Implantation of Irradiated Embryonic Stem Cells into the Heart Improves Heart Function After a Heart Attack


Adult stem cell transplantation has been used to treat heart attack patients in several different clinical trials. While the results have not been consistent, adult stem cells, it is clear that adult stem cells, primarily from bone marrow, and in some cases fat, help improve heart function. However, a major criticism of the use of adult stem cells is that they do not differentiate into heart muscle cells, but only improve the heart through “paracrine mechanisms,” which means that they secrete molecules that help heal the heart. This criticism is only represent part of the picture, since bone marrow stem cells transdifferentiate into heart muscle and blood vessel cells, albeit at a rather low rate, and fuse with endogenous cells to form hybrid cells that show improved function (Strauer BE, Steinhoff G. J Am Coll Cardiol. 2011 Sep 6;58(11):1095-104. doi: 10.1016/j.jacc.2011.06.016). In addition, adult stem cells activate endogenous cardiac stem cells to divide and replace lost heart muscle cells and make new blood vessels (Loffredo FS, et al., Cell Stem Cell. 2011 Apr 8;8(4):389-98. doi: 10.1016/j.stem.2011.02.002).

Embryonic stem cells, on the other hand, are thought to differentiate into heart muscle cells that integrate into the heart and directly replace the dead heart muscle cells, Animal studies do show such improvements (Caspi O, et al., J Am Coll Cardiol. 2007 50(19):1884-93). However, there is a caveat to all this: Most of the animal experiments with heart muscles derived from embryonic stem cells have only analyzed heart function for up to four weeks after transplantation. Experiments that examined heart function for longer than four weeks have not been able to show that these improvements are sustained after four weeks (van Laake LW, et al., Stem Cell Res. 2007 Oct;1(1):9-24. doi: 10.1016/j.scr.2007.06.001). Therefore, could it be possible that embryonic stem cell-derived cells also help the heart mainly through paracrine mechanisms?

A new paper from Piero Anversa’s and Richard Burt’s laboratories has shown that implantation of embryonic stem that were hit with radiation so that they cannot divide significantly improves heart function after a heart attack.

Experiments were conducted with mice and rhesus monkeys, and mouse and human embryonic stem cells (ESCs) were used. The ESCs were treated with 20 to 100 Grays of radiation, which completely abolished their ability to divide (a gray is the absorption of one joule of energy, in the form of ionizing radiation, per kilogram of matter).

The irradiated ESCs or iESCs were implanted into mice and Rhesus monkeys that had suffered a heart attack. Control animals were implanted with conditioned culture media from the ESC culture dishes.

In the mice and the Rhesus monkeys, the control animals showed little improvement and their hearts continued to deteriorate after the heart attack. However, the animals that had been implanted with the iESCs showed significant improvement of their heart function.

The authors in the discussion suggest that the iESCs might have suppressed the inflammatory response that occurs in the heart after a heart attack, but tissue sections of the hearts after the experiment showed that the iESC-implanted hearts had just as many immune cells infiltrating the tissue as the hearts of the control animals. Mesenchymal stem cells, however, do a very fine job of suppressing inflammation in the heart after a heart attack (see the recent paper by van den Akker et al., Biochimica et Biophysica Acta 1830 (2013): 2449-58). Therefore, the mechanisms by which ESCs improve heart function might be more paracrine-based than anything else. If this is the case, then why are embryonic stem cells being pursued for clinical purposes? Adult stem cells heal by means of paracrine mechanisms and can also sidestep the problem of immune rejection. Also, adult stem cells treatments do not require the dismemberment of young human beings at the embryo stage of their existence. Therefore, even though the present ESC lines are certainly appropriate for clinical and biological research, deriving more of them for clinical treatments is inappropriate, and even murderous.

Happy Easter to All My Readers


From Mark 16:1-8:

1 When the Sabbath was over, Mary Magdalene, Mary the mother of James, and Salome bought spices so that they might go to anoint Jesus’ body. 2 Very early on the first day of the week, just after sunrise, they were on their way to the tomb 3 and they asked each other, “Who will roll the stone away from the entrance of the tomb?”

4 But when they looked up, they saw that the stone, which was very large, had been rolled away. 5 As they entered the tomb, they saw a young man dressed in a white robe sitting on the right side, and they were alarmed.

6 “Don’t be alarmed,” he said. “You are looking for Jesus the Nazarene, who was crucified. He has risen! He is not here. See the place where they laid him. 7 But go, tell his disciples and Peter, ‘He is going ahead of you into Galilee. There you will see him, just as he told you.’”

8 Trembling and bewildered, the women went out and fled from the tomb. They said nothing to anyone, because they were afraid

Stem Cells From Gum Tissue Help Replace Missing Teeth


Researchers from King’s College London, UK have developed a new method that replaced missing teeth with bioengineered material made from a patient’s own gum cells.

If a patient loses a tooth, the dentist or oral surgeon will typically replace it with an implant. The vast majority of dental implants used today are root-form endosseous implants. Such implants have a similar look to an actual tooth root and are placed within the bone of the jaw. The bone of the jaw fuses the surface of the implant with the surrounding bone (a process known as osseointegration). Because dental implants lack the periodontal ligament they will feel slightly different from natural teeth during chewing. Also, the friction from chewing and from other jaw movements can cause loss of bone around the implant.

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Research by members of Paul Sharpe‘s laboratory at King’s College London has brought us closer to the reality of bioengineered teeth to replace toss teeth. Bioengineered tooth research has focussed primarily on producing immature teeth that can grow into adult teeth. Typically, such tooth buds are grown in culture and then transplanted into the gums. The gum actually provides and adequate environment for embryonic tooth buds to develop and form adult teeth. Therefore, the prospect of forming bioteeth certainly seems viable. The only question is identifying the cells and materials that can combine to properly form a normal adult tooth.

TOOTH DEVELOPMENT

Sharpe noted, “What is required is the identification of adult sources of human epithelial and mesenchymal cells that can be obtained in sufficient numbers to make biotooth formation a viable alternative to dental implants.”

Sharpe and his colleagues surmised that gum tissue might provide the right cells for this project. Therefore, they isolated adult human gum tissue samples from patients at the Dental Institute at King’s College and grew it in culture in the laboratory. Next, Sharpe’s group combined this gum tissue with mouse embryonic tooth mesenchyme cells, which are stem cells that can induce tooth formation.  This gum-tooth combination created teeth with surrounding gum tissue that could be transplanted into the mouths of mice. The teeth had dentine, enamel and viable roots.

The epithelial cells from human gum were able to respond to tooth-inducing signals from the embryonic tooth mesenchymal cells in a manner that allowed them to contribute to the tooth crown and the roots, and formed all the available cell types necessary for normal tooth formation. Thus, it appears that gum biopsies can provide a realistic source for human biotooth production.

The next step in this research is the formidable challenge of finding a mesenchymal stem cell population that can induce tooth formation. Presently, only embryonic mesenchymal cells can do this, according to Sharpe, but it is possible that adult mesenchymal stem cells can be manipulated to become tooth-inducing cells.

Polycomb Proteins Pave the Way for Proper Stem Cell Differentiation


Embryonic stem cells have the ability to differentiate into one of the more than 200 cell types. Differentiation requires a strictly regulated program of gene expression that turns certain genes on at specific times and shuts other genes off. Loss of this regulatory circuit prevents stem cells from properly differentiating into adult cell types, and an inability to differentiate has also been linked to the onset of cancer.

Researchers at the BRIC, University of Copenhagen have identified a crucial role of the molecule Fbx110 in embryonic stem cell differentiation. Kristian Helin from the BRIC said, “Our new results show that this molecule is required for he function of one of the most important molecular switches that constantly regulated the activity of our genes. If Fbx110 is not present in embryonic stem cells, the cells cannot differentiate properly and this can lead to developmental defects.”

What is the function of Fbx110? Fbx110 recruits members of the “Polycomb” gene family to DNA. Polycomb proteins, in particular PRC1 & 2, are known to modulate the structure of chromatin, even though they do not bind DNA. Fbx110,, however binds DNA, but it also binds PRC1 . Therefore, Fbx110 seems to serve as an adapter that recruits Polycomb proteins to DNA.

Polycomb proteins bound to nucleosomes
Polycomb proteins bound to nucleosomes

Postdoctoral fellow Xudong Wu, who led the experimental part of this investigation, said, “Our results show that Fbx110 is essential for recruiting PRC1 to genes that are to be silenced in embryonic stem cells. Fbx110 binds directly to DNA and to PRC1, and this way it serves to bring PRC1 to specific genes. When PRC1 is bound to DNA it can modify the DNA associated proteins, which lead to silencing of the gene to which it binds.”

Timing of gene activity is crucial during development and must be maintained throughout the lifespan of any cell. Particular genes are active at a certain times and inactive at other times, and PRC1 seems to be part of the reason for this coordination of gene activity. PRC1 is dynamically recruited to and dissociates from genes according to the needs of the organism.

When cancer arises, this tight regulation of gene activity is often lost and the cells are locked in an inchoate state. This loss of terminal differentiation causes increases cell proliferation and the accumulation of other mutations that allow the cancer cells to undergo continuous self-renewal through endless cell divisions. Such an ability is denied to mature cells because of their tightly controlled programs of gene expression.

Wu added, “Given the emerging relationship between cancer and stem cells, our findings may implicate that an aberrant activity of Fbx110 can disturb PRC function and promote a lack of differentiation in our cells. This makes it worth studying whether blocking the function of Fbx110 could be a strategy for tumor therapy.”

In collaboration with a biotech company called EpiTherapeutics, the BRIC researchers want to develop Fbx110 inhibitors as potential novel therapies for cancer.

The Role of Astrocytes in Lou Gehring’s Disease


A study from Columbia University and Harvard University has uncovered a complex interplay between neurons and support cells known as astrocytes that contributes to the pathology of ALS. Such an intricate interplay complicates regenerative therapies for this disease.

In the spinal cord, a group of neurons called motor neurons extend their axons to skeletal muscles and provide the neural signals for the muscles to contract, which allows movement. Motor neurons also have associated support cells known as glial cells, and a specific group of glial cells known as astrocytes associate with motor neurons in the spinal cord.

Astrocytes are star-shaped cells that surround neurons in the brain and spinal cord, and they outnumber neurons 50:1. Astrocytes are very active in the central nervous system, and serve to maintain, support, and repair the nervous tissue that they serve, and are responsible, in large part, for the plasticity of the nervous system.

astrocytes1 (1)

Motor neurons die off during the course of ALS, which leads to paralysis and death within two to fives years of diagnosis. ALS also affects neurons in the brain and it completely robs the individual of the ability to initiate movement or even breathe. There is, at present, no cure and no life-prolonging treatment for ALS.

Data from the ALS Association group suggests that astrocytes in ALS patients go from supporting neurons to strangling them. According to Lucie Bruijn, the chief scientist at the ALS Association in Washington D.C.,, these results seem to “strengthen the case that astrocytes are central to the ALS disease process.” She continued: “Furthermore, the results are based on an exciting new disease model system, one that will allow us to test important hypotheses and search for new therapeutic targets.”

In a cell culture system of ALS, in which neurons derived from embryonic cells were co-cultured with normal and ALS astrocytes, Bruijn’s team found that gene expression patterns in those neurons associated with ALS astrocytes were abnormal. In this experiment, neurons derived from embryonic stem cells with co-cultured with normal and ALS affected astrocytes. In a time course experiment in which gene expression profiles were analyzed from the neurons after specific amounts of time, the gene expression patterns from the normal astrocytes co-cultured with neurons were compared with those of the ALS-affected astrocytes co-cultured with neurons. From these experiments, it became clear that the ALS-affected astrocytes did not communicate properly with the nearby neurons.

Even though neurons communicated with each other by means of the release of neurotransmitters, astrocytes and other glial cells also communicate with each other by means of the release of various molecules. This astrocyte-neuron communication maintains healthy neuron function. However, in the case of ALS, the neuron-astrocyte communication is “profoundly disrupted” and is disruption is not neuron dependent, since in this experiment, the neurons were normal. Without proper communication with their astrocytes, motor neurons the spinal cords of ALS patients are not able to function properly.

According to Bruijn, “This study points out several potential points for treatment intervention.” The protection of motor neurons is the goal, since the astrocytes seem to be doing little to protect and support the neurons and also might be hurting them.

An added bonus to this study is that when spinal cords from mice with a disease that shows some similarities to ALS have their gene expression profiles compared to these gene expression profiles observed in the cultured neurons, the results are remarkably similar. This shows that culture system does recapitulate what goes on in the spinal cord.

The next step is to show that the molecular abnormalities discovered in this system mimics those that occur in human disease. This publication utilized mouse cells, and the human disease, while similar, is not exactly the same.

A Protein Responsible for Cancer Stem Formation Provides a Drug Target


Eighty-five percent of all tumors are carcinomas, which are tumors that form in layers of cells that line surfaces.  Such cell layers are known as an epithelium. When carcinomas form, they undergo an “epithelial-mesenchymal” transformation” or EMT.  EMT means that cells go from being closely aligned and tightly bound to each other in a an organized layer to cells that have little to do with each other and grow in unorganized clumps.  Is there a molecule that unites the carcinomas and if so is this molecule a potential drug target for cancer treatments?

Mammary Carcinoma
Mammary Carcinoma

Researchers at the University of Texas MD Anderson Cancer Center have identified a protein that seems to play a pivotal role in EMT.  This protein, FOXC2, may lay at the nexus of why some carcinomas resist chemotherapy and grow uncontrollably and spread.  FOXC2 could, conceivably represent a novel drug target for chemotherapy.

Sendurai Mani, assistant professor of Translational Molecular Pathology and co-director of the Metastasis Research Center at MD Anderson, said, “We found that FOXC2 lies at the crossroads of the cellular properties of cancer stem cells and cells that have undergone EMT, a process of cellular change associated with generating cancer stem cells.”

Cancer stem cells are fewer in number than other tumor cells, yet research has tied them to cancer progression and resistance to treatment.  Abnormal activation of EMT can actually create cancer stem cells, according to Mani.

Mani continued, “There are multiple molecular pathways that activate EMT.  We found many of these pathways also activate FOXC2 expression to launch this transition, making FOXC2 a potentially efficient check point to block EMT from occurring. ”  Mani’s research group used experiments with cultured cells and mice to discover these concepts, but the next step will require assessing the levels of FOXC2 expression in human tumors samples.

In the meantime, these new data from Mani’s research team may have profound implication for the treatment of particular types of carcinomas that have proven to be remarkably stubborn.  Breast cancers, for example, are typically carcinomas of the mammary gland ductal system.  A specific group of breasts cancers are very notoriously resistant to treatment, and FOXC2 seems to be at the center of such breast cancers.

The anti-cancer drug sunitinib, which is marketed under the trade name Sutent, has been approved by the US Food and Drug Administration (US FDA) for three different types of cancers.  In this study, sunitinib proved effective against these particularly stubborn types of breast cancer; the so-called “triple-negative, claudin-low” breast cancers.

Sunitinib
Sunitinib

Mani explained why such breast cancers are so resistant to treatment:  “FOXC2 is a transcription factor, a protein that binds to DNA in the promoter region of genes to activate them.  For a variety of reasons, transcription factors are hard to target with drugs.”

However, sunitinib seems to target these triple-negative breast cancers.  When mice with triple-negative breast cancer were treated with sunitinib, the treated mice had smaller primary tumors, longer survival, and fewer incidences of metastasis.  The cancer cells also showed a marked decreased in their ability to form “mammospheres,” or balls of cancer stem cells (this is an earmark of cancer stem cells).  Thus sunitinib seem to attack cancer stem cells.

As it turns out, FOXC2 activates the expression of the platelet-derived growth factor receptor-beta (PDGFRc-beta).  Activation of PDGFRc-beta drives cell proliferation in FOXC2-positive cells, and sunitinib inhibits PDGFRc-beta and inhibits cells that have active FOXC2 and undergoing EMT.

Triple-negative breast cancer cells lack receptors that are used by the most common anti-cancer drugs.  These deficiencies are responsible for the resistance of these cancers to treatment.  Such cancer cells also tend to under go EMT because they lack the protein claudin, which binds epithelial cells together.  Without claudin, these cancer cells become extremely aggressive.

Since cells undergoing EMT are heavily expressing FOXC2, Mani and his colleagues used a small RNA molecule that makes a short hairpin and inhibits FOXC2 synthesis.  Unfortunately, blocking FOXC2 had no effect on cell growth, but it did alter the physical appearance of the cells and reduced their expression of genes associated with EMT and increased the expression of E-cadherin, a protein necessary for epithelial cell organization.  Breast cancer cells also became less invasive when FOXC2 was inhibited, and they down-regulated CD44 and CD24, which are markers of cancer stem cells..  Additionally, triple-negative breast cancer cells that had FOXC2 inhibited had a reduced ability to make mammospheres.  Thus, FOXC2 expression is elevated in cancer stem cells, and inhibition of FOXC2 decreased the ability of the cancer stem cells to behave as cancer stem cells.

Mammospheres
Mammospheres

Mani’s group also approached these experiments from another approach by overexpressing FOXC2 in malignant mammary epithelial cells.  This forced FOXC2 expression drove cells to undergo EMT and become much more aggressive and metastatic (the cancer spread to the liver, hind leg, lungs, and brain).  Breast cancer cells without forced FOXC2 overexpression showed no tendency to metastasize.

Finally, Mani’s group examined metastatic mammary tumors that were highly aggressive when implanted into nude mice (mice that cannot reject transplants).  Two of the tumors were claudin-negative and both of these tumors showed elevated FOXC2 expression.  When FOXC2 expression was blocked by Mani’s hairpin RNA, the claudin-negative tumors became less aggressive and grew more as mesenchymal cells.  The cells that underwent EMT also showed high levels of PDGF-RC-beta expression.

Mani said of these data: “We thought PDGF-B might be a drugable target in these FOXC2-expressing cells.”  Mani’s group also showed that suppressing FOXC2 reduced the expression of PDGFRC-Beta.  Thus, this small molecule might be an effective therapeutic strategy for treating these hard-to-treat breast cancers.

MD Anderson has filed a patent application connected to this study.

See Hollier B.G., Tinnirello A.A., Werden S.J., Evans K.W., Taube J.H., Sarkar T.R., Sphyris N., Shariati M., Kumar S.V., Battula V.L., Herschkowitz J.I., Guerra R., Chang J.T., Miura N., Rosen J.M., and Mani S.A.,. FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer. Cancer Research. e-Pub 2/2013.

Mesenchymal Stem Cells Rarely Engraft But Work in a “Hit and Run” Manner


Even though this paper was published in 2012, it is a very important study that deserves a wide reading and lots of discussion.

The paper is “Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long-Term Engraftment and No Ectopic Tissue Formation” from Kathleen Le Blanc’ s laboratory, which was published in Stem Cells 2012;30:1575–1578.

In this paper, Le Blanc and her colleagues examined autopsies from patients who had received mesenchymal stem cell transplants. Since many scientists consider mesenchymal stromal cells (MSCs) a novel treatment for a variety of medical conditions, it is crucial that the fate of MSCs after infusion is better understood. Also, the long-term safety profile of MSCs is also quite important. DO they cause malignant transformation and ectopic tissue formation? Autopsies are an excellent way to address this questions.

The Le Blanc laboratory examined autopsy material from 18 patients who had received MSC transplants from people other than themselves. They analyzed 108 tissue samples from 15 patients by means of polymerase chain reaction (PCR) to search for the DNA of MSCs from donors in the tissue. If such foreign DNA was present in the tissues of the stem cell recipients, this would indicate that the MSCs had engrafted into the tissues of the patient. Unfortunately, MSC donor DNA was detected in only one or several tissues including lungs, lymph nodes, and intestine in eight patients at very low levels (from 1/100 to <1/1,000). Detection of MSC donor DNA was negatively correlated with time from infusion to sample collection, which simply means that the more time had elapsed since the time of the MSc transplant, the less likely it was that MSC DNA was found in the patient. For example, MSC DNA was detected in nine of 13 patients whose MSC infusions had been given within 50 days before sampling, in only two of eight of those infusions that had been given earlier.

On a more positive note, there were no signs of ectopic tissue formation or malignant tumors of MSC-donor origin upon macroscopic or histological examination of the tissues of the autopsied individuals.

What does all this mean? MSCs appear to mediate their healing capacities through the molecules that they secrete. This is called a “paracrine” mechanism. and MSCs seem to engraft into host tissues only very rarely. Instead MSCs come to a damaged tissue and stimulate the endogenous healing mechanisms already present. After doing this job, MSCs do not typically stick around. Thus, MSCs seem to work in, what Le Blanc calls a “hit and run” mechanism.

Because MSCs do not seem to engraft over a long period of time, the potential adverse reactions to these cells seems to be largely limited. Thus these cells are quite safe, but their effects are almost certainly indirect to some extent.