Cardiac Muscle Repair with Molecular Beacons

Pure heart muscle cells that are ready for transplantation. This is one of the Holy Grails of regenerative medicine. Of course when working with pluripotent stem cell lines, isolating nothing but beating heart muscle cells is rather difficult. A new technique makes the isolation of pure cultures of beating heart muscle cells that much easier.

Researchers at Emory and Georgia Tech have developed a method that utilizes molecules called “molecular beacons” to isolate heart muscle cells from pluripotent stem cells. Molecular beacons fluoresce when they come into contact with cells that express certain genes. In this case, the beacons target cells that express heart-specific myosin.

Physicians can use these purified cardiac muscle cells to heal damaged areas of the heart in patient that have suffered a heart attack or are suffering heart failure. This molecular beacon technique might also have applications in other fields of regenerative medicine as well.

“Often, we want to generate a particular cell population from stem cells for introduction into patients,” said Young-sup Yoon, professor of medicine and director for stem cell biology at Emory University School of Medicine. “But the desired cells often lack a readily accessible surface marker, or that marker is not specific enough, as is the case for cardiac muscle cells. This technique could allow us to purify almost any type of cell.”

Gang Bao pioneered he use of molecular beacons and was a co-author of this publication. Yoon and is colleagues and collaborators grew mouse and human embryonic stem cells and induced pluripotent stem cells and differentiated them into heart muscle cells (cardiomyocytes). They then used molecular beacons to label only those cells that expressed messenger RNAs with just the right sequences. These molecular beacons hybridized with the mRNAs and fluoresced. Bao and others then used flow cytometry to sort the fluorescent cells from the non- fluorescent cells. The fluorescent cells have differentiated into heart muscle cells and were isolated from all the other cells.

These purified heart muscle cells could engraft into the heart of a mouse that had suffered a heart attack and they improved heart function and formed no tumors. This proof-of-principle experiment shows that this technique is feasible.

“In previous experiments with injected bare cells, investigators at Emory and elsewhere found that a large proportion of the cells are washed away. We need to engineer the cells into compatible biomaterials to enhance engraftment and retention,” said Yoon,

RNA Molecule Protects Stem Cells During Inflammation

During inflammation and infection, bone marrow stem cells that make blood cells (so-called hematopoietic stem cells or HSCs) and progenitor cells are stimulated to proliferate and differentiate into mature immune cells. This especially the case for cells of the so-called “myeloid lineage.

Hematopoietic Stem Cells (HSCs) are able to differentiate into cells of two primary lineages, lymphoid and myeloid. Cells of the myeloid lineage develop during the process of myelopoiesis and include Granulocytes, Monocytes, Megakaryocytes, and Dendritic Cells. Circulating Erythrocytes and Platelets also develop from myeloid progenitor cells.

Hematopoiesis from Multipotent Stem Cell

Repeated infections and inflammation can deplete these cell populations, which leads to serious blood conditions and increased incidence of cancer.

A research team from the California Institute of Technology, led by Nobel Prize winner, David Baltimore, has discovered a small RNA molecule called microRNA-146a (miR-146a) that acts as a safety valve to protect HSCs during chronic inflammation. These findings also suggest that deficiencies for miR-146a might contribute to blood cancers and bone marrow failure.

Baltimore and his colleagues bred mice that lacked miR146a. MicroRNAs are very short RNA molecules (around 22 base pairs long) that regulate the activities of other genes. They control the expression of genes at the transcriptional and post-transcriptional level. In the case of miR146a(-) mice, whenever these mice were subjected to chronic inflammation, the total number and quality of their HSCs declined steadily. In contrast, miR-146a(+) mice were better able to maintain their levels of HSCs despite long-term inflammation.

The lead author of this work, Jimmy Zhao, said, “This mouse with genetic deletion of miR146a is a wonderful model with which to understand chronic inflammation-driven tumor formation and hematopoietic stem cell biology during chronic inflammation.”

Zhao also noted the surprising result that the deletion of one microRNA could cause such a profound and dramatic pathology. This underscores the critical and indispensable function of miR-146a in protecting the quality and longevity of HSCs. This work also establishes the connection between chronic inflammation and bone marrow failure and diseases of the blood.

Even more exciting is the prospect of synthesizing anti-inflammatory drugs that could treat blood disorders. In fact, it is possible that artificially synthesized miR146a might be an effective treatment if small RNAs can be effectively delivered to specific cells.

Zhao also noted the close resemblance that this mouse model has to the blood disorder human myelodysplastic syndrome or MDS. MDS is a form of pre-leukemia that causes severe anemia and a dependence on blood transfusions. MDS usually leads to acute myeloid leukemia. Further study of Zhao and Baltimore’s miR146a(-) mouse might lead to a better understanding of MDS and potential new treatments for MDS.

David Baltimore, senior author of this paper, said, “This study speaks of the importance of keeping chronic inflammation in check and provides a good rationale for broad use of safer and more effective anti-inflammatory molecules. If we can understand what cell types and proteins are critically important in chronic-inflammation-driven tumor formation and stem cell exhaustion, we can potentially design better and safer drugs to intervene.”

See Jimmy L Zhao, Dinesh S Rao, Ryan M O’Connell, Yvette Garcia-Flores, David Baltimore. MicroRNA-146a acts as a guardian of the quality and longevity of hematopoietic stem cells in mice.  DOI: May 21, 2013.  Cite as eLife 2013;2:e00537.

Postscript: This paper is especially meaningful to me because my mother died of MDS. The fact that a better model system for MDS has been established is an essential first step in finding a treatment for this killer disease.

Adult Stem Cells Isolated From Human Intestines

A laboratory at the University of North Carolina at Chapel Hill has, for the first time, isolated adult stem cells from human intestinal tissue. This achievement should provide a much-needed resource for stem cells researchers to examine the nuances of stem cell biology. Also, these new stem cells should provide stem cell researchers a new tool to treat inflammatory bowel diseases or to mitigate the side effects of chemotherapy and radiation, which often damage the gut.

Scott T. Magness, assistant professor in the departments of physiology at UNC, Chapel Hill, said, “Not having these cells to study has been a significant roadblock to research. Until now, we have not had the technology to isolate and study these stem cells – now we have the tools to start solving many of these problems.”

The study represents a leap forward for a field that for many years has had to resort to conducting experiments with mouse stem cells. While significant progress has been made using mouse models, differences in stem cell biology between mice and humans have kept researchers from investigating new therapeutics for human afflictions.

Adam Grace, a graduate student in Magness’ lab, and one of the first authors of this publication, noted, “While the information we get from mice is good foundational mechanistic data to explain how this tissue works, there are some opportunities that we might not be able to pursue until we do similar experiments with human tissue”

This study from the Magness laboratory was the first in the United States to isolate and grow single intestinal stem cells from mice. Therefore, Magness and his colleagues already had experience with the isolation and manipulation of intestinal stem cells. In their quest to isolate human intestinal stem cells, Magness and his colleagues also procured human small intestinal tissue for their experiments that had been discarded after gastric bypass surgery at UNC.

To develop their technique, Magness and others simply tried to recapitulate the technique they had developed in used to isolate mouse intestines to isolate stem cells from human intestinal stem cells. They used cell surface molecules found on in the membranes of mouse intestinal stem cells. These proteins, CD24 and CD44, were also found on the surfaces of human intestinal stem cells. Therefore, the antibodies that had been used to isolate mouse intestinal stem cells worked quite well to isolate human intestinal stem cells. Magness and his co-workers attached fluorescent tags to the stem cells and then isolated by means of fluorescence-activated cell sorting.

This technique worked so well, that Magness and his colleagues were able to not only isolated human intestinal stem cells, but also distinct types of intestinal stem cells. These two types of intestinal stem cells are either active stem cells or quiescent stem cells that are held in reserve. This is a fascinating finding, since the reserve cells can replenish the stem cell population after radiation, chemotherapy, or injury.

“Now that we have been able to do this, the next step is to carefully characterize these populations to assess their potential, said Magness. He continued: “Can we expand these cells outside the body to potentially provide a cell source for therapy? Can we use these for tissue regeneration? Or to take it to the extreme, can we genetically modify these cells to cure inborn disorders or inflammatory bowel disease? Those are some questions that we are going to explore in the future.”

Certainly more papers are forthcoming on this fascinating and important topic.

Human Amniotic Epithelial Cells – Remarkable Possibilities for a Small Price

My apologies to my readers for my inactivity. Many deadlines make for less blogging. Nevertheless, I hope to get back to a more regular blogging schedule once things quiet down a bit.

Today’s entry is about a fascinating group of cells found in the extraembryonic membranes of the fetus known as the amnion. The amniotic sac is a thin, transparent pair of membranes that is actually rather tough. This sac holds the fetus until shortly before birth. In inner membrane of the amnion sac contains the amniotic fluid and fetus and the outer membrane, the chorion, surrounds the amnion and is part of the placenta.

The amniotic membrane contains a remarkable cell type known as amniotic epithelial cells or hAECs (the “h” is for human). Upon isolation after birth, the amnion membrane and manually separated from the chorion membrane and washed in a saline (salt) solution in order to remove all the blood. Then the epithelial cells are liberated from the basement membrane upon which they sit by a product called TrypZean. TrypZean is a recombinant trypsin, which is very clean and devoid of animal products. Trypsin is one of the enzymes in your digestive system that degrades proteins. By expressing the human trypsin gene in bacteria and purifying the protein, Sigma-Aldrich corporation can sell it for a profit to scientists for various procedures.

A single amnion membrane can yield in the vicinity of 120 million viable hAECs, which can be maintained in serum-free culture conditions. After being grown for some time, hAECs will have normal chromosome compositions and will also maintain chromosomes that have nice, long ends (telomeres). This indicates that the cells are healthy and dying while they grow in culture (see Murphy et al., Current Protocols in Stem Cell Biology, 2010; Chapter 1: Unit 1E.6). .

In culture,. hAECs do not grow like weeds. Mesenchymal stem cells (MSCs) tend to grow better than their hAEC brethren, but hAECs possess a remarkable ability to differentiate into a wide variety of different cell types. Sivakami Ilancheran in the laboratory of Martin Pera at the University of Monash in Clayton, Australia showed that hAECs were able to differentiate into heart muscle, skeletal muscle, bone, fat cells, pancreatic cells, liver, and at least two kinds of nerve cells. Also, when injected into mice, hAECs never formed tumors (Ilancheran et al., Biology of Reproduction 77 (2007): 577-88). Murphy and others have also shown that hAECs can be isolated after collection and stored for clinical therapies.

Given that hAECs are accessible, what are they good for? When it comes to regenerative medicine, preclinical studies with hAECs have produced very solid results that may pave the way for other studies.

HAECs can differentiate into lung cells and this feature makes them an attractive candidate for lung diseases. Lung diseases cause inflammation of the lung and scarring that decreases overall lung capacity. Cystic fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary edema, and pulmonary hypertension are all lung diseases that could potentially be treated with hAECs.

In animal models of lung disease, particular chemicals are given to the animal that damage the lung. The wounded lung tissue initiates inflammation that brings white blood cells into the lung that augment the lung damage, which results in lung scarring. If hAECs are given to mice whose lungs have been damaged by the anti-cancer drug bleomycin, the signs of inflammation and the genes normally expressed during inflammation fade away. There is also less scarring in the lungs and the functional recovery of these animals is significantly better than those animals that do not receive hAECs (Murphy et al., Cell Transplantation 2011 20(6): 909-23). In fact, hAECs can differentiate into lung cells and integrate into lung tissue. The significance of this is not lost on respiratory specialists who treat patients with cystic fibrosis. Cystic fibrosis patients lack a functional copy of a ion transport protein and poor ion transport cause the production of thick, sticky mucous that clogs up the lung pathways and causes patients to suffocate to death. However, hAECs can differentiate into lung cells that express this ion transporter. Therefore, hAECs could be a potential treatment for cystic fibrosis. Clearly hAECs have great potential for tissue engineering applications with lung disease.

Lungs are not the only organ that hAECs can help heal. These cells can also differentiate into pancreatic insulin-making cells. In the laboratory, Wei and coworkers succeeded in stimulating hAECs to secrete insulin and express the main sugar transport protein found in pancreatic insulin-secreting cells (Wei et al., Cell Transplantation 2003 12(5): 545-552). When transplanted into diabetic mice, hAECs normalize their blood sugar levels and their weights returned to normal. This shows that hAECs might represent a major breakthrough in the management of diabetes.

Clearly these cells, which come from a tissue that is normally thrown out after birth, are brimming with possibilities for regenerative medicine. Hopefully more research will produce even more possibilities.

Injected Wnt Protein Helps With Muscular Dystrophy

Duchenne muscular dystrophy is a genetic disease that affects one of every 3,500 newborn males. Because the DMD gene is located on the X chromosome, loss-of-function mutations that cause Duchenne muscular dystrophy (DMD) tend to occur in males.

Muscular dystrophy or MS affects skeletal muscles and causes muscle weakness and muscle loss, and unfortunately, the disease often progresses to a state were the muscles are so weak and damaged that even the diaphragm, which is a voluntary muscle, becomes nonfunctional, and the patients dies from an inability to breath.

Recently, Michael Rudnicki, a MS researcher from the Ottawa Hospital Research Institute in Canada, has led a research team that discovered that injections of a protein called “WNT7a” into muscles can increase the size and strength of muscles in MS mice.

Rudnicki is the director of the Regenerative Medicine Program at Ottawa Hospital Research Institute (OHRI), Canada. The results of this work were published on the Nov. 26, 2012, in the Proceedings of the National Academy of Sciences (PNAS).

For these experiments, Rudnicki collaborated with a San Diego-based biotechnology firm known as Fate Therapeutics. Fate Therapeutics specializes in developing pharmaceuticals that are based on stem cell biology, and Rudnicki is one of the founders of this company. Rudnicki hopes to begin a clinical trial of WNT7a for DMD in the near future.

In 2009, Rudnicki and co-workers showed that WNT7a protein is able to stimulate muscle repair by increasing the available supply of a population of muscle stem cells known as “muscle satellite cells.” Muscle satellite cells are located near muscle fibers but they are dormant until they are needed for muscle repair or muscle fiber regeneration. When the muscle is stressed or damaged, the satellite cells increase in number (proliferate) and mature (differentiate).

Muscle Satellite Cells

These newly published findings build on these earlier results. Once injected into the muscles of mice afflicted with DMD, the WNT7a-injected muscles showed significant increases in fiber strength and size. However, Rudnicki and others also found that WNT7a stimulated a two-fold increase in the number of satellite cells in the injected mouse muscles.

Rudnicki was worried that WNT7a was pushing satellite cells to differentiate prematurely, which was disconcerting because such premature differentiation would deplete the muscle satellite population. However, no evidence of premature differentiation was observed. Additionally, WNT7a-injected mouse muscles showed far less contraction-related injury, suggesting that WNT7a has a kind of protective effect on the muscle.

Even though these experiments were done in a mouse model of DMD, would WNT7a also work in a similar fashion in human muscles? To answer this questions, Rudnicki and his colleagues analyzed human muscle tissue from healthy male donors that had been treated with WNT7a. The results showed that the effects of this protein in skeletal muscle are the same in humans as in mice.

To summarize from their own paper: “Our experiments provide compelling evidence that WNT7a treatment counteracts the significant hallmarks of DMD, including muscle weakness, making WNT7a a promising candidate for development as an ameliorative treatment for DMD.”

The remarkable conclusion is that increasing muscle strength by injecting WNT7a into specific, vital muscle groups, such as those involved in breathing, should be considered as a therapeutic approach for this debilitating disease.

Making Brain Cells from Urine

Every day people flush large quantities of cells down the toilet. We think nothing of it because these cells are sloughed into our urine and there is little that can be done about it. However, Chinese scientists have used cells from urine to make neurons that could eventually be used to treat neurodegenerative diseases.

The technique is described in a study that was published in the journal Nature Methods (Wang, et al., Nature Methods (2012) doi:10.1038/nmeth.2283). Unlike embryonic stem cells, which are derived through the destruction of embryos and have the potential to cause tumors, these neural progenitor cells do not form tumors and are made quickly and without the destruction of human embryos.

Stem cell biology expert Duanqing Pei and his co-workers from China’s Guangzhou Institutes of Biomedicine and Health, which is part of the Chinese Academy of Sciences, previously published a paper that showed that epithelial cells from the kidney that are sloughed into urine can be reprogrammed into induced pluripotent stem cells (iPSCs) (Ting Zhou, et al., Journal of the American Society of Nephrology 2011 vol. 22 no. 7 1221-1228, doi: 10.1681/ASN.2011010106). In this study, Pei and his colleagues used retroviruses to insert pluripotency genes into kidney-based cells to reprogram them. Retroviruses are efficient vectors for genes transfer, but they insert their virus genomes into the genomes of the host cell. This insertion event can cause mutations, and for this reason, retroviral-based introduction of genes into cells are not the preferred way to generate iPSCs for clinical purposes.

Researchers use retroviruses to routinely reprogram cultured skin and blood cells into iPSCs, and these iPSCs can be differentiated into any adult cell type. However, urine is a much more accessible source of cells.

In this present study, Pei’s team used a different technique to introduce genes into the cells from urine; they used “episomal vectors,” which is an overly fancy way of saying that they placed the pluripotency genes on small circles of DNA that were then pushed into the cells. Episomal vectors can reprogram adult cells into iPSCs, but they do so at lower levels of efficiency. Nevertheless, episomal vectors have an added advantage in that the vectors transiently express the pluripotency genes in cells and then are lost without inserting into the host cell genome. This makes episomal vectors inherently safer for clinical purposes.

In one of their experiments, perfectly round colonies of reprogrammed cells from urine that resembled pluripotent stem cells after only 12 days. This is exactly half the time typically required to produce iPSCs. When cultured further, the colonies assumed a rosette shape that is common to neural stem cells.

When Pei and others cultured his urine-derived iPSCs in a culture conditions that normally used for cultured neurons, these cells formed functional neurons in the lab. Could these cells work in the brain of a laboratory animal? Transplantation of these cells into the brains of newborn rats showed that, first of all, they did not form tumors, and, secondly, they took on the shape of mature neurons and expressed the molecular markers of neurons.

The beauty of this experiment is that neural progenitors cells (NPCs) grow in culture and researchers can generate buckets of cells for experiments. However, when cells are directly reprogrammed to neurons, even though they make neurons faster than iPSCs.

James Ellis, a medical geneticist at Toronto’s Hospital for Sick Children in Ontario, Canada who makes patient-specific iPSCs to study autism-spectrum disorders, said: “This could definitely speed things up.”

Another plus of this study is that urine can be collected from nearly any patient and banked to produce instant sources of cells from patients, according to geneticist Marc Lalande, who creates iPSCs to study inherited neurological diseases at the University of Connecticut Health Center in Farmington. Lalande is quite intrigued by the possibility of making iPSCs and NPCs from urine draw from the same patient. Lalande added: “We work on childhood disorders,” he says. “And it’s easier to get a child to give a urine sample than to prick them for blood.”

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.

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.

Training Stem Cells to Differentiate Properly

Pluripotent stem cells have the ability to differentiate into a whole host of adult cell types. Unfortunately this ability to differentiate into any adult cell type also comes with it the tendency to form tumors. Controlling stem cell differentiation requires that you give a little “push” in the right direction. What is the nature of that push? It varies from stem cell to stem cell and it also depends on what type of cell you want you stem cells to make. Therefore, pluripotent stem cell differentiation is sometimes a matter of art as much as a matter of science.

A research group at Stanford University School of Medicine have designed an experimental protocol that uses the signals in the body to direct the differentiation of stem cells to a desired end.

Stanford University professor Michael Longaker, who is also the director of the Institute for Stem Cell Biology and Regenerative Medicine at Stanford University, explained it this way: “Before we can use these cells, we have to differentiate or ‘coach,’ them down a specific developmental pathway.” Longaker continued: “But there’s always a question as to exactly how to do that, and how many developmental doors we have to close before we can use the cells. In this study, we found that, with appropriate environmental cues, we could let the body do the work.”

Allowing the patient’s body to direct differentiation of pluripotent stem cells could potentially speed approval of stem cell-based treatments by the US Food and Drug Administration (FDA). If Longeker’s protocol pans out, it could eliminate long period of extended laboratory manipulation in order to force stem cells to differentiate into the desired cell type.

“Once we identify the key proteins and signals coaching the tissue within the body, we can try to mimic then when we use the stem cells,” said Longaker. “Just as the shape of water is determined by its container, cells respond to external cues. For example, in the future, if you want to replace a failing liver, you could put cells in a scaffold or microenvironment that strongly promotes liver cell differentiation and place the cell-seeded scaffold into the liver to let them differentiate in the optimal macroenvironment.”

Longaker does not work on liver, but bone. As a pedatric plastic and reconstructive surgeon who specializes in craniofacial malformations, finding ways to coax pluripotent stem cells to make bone is his research Holy Grail. “Imagin being able to treat children and adults who require craniofacial skeletal reconstruction, not with surgery, but with stem cells,” opined Longaker.

In this experiment, Longaker and his colleagues removed a four-millimeter circle of bone taken from the skulls of anesthetized mice and implanted a tiny, artificial scaffold coated with a bone-promoting protein called BMP-2 (bone morphogen protein-2) that was seeded with one million human pluripotent stem cells.

According to Longaker, these implants formed bone and repaired the defect in the skulls of the mice even the original stem cells were not differentiated when added to the wound. These human stem cells made human bone that was then replaced by mouse bone as time progressed. This shows that the repair was physiologically normal.

This bone growth was stimulated by the presence of BMP-2 and the microenvironment that induced the stem cells to differentiate into bone-making cells that made normal bone.

In this experiment, Longaker and his group used human embryonic stem cells and induced pluripotent stem cells and both stem types seemed to work equally well at repairing the skull defect.

Teratomas (tumors made by pluripotent stem cells) were observed but only rarely (two of the 42 animals that received the stem cell implants developed tumors). Interestingly, the few teratomas that formed developed in two laboratory animals that received embryonic stem cell implants and not induced pluripotent stem cell implants. This is surprising, since most stem cells researchers consider induced pluripotent stem cells to be more tumorigenic than embryonic stem cells. Standard tests of these stem cells (implantation under the kidneys of immunodeficient mice) showed that they did produce teratomas under these conditions.

Longaker commented: “We still have work to do to completely eliminate teratoma formation, but we are highly encouraged.” Longaker also thinks that by combining this technique with other strategies, he and his group might be able to completely prevent teratoma formation. For example, including other cell types that can act as shepherds for the stem cells as they differentiate into the desired cell type can also increase differentiation into a desired cell type.

Longaker said, “I want to see how broadly applicable this technique may be.” He was referring to tissues that do not heal well. For example, cartilage heals very poorly if at all. Longaker wonders if you could “add some cells that can form replacement tissue in this macroenvironment while you’re already looking at the joint.”

Pluripotency in Embryonic Stem Cells is Linked to Open Chromatin Configuration

Within the cells of multicellular organisms is a structure called the nucleus. The nucleus houses the genome. The genome consists of a molecule called deoxyribonucleic acid or DNA. DNA stores genetic information, and in multicellular organisms, the DNA is organized into large, linear molecules known as chromosomes. In order to package the DNA into a compact structure that can successfully fit within the nucleus, the DNA is wrapped into a tight structure called chromatin (see Qiong Gan, et al., Stem Cells 25(1): 2-9).

Chromatin consists of special proteins called histones and the DNA. There are five types of histone proteins, and they are named rather uncreatively: H1, H2A, H2B, H3 & H4. Histones H2A, H2B, H3 and H4 assemble into an octomeric structure that is composed of two copies of H2A, two copies of H2B, two copies of H3 and two copies of H4. This octomer of histone proteins forms a kind of spool around which the DNA is wound. The histone octomer which its DNA wrapped around it is sometimes called a nucleosome and in the electron microscope, it looks very much like rosary beads.

The H1 histone pulls the nucleosomes together to form a spiral coil of nucleosomes that are known as a 30-nanometer solenoid.

Changes in chromatin structure can produce profound alterations to the genes that the cell expresses. Therefore, chromatin structure changes can cause the cell to take on a very different flavor even though the DNA sequence of the cell’s genome has not changed. Such changes, therefore, are known as “epigenetic” changes. Epigenetic mechanisms can play a central role in stem cell biology and several papers have borne this out. For example, a paper from the laboratory of Eran Meshorer from the Department of Genetics at Hebrew University has elucidated the mechanism of pluripotency.

Shai Melcer, a graduate student in Meshorer’s lab, examined the chromatin structures in embryonic stem cells, and he discovered that embryonic stem cells (ESCs) have a so-called “open” chromatin conformation. The chromatin in ESCs is less condensed, which allows them great flexibility to differentiate into just about any cell type.

How do cells achieve this open chromatin conformation? When cells need to loosen the grip of chromatin on DNA so that particular genes can be robustly expressed, cells chemically modify their histones to relax their grip on DNA. There are several types of enzymes involved in these chemical modifications and ESCs appear to have these enzymes in spades and the right modifications are also present.

There is a second difference within ESCs that also seems to predispose them to pluripotency and that is an absence of a protein called lamin A. Lamins are nuclear proteins that form a  filigree just underneath the nuclear membrane. Lamins provide structure to the nuclear membrane and lamin A binds to bundles of chromatin and keep them compacted. Because ESCs are devoid of lamin A, their chromatin bundles are not nearly as compacted. This leads to a more dynamic, freer chromatin state for ESCs and is the key to the functional plasticity of these cells.

According to Meshorer: “If we can apply this new understanding about the mechanisms that give embryonic stem cells their plasticity, then we can increase or decrease the dynamics of the proteins that bind DNA and thereby increase or decrease the cell’s differentiation potential.”