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.

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.

Stimulating Stem Cell Activity to Prevent Aging-Related Mental Decline


Aging tends to rob us of our ability to concentrate, recall facts, and reason, and this decline seems to stem from the fact that older brains generate fewer neurons than they did when they were younger. However, German researchers have discovered a molecule that accumulates with age that inhibits the formation of new neurons. This finding might help scientists design therapies to prevent age-related mental decline.

This molecule, Dkk1 or Dickkopf-1, accumulated in the brains of aged mice. If Dkk1 production was blocked, neurons were born at much higher rates. Dr. Ana Martin-Villalba, the senior author of this work and a member of the German Cancer Research Center in Heidelberg. Said, “We released a brake on neuronal birth, thereby resetting performance in spatial memory tasks to levels observed in younger animals.”

Aged mice that lacked Dkk1 performed just as well in cognitive tests that included memory and recognition tests as younger mice because of the ability of their neural stem cells to self-renew and generate immature neurons.

Younger mice that lacked Dkk1 were less susceptible to developing acute stress-induced depression than normal mice. This seems to indicate that in addition to slowing memory loss during aging, neutralizing Dkk1 could be beneficial in counteracting symptoms of depression.

Martin-Villalba said that there are ongoing clinical trials to test inhibitors of Dkk1 for other medical purposes. “The design of inhibitors that reach the brain might enable the prevention of cognitive decline in the aging population and depression in the general population,” she said.

Making Preneurons from White Blood Cells for ALS Patients


ALS or amyotrophic lateral sclerosis is a disease that results in he death of motor neurons. Motor neurons enable skeletal muscles to contract, which drives movement. The death of motor neurons robs the patient of the ability to move and ALS patients suffer a relentless, progressive, and sad decline that culminates in death from asphyxiation. Treatments are largely palliative, but stem cells treatments might delay the onset of the disease, or even regenerate the dead neurons.

To this end a Mexican group from Monterrey has used a protocol to isolate white blood cells from the circulating blood of ALS patients, and differentiate a specific population of stem cells from peripheral blood into preneurons. Although these cells were not used to treat the patients in this study, such cells do show neuroprotective features and using them in a clinical study does seem to be the next step.

In this study, CD133 cells were isolated from peripheral blood and subjected to a special culture system called a neuroinduction system. After 2-48 hours in this system, the cells showed many features that were similar to those of neurons. The cells express a cadre of neural genes (beta-tubulin III, Oligo 2, Islet-2, Nkx6.1, and Hb9). Some of the ells also grew extensions that resemble the axons of true neurons.

Interestingly, the conversion of the CD133 cells into preneurons showed similar efficiency regardless of the age, sex, or health of the individual. Even those patients with more advanced ALS had CD133 cells that differentiated into preneurons with efficiencies equal to those of their healthier counterparts. While each patient showed variation with regards to the efficiency at which their CD133 cells differentiated into preneurons, these variations could not be correlated with the age, health or sex of the patient.

The fact that these preneurons expressed Oligo2, suggests that they could differentiate into motor neurons. Therefore, even though this study was small (13 patients), it certainly shows that cells that might provide treatment possibilities for ALS patients can be made from the patient’s own blood cells.

See Maria Teresa Gonzalez-Garza et al., Differentiation of CD133+ Stem Cells from Amyotrophic Lateral Sclerosis Patients into Preneuron Cells. Stem Cells Translational Medicine 2013;2:129-35.

Treating Diabetic Retinopathy with Stem Cells


Scientists at Queen’s University Belfast hope to design a new approach for treating the eyesight of diabetic patients by using adult stem cells.

Millions of diabetics every year are at risk for losing their eyesight due to diabetic retinopathy. When high blood sugar causes blood vessels in the eye to leak or become blocked, failed blood flow damages the retina and lead to vision impairment. If left untreated, diabetic retinopathy can lead to blindness.

The Queen’s University Belfast group have initiated the REDDSTAR study, which stands for Repair of Diabetic Damage by Stromal Cell Administration, and this study involves researchers from the Queen’s Center for Vision and Vascular Science in the School of Medicine, Dentistry and Biomedical Sciences. REDDSTAR begins with the isolation of stem cells from patients and expanding them in the laboratory. Then these patient-specific cells are delivered to the patient from whom they were originally drawn in order to repair the blood vessels in the eye. This blood vessel repair is especially useful in patients with diabetic retinopathy.

Presently, diabetic retinopathy is treated with laser ablation of new blood vessels that grow in response to damage. These new blood vessels become so dense that they obscure vision. However, presently, there are no treatments to control the progression of diabetic complications.

Alan Stitt, the director of the Centre for Vision and Vascular Science at Queen’s and lead scientist for the REDDSTAR study, said, “The Queen’s component of the REDDSTAR study involves investigating the potential of a unique stem cell population to promote repair of damaged blood vessels in the retina during diabetes.” Professor Stitt continued: “The impact could be profound for patients, because regeneration of damaged retina could prevent progression of diabetic retinopathy and reduce the risk of vision loss.”

“Treatments for diabetic retinopathy are not always satisfactory. They focus on end-stages and fail to address the root causes of the condition. A novel, alternative therapeutic approach is to harness adult stem cells to promote regeneration of the damaged retinal blood vessels and thereby prevent and/or reverse retinopathy.”

Stitt said the new research project is one of several regenerative medicine approaches ongoing at his research center. Their approach is to isolate a rather well-defined population of stem cells and then deliver those stem cells to sites in the body that have been ravaged by diabetes. In particular patients, these strategies have produced remarkable benefits from stem cell-mediated repair of their blood vessels. Treatments such as this one are simply the first step in the quest to develop exciting, effective and new therapies in an area of medicine where such therapies are desperately needed.

In the REDDSTAR study, stem cells from bone marrow are used and these stem cells are provided by Orbsen Therapuetics, which is a spin-off from the Science Foundation Ireland-funded Regenerative Medicine Institute (REMEDI) at NUI Galway.

This project will design protocols for growing these bone marrow-derived stem cells and they will be tested in several preclinical models of diabetes and diabetic complications at research centers in Belfast, Galway, Munich, Berlin, and Porto before human clinical trails take place in Denmark.

Queen’s Centre for Vision and Vascular Science is a key focus of the University’s ambitious 140-million pound “together we can go Beyond” fundraising campaign. This campaign is due to expand the Vision Science program further when the University’s new 32-million pound Wellcome-Wolfson Centre for Experimental Medicine opens in 2015. Along with vision, two new programs in Diabetes and Genomics will also be established in the new Center. These Center should stimulate further investment and global collaborations between biotech and health companies in Ireland.

Reprogramming Neurons into New Cells


Researchers from Harvard’s Department of Stem Cell and Regenerative Biology have succeeded in reprogramming one type of neuron into a different type of neurons in a living animals.  Such an experiment has never been done before.  These researchers, Paola Arlotta and Caroline Rouaux said that their work “tells you that maybe the brain is not as immutable as we always thought, because at least during an early window of time one can reprogram the identity of one neuronal class into another”  Arlotta, an associate professor in Harvard’s Department of Stem Cell and Regenerative Biology (SCRB).

Direct lineage reprogramming of differentiated cells within the body was first proven by the SCRB co-chair and Harvard Stem Cell Institute  (HSCI) co-director Doug Melton and colleagues five years ago.  Workers in Melton’s lab succeeded in reprogramming exocrine pancreatic cells directly into insulin-producing beta cells.  Now Arlotta and Rouaux now have shown that neurons can change too.  Their work has been published in the journal Nature Cell Biology

In their experiments, Arlotta and Rouaux targeted a group of neurons known as callosal projection neurons.  Collosal projection neurons connect the two hemispheres of the brain.  After specific treatments, the collosal projections neurons in this study were converted into corticofugal projection neurons.  The significance of corticofugal projection neurons are not lost on Arlotta and Rouaux because they are a type of corticospinal motor neuron, which is one of two populations of neurons destroyed in Amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease.

To achieve such reprogramming of neuronal identity, the researchers inserted a gene for a transcription factor known as Fezf2 into the collosal neurons.  Fexf2 plays a central role in the development of corticospinal neurons in the embryo.  The collosal neurons retracted their connects to the other hemisphere and made connections with neurons in the lower layers of the cerebral cortex.

Luci Bruijn, a neuroscientist who was not directly involved in this work noted, “This discovery tells us again that the brain is a somehow flexible system and gives us more evidence that reprogramming neurons to take on new identities and, perhaps, that new functions are possible. For those working to treat neurodegenerative diseases, that is reassuring.”

This work did not take take place in a culture dish in a laboratory.  Instead it was done in the brains of living mice.  The mice were young, so it is still not certain if such reprogramming could occur in older animals or even humans.  If such reprogramming is possible, the implications for the treatment of neurodegenerative diseases could be enormous.

“Neurodegenerative diseases typically affect a specific population of neurons, leaving many others untouched. For example, in ALS it is corticospinal motor neurons in the brain and motor neurons in the spinal cord, among the many neurons of the nervous system, that selectively die,” Arlotta said. “What if one could take neurons that are spared in a given disease and turn them directly into the neurons that die off? In ALS, if you could generate even a small percentage of corticospinal motor neurons, it would likely be sufficient to recover basic functioning.”

Bruijn said of this work, “Understanding the constraints and possibilities of nervous system development allows us to consider new experiments and new strategies for therapy development. The most immediate importance of this finding is likely to be in the laboratory, where it will help us understand more about how the nervous system may respond when neurons are injured as they are in ALS.”