Induced Pluripotent Stem Cells Form Limbal-Like Stem Cells

Limbal epithelial stem cells or LESCs are found at the periphery of the cornea and they continuously renew the corneal epithelium. Loss of this stem cell population can cause loss of corneal transparency and eventual loss of vision.

Genetic conditions can cause LESC deficiency, such as congenital aniridia, Stevens-Johnson syndrome or Ocular cicatricial pemphigoid. Other causes of LESC deficiency include chemical or thermal burns to the eye, microbial infections, extended contact lens wear, sulfur mustard gas poisoning, or chronic inflammation of the eye,

Limbal epithelial stem cells reside in the basal layer of the epithelium (Ep), which undulates at the limbus. Daughter transient amplifying cells (TACs) divide and migrate towards the central cornea (arrowed) to replenish the epithelium, which rests on Bowman's layer (BL). The stroma (St) of the limbal epithelial stem cell niche is populated with fibroblasts and melanocytes and also has a blood supply.
Limbal epithelial stem cells reside in the basal layer of the epithelium (Ep), which undulates at the limbus. Daughter transient amplifying cells (TACs) divide and migrate towards the central cornea (arrowed) to replenish the epithelium, which rests on Bowman’s layer (BL). The stroma (St) of the limbal epithelial stem cell niche is populated with fibroblasts and melanocytes and also has a blood supply.

Treatments of LESC deficiency include limbal stem cell grafts from one eye to another, but these grafts have a 3-5-year graft survival of only 30%-45%. If LESCs are expanded in culture on human amniotic membrane, then 76% of the grafts will successfully take 1-3 years after grafting. This procedure is not standardized. If LESCs are grafted from a cadaver, their survival is low.

Given these less than optimal treatments for LESC deficiencies, Alexander Ljubimov and his team from UCLA have used induced pluripotent stem cells (iPSCs) to make cultured LESCs. Ljubimov and his coworkers derived iPSCs from the skin cells of volunteers with non-integrating plasmids. Then they grew these cells on corneas that have been stripped of their cells and human amniotic membranes and these cells differentiated into LESC-like cells.

Ljubimov and others also made iPSCs from human LESCs, and when they cultured these iPSCs derived from LESCs on human amniotic membranes for two weeks, the cells differentiated into LESCs that made LESC-specific genes, and had the epigenetic characteristics of LESCs.

These experiments show that the cell source for iPSC derivation can greatly influence the epigenetic characteristics of the iPSC line. Also these experiments show that iPSCs can be used to make LESCs that can potentially be used for therapeutic purposes.


Making Better Induced Pluripotent Stem Cells

On July 2nd of this year, a paper appeared in the journal Nature that performed complete genomic analyses of embryonic stem cells derived from embryos or cloned embryos, and induced pluripotent stem cells (iPSCs), which are made from reprogrammed adult cells.  They found that both embryonic stem cells made from cloned embryos and iPSCs derived from the same types of adult cells contained comparable numbers of newly introduced mutations.  However, when it came to the epigenetic modification of the genome (the small chemical tags attached to specific bases of DNA that gives the cell hints as to which genes to turn off), the epigenetic pattern of the embryonic stem cells made from cloned embryos more closely resembled that from embryonic stem cells.  The iPSCs still had some similarities with the adult cells from which they were derived whereas the embryonic stem cells made from cloned embryos were more completely reprogrammed.  From this the authors claimed that making embryonic stem cells by means of cloning is ideal for cell replacement therapies.

There is a big problem with this conclusion:  This was tried in animals and it did not work because of immunological rejection of the products from the stem cells.  For more information on this, see my book, The Stem Cell Epistles, chapter 18.

Despite this “bad news” for iPSCs, two recent papers have actually provided some good news for stem cells that can heal without destroying embryos.  The first paper comes from Timothy Nelson’s laboratory at the Mayo Clinic in Rochester, Minnesota.  Differentiation of iPSCs is, in some cases, rather efficient and the isolation procedures fail to effectively isolate the differentiated cells from potentially tumor-causing cells.  However, in other cases, the differentiation is inefficient and the isolation procedures are also rather poor, which leaves a large enough population of undifferentiated tumor-causing cells.

Nelson’ group has discovered that treating iPSCs and their derivatives with anti-cancer drugs like etoposide (a topoisomerase II inhibitor for those who are interested) increases engraftment efficiency and decreases the incidence of tumors.  My only problem with Nelson’s paper is that he and his colleagues used lentiviral vectors to make their iPSCs.  These vectors tend to produce iPSCs that are rather good at causing tumors.  I would have rather that he tried making iPSCs with other methods that do not leave permanent transgenes in the cells.  Nelson and his group transplanted their iPSC-derived cells into the hearts of mice where they could use high-resolution imaging to determine the number of cells that integrated into the heart and the presence of cell masses that were indicative of tumors.  None of the ectoposide-treated cell transplants caused tumors whereas 4 of the 5 transplants not treated with ectoposide caused tumors.  This paper appeared in Stem Cells and Development.

The second “good news” paper for iPSCs comes from Junji Takeda at the University of Osaka and Ken Igawa from the Tokyo Medical and Dental University, Japan.   In their paper from Stem Cells Translational Medicine, the Japanese groups collaborated to make iPSCs from skin based fibroblasts and then differentiate them into skin cells (keratinocytes).  However, they made the iPSCs in two different ways.  The first protocol utilized the piggyBac transposon system to make iPSCs.  The piggyBac system comes from moths, but it is highly active in mammalian cells.  It can deliver the genes to the cells, but the segment of DNA is then easily excised from the host cells without causing any mutations.  This system, therefore, will generate iPSCs that do not have any transgenes in them.  The second protocol used a system based on cytomegalovirus that leaves the transgenes in the cells but gradually inactivates their expression.

When these two types of iPSCs were compared, they seems to be essentially identical when grown in culture.  Thus in the pluripotent state, the cells were equivalent for the most part.  But once the iPSC lines were differentiated into skin cells, the transgene-free iPSCs formed skin cells that looked, behaved and had the same gene expression profile as normal human skin cells.  The transgene-containing iPSCs differentiated into skin cells, but they did not look quite like skin cells, did not have the same gene expression profile as normal human skin cells, and did not behave like normal human skin cells.

The moral of this story is that not all iPSC lines are created equally and the way you derive them is as important as the cell type from which they were derived.  Also, even incomplete differentiation does not need to be an obstacle for iPSCs, since the cancer-causing cells can be removed by means of specific drugs.  Finally, not all that glitters is gold.  Cloned embryos may give you stem cells that look more like embryonic stem cells, but so what.  These might still suffer from many of the same set backs.  Add to that the ethical problems with getting women to give up their eggs for research and cures (see Jennifer Lahl’s movie Eggsploitation for more disturbing information about that), and you have a losing combination.

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.

Modulating Gene Expression to Repair Lungs

According to the American Lung Association and the National Institutes of Health, lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are on the rise. These are chronic ailments that affect the small airways of the lung. Asthma and COPD involve an injury-repair cycle that leads to the destruction of normal airway structure and function. Presently, drug treatments for COPD only treat the symptoms.

“A healthy lung has some capacity to regenerate itself like the liver,” noted Ed Morrisey, professor of Medicine and Cell and Developmental Biology and the scientific director of the Penn Institute for Regenerative Medicine in the Perelman School of Medicine, University of Pennsylvania. “In COPD, these reparative mechanisms fail.”

Morrisey and his colleagues are examining how epigenetic mechanisms control lung repair and regeneration. Epigenetics consists of chemical modifications to DNA and its supporting proteins that affect gene expression. Previous studies have discovered that smokers with COPD had the most significant decrease in one of the enzymes that controls these modifications, called HDAC2.

“HDAC therapies may be useful for COPD, as well as other airway diseases,” he explained. “The levels of HDAC2 expression and its activity are greatly reduced in COPD patients. We believe that decreased HDAC activity may impair the ability of the lung epithelium to regenerate.”

By using genetic and pharmacological approaches, Morrisey and others showed that the development of progenitor cells in the lung is specifically regulated by the combined function of two highly related HDACs, HDAC/1 and /2. Morrisey and his colleagues published their findings in the prestigious journal Developmental Cell.

By studying how HDAC activity and other epigenetic regulators control lung development and regeneration, they hope to develop new therapies to alleviate the unmet needs of patients with asthma and COPD.

HDAC1/2 deficiency leads to a loss of expression of the an essential transcription factor, a protein called Sox2, which in turn leads to disruption of airway epithelial cell development. This is affected in part by increasing the expression of two genes, Bmp4 and the tumor suppressor Rb1, both of which are inhibitors of cell proliferation including the proteins p16, and p21. This results in decreased epithelial proliferation in lung injury and inhibition of regeneration.

Together, these data support a critical role for HDAC-mediated mechanisms in regulating both development and regeneration of lung tissue. Since HDAC inhibitors and activators are currently in clinical trials for other diseases, including cancer, such compounds could be tested in the future for efficacy in COPD, acute lung injury and other lung diseases that involve defective repair and regeneration, said Morrisey.