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.


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.”

Georgetown Team Discovers New Type of Stem Cell

A research group a Georgetown Lombardi Comprehensive Cancer Center has developed a new and powerful stem cell in the laboratory that grows in sheets and has many characteristics desirable for regenerative medicine.

The senior author of this paper, Richard Schlegel, M.D., Ph.D., chairman of the department of pathology at Georgetown Lombardi, a part of Georgetown University Medical Center, said of these new stem cells: “These seem to be exactly the kind of cells that we need to make regenerative medicine a reality.”

The results of his lab’s research has been published in the November 19 online early edition of the Proceedings of the National Academy of Sciences (PNAS). In this publication, they report that their new stem-like cells do not express the same genes as embryonic stem cells and induced pluripotent stem cells (iPSCs). Thus, they do not produce tumors when injected into laboratory animals. Also, these cells are stable, since they differentiate into the cell types desired by researchers.

This publication is a continuation of a study published in December 2011, when Schlegel and his colleagues invented a laboratory technique that could maintain both normal and cancer cells alive indefinitely. Previously such a technique did not exist and it was simply not possible to keep such cells alive in the laboratory indefinitely.

Schlegel and others showed that if they added two different substances to their cells in culture – fibroblast feeder cells and a chemical that inhibits the Rho kinase – they could push the cells to assume a kind of stem-like state. While in this stem cell-like state, the cells would stay alive indefinitely. Once the feeder cells and the inhibitor were withdrawn, the cells reverted back to their original state. In this paper, Schlegel and his team called these laboratory-derived cells “conditionally reprogrammed cells” or CRCs. See Liu X et al. Am J Pathol. 2012 Feb;180(2):599-607.

Could CRCs be used for personalized medicine? A follow-up study suggested that they could. Published in the New England Journal of Medicine in September 2012, they found a patient who had a 20-year history of recurrent respiratory “papillomatosis” (a type of tumor) that had invaded the lung tissue in both lungs. The tumor was difficult to treat and slow-growing, but it stubbornly resisted treatment. Schlegel and his team made CRCs from this patient’s normal and tumorous lung tissue. By utilizing this technique, they discovered that the tumor cells were infected the same virus that causes warts; the human papillomavirus. They then used these cultured tumor CRCs to determine which cancer drug would work the best. They identified a drug called vorinostat as the best candidate, and 3 months after starting treatment, the tumors stopped growing and the prognosis looked substantially better for this patient (see Yuan H, et al. N Engl J Med. 2012 Sep 27;367(13):1220-7).

Of this paper, Schlegel said, “Our first clinical application utilizing this technique represents a powerful example of individualized medicine. It will take an army of researchers and solid science to figure out if this technique will be the advance we need to usher in a new era of personalized medicine.”

The present study is study was published in PNAS compared CRCs to embryonic stem cells and iPSCs. Both embryonic stem cells and iPSCs have been investigated for use in regenerative medicine, but both cells have the drawback to potentially producing tumors when injected into mice and “it is difficult to control what kind of cells these cells differentiate into,” Schlegel says. “You may want them to be a lung cell, but they could form a skin cell instead.”

In contrast, if lung cells are treated to make lung-specific CRCs, they can be expanded in culture to make a huge quantity of lung-specific cells, but when these conditions are withdrawn, the lung-specific CRCs will revert to mature lung cells. This transformation is rather rapid, since the cells become CRCs within three days of adding the inhibitor and the feeder cells. Once the cells lose their stem-like properties and potentially can be safely implanted into tissue.

A comparison of gene expression patterns from CRCs and embryonic stem cells (ESCs) or iPSCs showed that CRCs do not overexpress the same critical genes that embryonic stem cells and iPSCs express. “Because they don’t express those genes, they don’t form tumors and they are lineage committed, unlike the other cells,” Schlegel says. “That shows us that CRCs are a different kind of stem-like cell.”

In this study, Schlegel’s team used cervical cells and made CRCs from them. However, then they placed the cervical cell-derived CRCs on a three-dimensional platform, they grew into a canal-like structure that looked startlingly like a cervix. A very similar result was seen with cells extracted from the trachea. When the trachea-derived CRCs were grown on a 3-D platform, they begin to look like a trachea.

If and when use of CRCs are perfected for the clinic, which will require considerably more work, they have the potential to be used in a wide variety of novel ways. “Perhaps they could be used more broadly for chemosensitivity, as we demonstrated in the NEJM study, for regenerative medicine to replace organ tissue that is damaged, for diabetes — we could remove remaining islet ells in the pancreas, expand them, and implant them back into the pancreas —and to treat the many storage diseases caused by lack of liver enzymes. In those cases, we can take liver cells out, expand them and insert normal genes in them, and put them back in patients,” Schlegel says.

Schlegel added: “The potential of these cells are vast, and exciting research to help define their ability is ongoing.”

Leukemia Gene is a Key Factor for Nerve Cell Differentiation

Research from the laboratory of Pierre Vanderhaeghen from the Universite’ Libre de Bruxelles has provided a new perspective on brain development and neural stem cell biology.

The cerebral cortex is the most complex structure in the brain. It is the seat of such higher cortical functions as consciousness, learning and memory, emotion, motor control, and language. To execute these functions, the cerebral cortex is composed of an array of cortical neurons, and these cells are adversely affected in cases of neurological or even psychiatric disorders.

According to work from Vanderhaeghen’s laboratory, a gene known as BCL6 is a key element in the development of cortical neurons during development. BCL6 acts as a transcription factor, which is to say that it plays a role in gene expression. In the case of BCL6, this gene product prevents gene expression (functions as a repressor). In the immune system, BCL6 is made in antibody-producing cells (B cells) and it controls the response of B cells to a signaling protein called Interleukin 4 (IL-4). IL-4 drives the differentiation of B cells into antibody-making plasma cells and drives the maturation of plasma cells into those that make distinct types of antibodies. Even more interestingly, BCL6 is frequently abnormal in a blood cancer known as diffuse large B cell lymphoma (DLBCL),

Two members of Vanderhaeghen’s lab discovered BCL6 in a search for genes that modulate the production of new nerve cells from mouse embryonic stem cells. If they overexpressed BCL6 in neural stem cells made from mouse embryonic stem cells, these stem cells transformed en mass into cortical neurons. Because BCL6 is normally known for its role in blood cancers (lymphomas), this BCL6-medicated function was a complete surprise.

Because data from overexpression studies is always suspect without verification, Vanderhaeghen and his colleagues used mouse genetics to confirm the role of BCL6 in the production of cortical neurons. Vanderhaeghen’s team made mutant mice embryos that had lost a functional copy of the BCL6 gene. When these mice developed to the fetal stage, it was clear that they had small cerebral cortexes that consisted of far fewer cortical neurons. Therefore, BCL6 overexpression increases cortical neuron production and the absence of it decreases cortical neuron production. This certainly confirms the role of BCL6 in cortical neuron development.

Next, Vanderhaeghen’s lab determined how BCL6 was influencing the development of cortical neurons. A protein that is encoded by the Notch gene are essential in the self-renewal of neural stem cells. BCL6 works with another protein called SIRT1 to repress the Notch pathway, and this repression moves the progeny of neural stem cells to differentiate into cortical neurons.

Because cortical neurons are the main entities affected by neurological and psychiatric disorders, this understanding of cortical neuron development might provide insights into inherited forms of dementia, behavioral problems or other types of neurological problems. Also, Vanderhaeghen’s work bring together three major players involved in cancer BCL6), aging, Alzheimer’s disease, metabolism and diabetes (SIRT1), and brain and heart development and cancer (Notch). Because these three genes were not know to interact with each other prior to this work, Vanderhaeghen’s findings have opened up a new avenue of possible targets for therapies and model systems for understanding stem cell renewal and differentiation.

Embryonic Stem Used to Make A Thyroid from Scratch

Scientists from the Universite´ Libre de Bruxelles, Belgium in collaboration with scientists from the Lillehei Heart Institute at the University of Minnesota, University of Chicago, and Ghent University, in Merelbeke, Belgium have differentiated engineered mouse embryonic stem cells into thyroid cells that make thyroid hormone, organize themselves into a thyroid, and even rescue thyroid deficient mice.

In a paper published in the international journal Nature, lead author Francesco Antonica and her colleagues used mouse embryonic stem cells for these experiments. Antonica and others engineered these cells to express two transcription factors; NKX2-1 and PAX8. They used a trick to engineer these cells so that they would only express these genes if they were treated with the drug doxycycline. A variety of experiments showed that the genetic manipulation of the cells did not affect their pluripotency.

After genetically engineering their mouse embryonic stem cells, they grew half of them without doxycycline and the other half in the presence of doxycycline. Three days after growing cells on doxycycline, the cells expressed high levels of Pax8 and NKX2-1, and also showed high levels of expression of thyroid-specific genes such as thyroid-stimulating hormone (TSH) receptor (Tshr), the sodium/iodide symporter NIS (Slc5a5) and thyroglobulin (Tg), as well as Foxe1, which is yet another key transcription factor for thyroid development. In contrast, the cultures without doxycycline showed no such changes in gene expression.

These cells, however, did not stop there. 22 days after being grown on doxycycline, the cells rounded up and formed clusters that exactly resemble those found in a living thyroid gland. The resemblance to thyroid glands, however, was not superficial. Thyroid-specific proteins were detected in these clusters. Those cells formed a circle that surrounded a space and it also showed proper localization of thyroid-specific proteins. There is a protein found on the bottom of the thyroid celll called NIS, which stands for sodium/iodide symporter. This proteins transports two sodium cations (Na+) for each iodide anion (I–) into thyroid cells. The uptake of iodide into follicular cells of the thyroid gland is the first step in the synthesis of thyroid hormone. Another protein found at the bottom of thyroid cells called E-cadherin helps the cells stick to each other.

Thyroid hormone production

These ESC-derived thyroid cells also express E-cadherin at the bottom of the cell. Also, at the other end of the cell (the apical end) thyroid cells express a protein called zona occludens 1 (ZO-1). These ESC-derived thyroid cells also express ZO-1 at the top of the cell. Finally, thyroglobulin, which is the precursor version of thyroid hormone was also expressed in these cells and was also found in the space at the center of the cell clusters – just like in a thyroid gland.

a, Schematic diagram of the thyroid gland organized in follicles. b, Immunostaining of NIS in adult thyroid tissue. c–f, Immunofluorescence at day 22 of thyroid follicles derived from ESCs on ectopic expression of Nkx2-1 and Pax8 for NKX2-1 and NIS (c), NKX2-1 and E-cadherin (E-cad.) (d), NKX2-1 and ZO-1 (e) and NKX2-1 and TG (f). g, Immunodetection of TG-I in the luminal compartment of NKX2-1-positive follicles. h–j, Iodide-organification assay in cells differentiated after Dox induction of Nkx2-1-Pax8 (h), Nkx2-1 (i) and Pax8 (j). Histograms show the organification percentage of iodine-125 at day 22 in cells differentiated without Dox and rhTSH (left column), in the presence of Dox only (centre column) and on Dox and rhTSH treatment (right column). Data are mean ± s.e.m. (n = 3). Tukey’s multiple comparison test was used for statistical analysis. ***P < 0.001. Scale bars, 200 μm (b) and 20 μm (c–g). PBI, protein-bound 125I.
a, Schematic diagram of the thyroid gland organized in follicles. b, Immunostaining of NIS in adult thyroid tissue. c–f, Immunofluorescence at day 22 of thyroid follicles derived from ESCs on ectopic expression of Nkx2-1 and Pax8 for NKX2-1 and NIS (c), NKX2-1 and E-cadherin (E-cad.) (d), NKX2-1 and ZO-1 (e) and NKX2-1 and TG (f). g, Immunodetection of TG-I in the luminal compartment of NKX2-1-positive follicles. h–j, Iodide-organification assay in cells differentiated after Dox induction of Nkx2-1-Pax8 (h), Nkx2-1 (i) and Pax8 (j). Histograms show the organification percentage of iodine-125 at day 22 in cells differentiated without Dox and rhTSH (left column), in the presence of Dox only (centre column) and on Dox and rhTSH treatment (right column). Data are mean ± s.e.m. (n = 3). Tukey’s multiple comparison test was used for statistical analysis. ***P < 0.001. Scale bars, 200 μm (b) and 20 μm (c–g). PBI, protein-bound 125I.

So, it looks like a thyroid, it makes the same genes as a thyroid, but is it a thyroid functionally speaking? To answer this question, Antonica and colleagues took normal mice and feed them radioactive iodine. This destroys the thyroid and they were able to confirm that these mice were devoid of thyroid activity and showed the symptoms of hypothyroidism. Then they grafted their ESC-derived thryoid cells into the kidney capsule of the hypothyroid mice. The grafts took and tissue examination showed that the grafts looked like thyroid tissue and also expressed thyroid-specific proteins. Therefore, transplantation of the ESC-derived thyroid tissue does not change its characteristics.

Amazingly, 8 or 9 hypothyroid mice that had received the grafts recovered full thyroid function. Those hypothyroid mice transplanted with ESCs that were not grown in the presence of doxycycline showed no signs of recovery from hypothyroidism.

These experiments show that ESCs can be differentiated into thyroid follicles that can serve as an excellent model for thyroid physiology and development. Such a model system can also be used to test thyroid drugs and model thyroid diseases. Additionally, such cells can also potentially be used to treat thyroid diseases. If this technology can be recapitulated with human pluripotent stem cells – particularly with induced pluripotent stem cells and might be patient specific, then a ready-made treatment for patients who have lost their thyroids as a result of surgery is potentially at hand.

Making Cardiovascular Progenitor Cells from Induced Pluripotent Stem Cells

In fetal heart, stem cells known as cardiovascular progenitor cell (CPC) differentiates into smooth muscle cells for blood vessels, blood vessel wall cells, and heart muscle cells. Making CPCs from stem cells has proven to be rather difficult because CPCs do not express any known surface molecules that distinguishes them from other cell types. Therefore, if you want to differentiate pluripotent stem cells into CPCs, determining that you have made CPCs is very difficult.

This problem has been addressed by an international research team led by a team from Stuttgart, Germany who have discovered cell surface molecules that allow the identification and isolation of CPCs. With this knowledge, it will be possible to derive CPCs from induced pluripotent stem cells, which can be implanted into damaged hearts, differentiate into heart-specific cell types and integrate into the heart.

Heart attacks are the most frequent cause of death in the developed world. The cause of a heart attack is usually a clogged coronary vessel, which prevents sufficient blood flow through the heart and kills off heart tissue as a result of ischemia. There are some 17 million people who die from cardiovascular disease each.

Heart muscle cells (cardiomyocytes) do not have the ability to regenerate sufficiently after a heart attack. A heart attack causes a huge loss of cells and further impairs blood supply through the heart. This causes the heart to deteriorate further. To fix the heart, new heart muscle cells are required to replace to dead ones.

This now seems to be a distinct possibility. A research team led by Dr. Katja Schenke-Layland from the Frauhofer Institute for Interfacial engineering and Biotechnology IGD in Stuttgart, in collaboration with Dr. Ali Nasar from the University of California and Dr. Robb MacLean from the University of Washington in Seattle have used cultured CPCs to make heart muscle cells.

To identify CPCs, two proteins of the surfaces of CPCs were identified; a receptor called Flt1 and another called Flt4. By exploiting these two surface proteins, scientists can identify and isolate CPCs from a culture of differentiating pluripotent stem cells. To exploit this new finding, these groups, made induced pluripotent stem cells (iPSCs) from a mouse strain that expressed a green fluorescent protein. They then used skin cells from these mice to make iPSCs.

Japanese stem cell researcher Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs. To make iPSCs, adult cells are genetically engineered with four different genes and these genes de-differentiate the adult cells to a pluripotent stem cells state.

The iPSCs made from the green fluorescent mice were then differentiated into CPCs. They were able to isolate and identify CPCs by means of capturing all the cells that made Flt1 and Flt4.

According to Schenke-Layland, “Using our newly established cell surface markers, we could detect and isolate the Flt1- and Flt4-positive CPCs in culture. When we cultured the isolated mouse CPCs then in vitro, they actually developed – as well as the embryonic stem cell-derived progenitor cells – into endothelial cells, smooth muscle cells and more interestingly into functional heart muscle cells.”

To determine if these iPSC-derived CPCs could integrate into a living heart, they injected them into the hearts of living mice. 28 days later, the noticed that the injected hearts were loaded with green fluorescent cells that had differentiated into beating heart muscle that were fully integrated into the heart muscle tissue of the heart.

The next step is to determine if these CPCs can help heal a heart after a heart attack. Bone marrow-derived stem cells have been used to help heal the hearts of heart attack patients, and to date, these stem cells are safe, but only seem to help most people just little, even though they seem to help some patients more than others. However, iPSC-derived CPCs could potentially heal the heart to a greater degree.

According to Schenke-Layland, “We are currently focusing on research with human iPS cells. If we can show that cardiovascular progenitor cells can be derived from human iPS cells that have the ability to mature into functional heart muscle, we will have discovered a truly therapeutic solution for heart attack patients.”

See “Characterization and Therapeutic Potential of INduced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells;” Ali Nasar et al: PLoS ONE, 2012; 7 (10): e45603 DOI: 10.1371/journal.pone.0045603.

Keeping Stem Cells Stem Cells

Chengcheng Zhang is an assistant professor in the UT Southwestern Medical Center departments of physiology and developmental biology in Dallas, Texas. His lab has identified a receptor on the surface of cancer stem cells that, when activated, prevents them from differentiating.

Zhang explains his work this way: “Cancer cells grow rapidly in part because they fail to differentiate into mature cells. Drugs that induce differentiation can be used to treat cancers.” In his however has identified a new target for cancer: “Our research has identified a protein receptor on cancer cells that induces differentiation, and knowing the identity of this protein should facilitate the development of new drugs to treat cancers.”

The receptor to which Zhang is referring is a member of a family of proteins known as the “leukocyte immunoglobulin-like receptors.” These LIRs, as they are called, have bits located outside the cell and help regulate cells of the immune system. The LIR that Zhang’s lab found is called the subfamily B member 2 or LILRB2. LILRB2 is found on the surface of immune cells where it binds to molecules on the surface of cells that process antigens (foreign substances in the body) and prevents the initiation of an immune response. LILRB2 also has a newly-described role in stem cell biology.

Zhang again: “The receptor we identified turned out to be a protein called a classical immune inhibitory receptor, which is known to maintain stemness of normal adult stem cells and to be important in the development of leukemia.”

What does Zhang mean by “stemness?” He is referring to the potential of a bone marrow stem cell that makes blood cells to develop into different kinds of cells and replenish red blood cells lost to wear and tear or injury. Once stem cells differentiate into adult cells, they cannot return to their original stem cell state. The body seems to only have a finite number of stem cells and, therefore, depleting them is unwise.

Before Zhang’s study, there was no indication that LILRB2 could bind to anything but surface proteins on antigen-presenting cells, but Zhang and his team has discovered a new function for LILRB2. LILRB2 can bind to members of a poorly understood group of proteins known as angiopoietic-like proteins that support stem cell growth. By binding angiopoietic-like proteins, LILRB2 sends a signal to the interior of the stem cell to not differentiate. This inhibition keeps cancer stem cells from differentiating. By not differentiating, the stem cells divide furiously and never differentiate and make progeny cells that also divide many times and do not differentiate. This is the main mechanism that drives the progression of leukemia.

Zhang said that this inhibition does not cause cancer stem cells to make new stem cells but does not preserve their potential to do so. Also, making inhibitors that prevent the interaction between angiopoietin-like proteins and LILRB2 can force cancer stem cells to differentiate. Thus these new findings may give us a target for fighting leukemia.