Molecular Signature Distinguished Old Stem Cells from New Stem Cells

Eukaryotic organisms include every living thing with the exception of bacteria, Bacteria are known as prokaryotes, and they do not have an organized nucleus. Eukaryotic cells, on the other hand, have an organized nucleus in which that houses the chromosomes, which are linear molecules of DNA.

DNA is the molecule that stores genetic information. The chromosomes of eukaryotic organisms are sometimes rather long. How then does the cell manage to store all that DNA in such a small compartment such as the nucleus? The answer is that DNA in eukaryotic cells is wound into a tight configuration known as chromatin.

Chromatin consists of DNA molecules that are spooled around a cylindrical structure made of histone proteins. There are four so-called “core histones” that compose the cylinders and the DNA winds around these histone cores. Then a non-core histone called H1 pulls the histone cylinders with their DNA wound about them together to form higher-order structures. The histone cylinders wound about with DNA are called “nucleosomes” or “core particles.” The assembled clusters to nucleosomes are called “30 nanometer solenoids.”


You might think that DNA all wound into chromatin would be difficult to access and transcribe.  If you think that, then you are correct.  How then does the cell access DNA wound into chromatin? It modifies the histones so that the grip the histones have on the DNA is loosened.  Since histones are positively charged and DNA is negatively charged (lots of phosphate), the two molecules bind to each other rather tightly.  However, If histones are decorated with acetate groups, they become less positively charged and bind to DNA less tightly.  This opens up the chromatin for gene expression.  However, if histones are decorated with methyl groups (CH3), then proteins bind the histones and cinch the DNA even more tightly so that nothing is expressed.  This is known as the “histone code,” since geneticists can use the chemical modifications of histones to make highly educated guesses about if genes will be expressed and the levels at which they will be expressed.

A research team at Stanford University in Palo Alto, CA, led my Thomas Rando, professor of neurological sciences and chief of the Veterans Affairs Palo Alto Health Care System’s Neurology service, has identified characteristic differences in histone modifications between stem cells from the muscles of young mice and old mice.  Rando’s team also identified histone signatures characteristic of sleeping or quiescent and active stem cells in the muscles of young mice.

Rando said, “We’ve been trying to understand both how the different states a cell finds itself in can be defined by the markings on the histones surrounding its DNA, and to find an objective way to define the ‘age’ of a cell.”

All the cells of our body share the same genes, but these cells can be remarkably different in their function, structure, shape, and metabolism.  Only a fraction of a cell’s genes are actually turned one and are actively making proteins.  A muscle cells produced muscle-specific proteins and a liver cell makes liver cell specific proteins.  Rando’s team has generated data that suggests that these same kinds of on/off differences may distinguish old stem cells from young stem cells.

First a little background in necessary.  In 2005, Rando and others published a study that demonstrated that stem cells in several tissues from older mice, including muscle, seemed to act younger after continued exposure to the blood of a younger mouse.  The capacity of these stem cells in older mice to divide, differentiate, and repopulate tissues declines with advancing age.  However, after these stem cells from older mice were exposed to younger mouse’s blood, their ability to proliferate and repair tissues resembled those of their stem-cell counterparts in younger animals (see Conboy IM et al., Nature. 2005 433(7027):760-4).

Rando and his group asked the next question: “What is happening inside these cells that make them act as though they are younger?”  The first place Rando and others decided to look was the chemical modifications of their histones.  The cell population they examined was muscle satellite cells, which are relatively easy to isolate and grow in culture.  Normally, muscle satellite cells sit within skeletal muscles and do well little.  However, once the muscle is damaged, muscle satellite cells wake up, swing into action, and divide and fuse with damaged muscle fibers to repair them.

Muscle Satellite Cells in green
Muscle Satellite Cells in green

In mice that are old, histones in muscle satellite cells are a mixture of signals that tell expression to stop and signals that tell gene expression to go.  However, in satellite cells from younger mice, the histones are largely a collection of go signals with only few stop signals.  According to Rando, “Satellite cells can sit around for practically a lifetime in a quiescent state, not doing much of anything.  But they’re ready to transform to an activated state as soon as they get the word that the tissue needs repair.  So you might think that satellite cells would be already programmed in a way that commits them solely to the ‘mature muscle cell’ state.”  Thus you would expect those genes specific for other tissues like skin, brain or fat would be marked with stop signals.

Instead quiescent satellite cells taken from the younger mice contained histones with a mixture of stop and go signals in those genes ordinarily reserved for other tissues.  This was similar to what was observed in mature muscle-specific genes.  Satellite cells from older mice were pockmarked with stop signals interspersed with go signals.

Are these changes typical of those that occur in other types of stem cells in other tissues?  That is presently unknown.  Also, what is the signal in the blood from the younger mice that causes the satellite cells function as though they are young?”  Rando said, “We don’t have the answers yet.  But now that we know what kinds of these changes occur as these cells age, we can ask which of these changes reverse themselves when an old cell goes back to becoming a young cell.”

Rando’s group is presently examining if the signatures they have identified in satellite cells generalize to other kinds of adult stem cells as well.

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.