’s Hospital.

This chaotic state is called aneuploidy. It stems from errors during cell division causing the daughter cells to have abnormal numbers of chromosomes or chromosome fragments. Aneuploidy affects hundreds or thousands of genes and can wreak all kinds of havoc, including miscarriages, lethal birth defects, and disorders like Down syndrome.

Based on his group’s latest research, Elledge says these massive alterations have evolved because they give malignant cells an edge in the “brutal competition” to win out over normal cells.

Because chromosomes exist in pairs, the loss of single chromosomes affects only one copy of a given gene. The second copy on the partner chromosome remains intact. As a result, these “hemizygous” losses have a weaker effect on cancer growth than the mutation of both copies of a tumor suppressor gene. But the additive combination of groups of hemizygous losses can have a large impact.

We have basically answered the question: Does aneuploidy drive cancer? We believe it does” says Stephen Elledge.

To those familiar with the “two-hit” model of cancer, it may come as a surprise that loss of a single gene copy can have an effect. According to this model, a mutation in a single copy of a tumor suppressor gene does nothing because the second copy compensates, and only if that second copy is subsequently “hit,” or mutated, does the cell begin its malignant journey.

However, Elledge cites evidence that a large proportion of cancer-suppressing genes are “haploinsufficient”—loss of even one copy can contribute to cancer development. In fact, Elledge estimates that 30 percent of all genes in humans are haploinsufficient, which has important implications for human development and disease.

“Losing or gaining single copies of genes on their own may have small effects, but altering many at the same time gives the cancer cell an advantage,” says Angelika Amon, a biologist and HHMI investigator at the Massachusetts Institute of Technology who studies aneuploidy. “Once you see [Elledge’s findings], you realize these losses and gains are not random noise in tumors, and we can begin to understand them.” 

Precise division of a bacterial cell

The recent development of cell biology techniques for bacteria to allow visualization of fundamental processes in time and space, and their use in synchronous populations of cells, has resulted in a dramatic increase in our understanding of cell division and its regulation in these tiny cells. Cell division in bacteria is driven by a cytoskeletal ring structure, the Z ring, composed of polymers of the tubulin-like protein FtsZ, at the division site precisely at midcell. Z-ring formation must be tightly regulated to ensure faithful cell division, and several mechanisms that influence the positioning and timing of Z-ring assembly have been described. Several membrane-associated division proteins are then recruited to this ring to form a complex, the divisome, which causes invagination of the cell envelope layers to form a division septum.

Another important but as yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. How bacteria coordinate cell cycle processes with nutrient availability and growth is a fundamental yet unresolved question in microbiology. The deletion of the gene encoding pyruvate kinase (pyk), which produces pyruvate in the final reaction of glycolysis, rescues the assembly defect of a temperature-sensitive ftsZ mutant and has significant effects on Z-ring formation in wild-type B. subtilis cells. Addition of exogenous pyruvate restores normal division in the absence of the pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the coordination of bacterial growth and division (mBio 2014).


Fructose vs Glucose

Fructose and glucose have the same caloric value, but the two sugars are metabolized differently. It emerges that mice that cannot metabolize fructose are healthier when placed on carbohydrate-rich diets.

A drastic increase in dietary sugar consumption in the western world during the past four decades has been paralleled by epidemics of obesity and metabolic syndrome, suggesting a cause-and-effect relationship. Yet the relative contribution of individual sugars — as opposed to total caloric intake — to this epidemic remains controversial. For instance, increased intake of fructose, which is enriched in soft drinks and processed foods, has been proposed to greatly contribute to these disorders. However, this proposal has not been universally embraced.

Dietary sugar encompasses several carbohydrates. Most often, however, it describes starch, sucrose and high-fructose corn syrup, each of which is composed of glucose with or without fructose: starch, found in bread and rice, is a glucose polymer; sucrose (table sugar) is a disaccharide made up of glucose and fructose; and high-fructose corn syrup, a common constituent of soft drinks, is a mixture of approximately 40% glucose and 60% fructose. From an energetic standpoint, a molecule of glucose has the same caloric value as a molecule of fructose. However, the human body treats these carbohydrates quite differently, raising questions about their individual roles in obesity and metabolic syndrome.

In general, glucose is used directly by tissues such as the muscles and brain as an energy source. Excess glucose is stored in the liver as glycogen (a glucose polymer) but can also be converted into fructose by the polyol biochemical pathway. By contrast, fructose is almost exclusively metabolized by the liver. In this organ, ketohexokinase (KHK) — a liver-specific fructose-metabolizing enzyme also known as fructokinase — traps fructose in liver cells as fructose 1-phosphate. Unlike fructose 6-phosphate (an isomer of fructose 1-phosphate that participates in the biochemical pathway of glycolysis), fructose 1-phosphate can bypass a major regulatory step in glycolysis that generates fructose 1,6-bisphosphate through the action of the energy-sensitive enzyme phosphofructokinase. Thus, fructose can be converted into fat, unfettered by the cellular controls that prevent unrestrained lipid synthesis from glucose.

By this logic, diets high in fructose could cause excess fat accumulation in the liver, leading to the liver disorders fatty liver disease, steatohepatitis and, ultimately, cirrhosis. Liver fat could also be released into the circulation and taken up by fat cells in other tissues, resulting in obesity. Furthermore, the circulating fat could accelerate the onset of cardiovascular disease, insulin resistance and type 2 diabetes. So fructose over-consumption may be at the heart of metabolic syndrome, which has also been linked to poor outcome of a wide range of cancers.(Nature 502, 181–182 (10 October 2013) doi:10.1038/502181a)

The Map of Health

The Map of Health

When seen through the microscope, the tissues that form our organs and body parts can be stunningly beautiful, with all the complex structures that determine and enable their function forming beguiling, literally organic, patterns.

What are the main causes of death around the world? If we need to

take a single cause, cardiovascular disease is the winner. And if we look into it in a little more detail, we see differences from area to area, not only in the causes of death but also in the diseases that are the greatest burden for those societies.

North America struggles with rising obesity, and this adipose tissue (fat) is more beautiful close up than you would imagine.

Central and South America are represented here by pulmonary tissue (lungs); smoking and respiratory infections are a leading cause of death.

Europe, with its ageing population, suffers greatly from neurodegenerative diseases, including dementia (neurones, brain tissue).

Great swathes of the middle East and central Asia are shown here as cardiac muscle (heart), as these regions are afflicted with rising levels of hypertension and other causes of heart and cardiovascular failure.

The far East and the Pacific look beautiful in pancreatic acinar tissue; its failure causes diabetes, a major problem in this area, frequently described as a diabetes epidemic.

And Africa is made of blood here. The only continent where the leading cause of death are transmittable diseases (infections), notably malaria and HIV.

The good news is, we have medicines and other treatments to cure, alleviate, prevent or slow down the progression of all these diseases. And many people around the world are doing research to make progress on all these fronts.

Can you see the mitochondria? There are 5 hidden in the map. They are the organelles inside our cells responsible for energy generation. Mitochondrial research will play an important role over the coming years!

Data from WHO 2008 (accessed in February 2013)

Cancer prevention through a diet that affects your epigenome

Cancer, the leading cause of death worldwide, caused 8.2 million deaths in 2012. With 575,000 deaths attributable to cancer in 2010 in the United States, cancer-related deaths in the US are second only to those caused by heart disease, which caused 594,000. How can we end cancer? First and foremost, focus on prevention—the most viable option as a cure.

Historically, cancer has been perceived as a disease in which our genetic makeup dictates our likelihood of developing cancer. Presently, it has become broadly recognized that the initiation and progression of cancer is an intricate web of both genetic makeup and epigenetic events that alter our gene expression. Many studies have proven that epigenetic alterations are key components of the initiation and progression of cancer. These epigenetic processes—including DNA methylation, histone modification, and microRNA expression—are potentially reversible.

CpG island hypermethylation and down-regulation, histon acetylation and the resulting up-regulation of genes are common for many genes involved in a broad range of functions that are deregulated in cancer.

Dietary compounds have been shown to elicit epigenetic changes in cancer cells. To fully understand how we can modulate cancer prevention through lifestyle, research must focus on how diet and bioactive food components specifically impact epigenetic processes. Antioxidants such as carotenoids and fiber found in many vegetables and fruit offer a variety of anti-cancer benefits. Increased dietary folate, a soluble form of B6 vitamin, consumption has been linked to a decrease in colorectal cancer through its affect on DNA methylation. Dietary phytochemicals, that act as anti-cancer agents (including polyphenols, genistein, sulforaphane, resveratrol, and curcumin) have been shown to act through epigenetic mechanisms.

Cancer prevention is the best way to ultimately cure the disease. To work towards cancer prevention, we must further explore how dietary modifications may achieve epigenetic reprogramming, resulting in the maintenance of normal gene expression and reversal of tumor progression. (Scizzle, FEBRUARY 4, 2014 •  KELLY JAMIESON THOMAS)

Healing cancer cells and aerobic glycolysis

Instead of relying on mitochondrial oxidative phosphorylation, most cancer cells rely heavily on aerobic glycolysis, a phenomenon termed as “the Warburg effect. This effect may be is a direct consequence of damage and it persists in cancer cells that recover from damage. Glycolysis and rate of cell proliferation in cancer cells that recovered from severe damage show that such in vitro Damage-Recovered (DR) cells exhibit mitochondrial structural remodeling, display Warburg effect, and show increased in vitro and in vivo proliferation and tolerance to damage.

To test whether cancer cells derived from tumor microenvironment can show similar properties, (DR) cells from tumors show increased aerobic glycolysis and a high growth rate. These findings show that Warburg effect and its consequences are induced in cancer cells that survive severe damage. (Biochemical and biophysical research communications. 2014 May 24, PMID: 24802411).