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Neural stem cells shown in green, neurons in red
Photo: University of California-Irvine

Scientists at Schepens Eye Research Institute have identified specific molecules in the brain that are responsible for awakening and putting to sleep brain stem cells, which, when activated, can transform into neurons (nerve cells) and repair damaged brain tissue. Their findings were recently published online in the Proceedings of the National Academy of Science (PNAS).

An earlier paper (published in the May 2008 issue of Stem Cells) by the same scientists laid the foundation for the PNAS study findings by demonstrating that neural stem cells exist in every part of the brain, but are mostly kept silent by chemical signals from support cells known as astrocytes.

“The findings from both papers should have a far-reaching impact,” says principal investigator, Dr. Dong Feng Chen, who is an associate scientist at Schepens Eye Research Institute and an assistant professor of ophthalmology at Harvard Medical School. Chen believes that tapping the brain’s dormant, but intrinsic, ability to regenerate itself is the best hope for people suffering from brain-ravaging diseases such as Parkinson’s or Alzheimer’s disease or traumatic brain or spinal cord injuries.

Until these studies, which were conducted in the adult brains of mice, scientists assumed that only two parts of the brain contained neural stem cells and could turn them on to regenerate brain tissue– the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ). The hippocampus is responsible for learning and memory, while the SVZ is a brain structure situated throughout the walls of lateral ventricles (part of the ventricular system in the brain) and are responsible for generating neurons responsible for smell. So scientists believed that when neurons died in other areas of the brain, they were lost forever along with their functions.

In the first study, Chen’s team learned that stem cells existed everywhere in the brain by testing tissue from different parts of adult mice brains in cultures containing support cells (known as astrocytes) from the hippocampus, where stem cells do regenerate. In the cultures the stem cells from other brain regions came to life and turned into neurons.

When they compared the chemical makeup of the areas known to generate new neurons in the hippocampus with other parts of the brain, the team discovered that astrocytes in the hippocampus were sending one signal to the stem cells and that those from the rest of the brain were sending a different signal to stem cells.

In the second (PNAS) study, the team went on to discover the exact nature of those different chemical signals. They learned that in the areas where stem cells were sleeping, atrocytes were producing high levels of two related molecules–ephrin-A2 and ephrin-A3. They also found that removing these molecules (with a genetic tool) activated the sleeping stem cells.

The team also found that astrocytes in the hippocampus produce not only much lower levels of ephrin-A2 and ephrin-3, but also release a protein named sonic hodghoc that, when added in culture or injected into the brain, stimulates neural stem cells to divide and become new neurons.

“These findings identify a key pathway that controls neural stem cell growth in the adult brain and suggest that it may be possible to reactivate the dormant regenerative potential by adding sonic hedgehoc, or blocking ephrin-A2 or ephrin-A3,” says Dr. Jianwei Jiao, the first author of the two papers,

The next step for the team will be to stimulate the sleeping stem cells in animals who are models of neurodegenerative disorders, such as Parkinson’s disease, to see if the brains can repair themselves and restore their damaged functions.

Adapted from materials provided by Schepens Eye Research Institute

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During development, a small number of endothelial cells express Runx1 (blue cells), signaling the production of grapelike clusters of hematopoietic (or blood) stem cells along the interior walls of several major blood vessels in the mouse embryo. This cluster of endothelial cells is in the lumen of the vitelline artery. (Credit: Nancy Speck, PhD, University of Pennsylvania School of Medicine)

Univ. of Pennsylvania School of Medicine, January 2009 — A research team led by Nancy Speck, PhD, Professor of Cell and Developmental Biology at the University of Pennsylvania School of Medicine, has identified the location and developmental timeline in which a majority of bone marrow stem cells form in the mouse embryo.

The findings, appearing online the week of Jan 5 in the journal Nature, highlight critical steps in the origin of hematopoietic (or blood) stem cells (HSCs), says senior author Speck, who is also an Investigator with the Abramson Family Cancer Research Institute at Penn.

Because HSCs, found in the bone marrow of adult mammals, generate all of the blood cell types of the body, unlocking the secrets of their origin may help researchers to better manipulate embryonic stem cells to generate new blood cells for therapy.

“The ultimate goal for stem cell therapies is to take embryonic stem cells and push them down a particular lineage to replace diseased or dead cells in human adults or children,” says Speck. For instance, in theory embryonic stem cells could be tweaked in a lab to provide a patient with bone marrow failure a fresh supply of compatible HSCs.

To date, however, Speck says scientists have been unable to coax embryonic stem cells to become HSCs without significant genetic manipulations that are too risky for clinical therapies. First things first, Speck says: “You have to understand what’s happening in the embryo.”

Previous studies hinted that HSCs originated from a small population of cells lining the blood vessels, called endothelial cells. But, it was unclear how endothelial cells transitioned to blood stem cells during early development.

Before joining Penn in September 2008, Speck, then at Dartmouth Medical School, led a team that confirmed that HSCs in bone marrow were originating from the endothelial cells and determined whether the activity of a protein called Runx1, which is known to be critical in the formation of blood cells, was responsible for this important transition.

First, the researchers inactivated the gene that codes for the protein Runx1 in the endothelial cells of mouse embryos. During development, some endothelial cells express Runx1, signaling the production of grapelike clusters of HSCs along the interior walls of several major blood vessels. Upon release from the vessel walls HSCs enter the blood circulation and travel to the fetal liver, and upon birth they relocate to the bone marrow.

By selectively blocking the ability of endothelial cells to express Runx1 during embryo development, the researchers halted HSC production, demonstrating that Runx1 is vital to the endothelial cell to HSC transition.

Next, Speck’s team shut off Runx1 expression in mouse embryos at day 11.5 of gestation — a time when most newly born HSCs have detached from the vessel wall and migrated to the fetal liver. The researchers found that blocking Runx1 expression had no effect on HSC formation, suggesting while Runx1 is required for the transition from endothelium to HSCs, the process is complete by the end of the 11th day of gestation.

The researchers also showed that at least 95 percent of all adult HSCs (and therefore almost all adult blood) originate in the endothelium, during this short window of time during development.

“This study helps illustrate a very important step in the transitional stage from embryonic stem cells to HSCs – the need to move through endothelial cells as an intermediary,” Speck says.

Understanding the location and developmental timeline of the origin of blood stem cells will help guide future efforts to coax embryonic stem cells to produce mature blood cells, she says.

Co-authors include Michael Chen and Brandon Zeigler from Dartmouth Medical School (Departments of Biochemistry and Genetics) and Tomomasa Yokomizo and Elaine Dzierzak from Erasmus Medical Center in Rotterdam, Netherlands.

This work was funded by the National Institutes of Heart, Lung and Blood and Diabetes and Digestive and Kidney Diseases

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The above image shows brain tumour stem cells that have been isolated from human brain tumours. The brain tumour stem cells are the only cells within the brain tumour that can grow are capable of forming a new brain tumour. These cells can differentiate into mature brain cells of different types such as astrocytes (red) and neurons (green). (Image courtesy of The Hospital For Sick Children)

The Hospital for Sick Children-TORONTO – Researchers at The Hospital for Sick Children (Sick Kids) and the University of Toronto (U of T) have confirmed that childhood and adult brain tumours originate from cancer stem cells and that these stem cells fuel and maintain tumor growth. This discovery has led to development of a mouse model for human brain tumors and opens the door for new therapeutic targets for the treatment of brain tumors.

“Now that we have confirmed that a small number of cancer stem cells initiates and maintains human brain tumor growth in a mouse model, we can potentially use the mouse model with each patient’s tumor cells to see if therapies are working to conquer that patient’s tumor,” said Dr. Peter Dirks, the study’s principal investigator, a scientist and neurosurgeon at Sick Kids, and an assistant professor of Neurosurgery at U of T. “A functional analysis of the brain tumor stem cell may also give new insight into patient prognosis that may then warrant individual tailoring of therapy.”

Dr. Dirks’ laboratory was able to regrow an exact replica of patients’ brain tumors in a mouse from the isolated cancer stem cells, or brain tumor initiating cells. They were then able to study the growth of the human brain tumor in the mouse model using the advanced imaging technology in the Mouse Imaging Centre (MiCE) at Sick Kids.

Brain tumors are the leading cause of cancer mortality in children and remain difficult to cure despite advances in surgery and drug treatments. In adults, most brain tumors are also among the harshest cancers with formidable resistance to most therapies.

“Next, we are going to study the gene expression of the brain tumor stem cells. Once we have identified what genes are expressed in those cells, we will then be able to target these genes using new drugs or genetic-type therapies,” said Dr. Sheila Singh, the paper’s lead author and Sick Kids neurosurgery resident and U of T graduate student who is enrolled in Sick Kids’ Clinician-Scientist Training Program. Dr. Singh was supported by a fellowship from The Terry Fox Foundation, as well as by funding from the Neurosurgical Research and Education Foundation and the American Brain Tumor Association.

“We have shown that it is really worthwhile to invest further in studying brain tumor stem cells, as we will be able to determine if current therapies are failing because they are not stopping the cancer stem cells,” added Dr. Dirks. “It also looks like cancer stem cells play a role in other solid tumors such as breast cancer, so we can all work together to develop new treatments for these cancers.”

Other members of the research team included Dr. Cynthia Hawkins, Dr. Ian Clarke, Dr. Takuichiro Hide and Dr. Mark Henkelman, all from Sick Kids, Dr. Jeremy Squire and Jane Bayani from the Ontario Cancer Institute, and Dr. Michael Cusimano from St. Michael’s Hospital.

This research was supported by the Canadian Cancer Society, the Canadian Institutes of Health Research and Sick Kids Foundation (including support from BrainChild, the Jack Baker family fund and the Jessica Durigon family fund). MiCE is supported with funding from the Canada Foundation for Innovation, Ontario Innovation Trust, the Ontario Research and Development Challenge Fund and Sick Kids Foundation.

The Hospital for Sick Children, affiliated with the University of Toronto, is Canada’s most research-intensive hospital and the largest center dedicated to improving children’s health in the country. Its mission is to provide the best in family-centered, compassionate care, to lead in scientific and clinical advancement, and to prepare the next generation of leaders in child health.

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Immunohistochemical image of human renal tissue (colours indicate transporter proteins). Red: BCRP in the membrane of the proximal tubulus. Green: the sodium transporter Na+,K+-ATPase in the basal membrane. G: glomerulus. Magnification 200x. (Credit: Image courtesy of Dutch Kidney Foundation)

ScienceDaily.com, January 28, 2009 — In a study funded by the Dutch Kidney Foundation (DKF) a research group at Radboud University Nijmegen Medical Centre in the Netherlands, found that stem cells and ABC transporter proteins are indispensable for tubular regeneration after acute kidney injury.

Said project leader Dr. Rosalinde Masereeuw: ‘To our surprise, our knockout mice for the ABC transporters P-gp and BCRP, P-gycoprotein and breast cancer resistance protein, were protected against acute kidney damage. This was the opposite of what we expected since the transporters usually have a protective function in excreting potentially toxic compounds, while these mice lack expression. Moreover, when we cross transplanted bone marrow between normal mice and the knockouts it turned out that bone marrow from the knockouts was the source of protection.’

Regeneration

Acute kidney injury is an important cause for the need of acute hemodialysis and a source of kidney failure. On the other hand, the kidney has a remarkable capacity for recovery. Stem cells seemed to have a limited share in the repair process, but now this study suggests otherwise.

‘It was known that stem cells from the bone marrow express P-gp and BCRP abundantly but will downregulate them at differentiation. Repair of tubular damage in the kidney depends primarily on local cells but stem cells are involved as well. Further, we observed an upregulation in the expression of the transporters during ischemic injury. .So we thought they might be important in renal regeneration.’

Transporter Proteins

ABC transporters (ATP binding cassette transporters) form a superfamily of highly conserved transporter proteins whose functions are not yet well understood. However, BCRP and especially P-gp have been studied in more detail in man. These cell membrane pumps are responsible for the transport of many substances, for instance drug molecules in the intestine. P-gp plays an important role in drug resistance of tumour cells.

Masereeuw: ‘Our new hypothesis claims a bigger role for bone marrow derived stem cells in kidney regeneration. A possible mechanism is the infiltration of macrophages. These large immune cells have subgroups one of which increases damage but another supports tissue regeneration.’

Also, the study showed that mice without P-gp expression lose renal tubular function in a way comparable to Fanconi syndrome in man. BCRP knockouts, on the other hand, have a normal kidney function.

Blocking P-gp and BCRP

There is a great need for novel therapies that limit kidney damage after acute injury by toxic substances or shortage of oxygen, as in transplant kidneys which have no blood supply during transport. The results from this DKF study are pointing at inhibition of the transporters in kidney or bone marrow to strengthen the regenerative power of stem cells.

‘Next, we will try to discover the mechanism by which stem cells and ABC transporters contribute to kidney repair’, concludes Dr. Masereeuw, ‘and we will test the effect of transporter blockers in our mouse models. We are convinced there are good opportunities here for new drug targets.’

Journal reference:

1. Huls et al. The Role of ATP Binding Cassette Transporters in Tissue Defense and Organ Regeneration. Journal of Pharmacology and Experimental Therapeutics, 2008; 328 (1): 3 DOI: 10.1124/jpet.107.132225

Adapted from materials provided by Dutch Kidney Foundation

Richard K Burt MD, Yvonne Loh MD, Bruce Cohen MD, Dusan Stefosky MD, Roumen Balabanov MD, George Katsamakis MD, Yu Oyama MD, Eric J Russell MD, Jessica Stern MD, Paolo Muraro MD, John Rose MD, Alessandro Testori MD, Jurate Bucha MD, Borko Jovanovic PhD, Francesca Milanetti MD, Jan StorekMD, Julio C Voltarelli MD, William H Burns MD

Summary

Background

Autologous non-myeloablative haemopoietic stem cell transplantation is a method to deliver intense immune suppression. We evaluated the safety and clinical outcome of autologous non-myeloablative haemopoietic stem cell transplantation in patients with relapsing-remitting multiple sclerosis (MS) who had not responded to treatment with interferon beta.

Methods

Eligible patients had relapsing-remitting MS, attended Northwestern Memorial Hospital, and despite treatment with interferon beta had had two corticosteroid-treated relapses within the previous 12 months, or one relapse and gadolinium-enhancing lesions seen on MRI and separate from the relapse. Peripheral blood haemopoietic stem cells were mobilised with 2 g per m2 cyclophosphamide and 10 μg per kg per day filgrastim. The conditioning regimen for the haemopoietic stem cells was 200 mg per kg cyclophosphamide and either 20 mg alemtuzumab or 6 mg per kg rabbit antithymocyte globulin. Primary outcomes were progression-free survival and reversal of neurological disability at 3 years post-transplantation. We also sought to investigate the safety and tolerability of autologous non-myeloablative haemopoietic stem cell transplantation.

Findings

Between January, 2003, and February, 2005, 21 patients were treated. Engraftment of white blood cells and platelets was on median day 9 (range day 8—11) and patients were discharged from hospital on mean day 11 (range day 8—13). One patient had diarrhoea due to Clostridium difficile and two patients had dermatomal zoster. Two of the 17 patients receiving alemtuzumab developed late immune thrombocytopenic purpura that remitted with standard therapy. 17 of 21 patients (81%) improved by at least 1 point on the Kurtzke expanded disability status scale (EDSS), and five patients (24%) relapsed but achieved remission after further immunosuppression. After a mean of 37 months (range 24—48 months), all patients were free from progression (no deterioration in EDSS score), and 16 were free of relapses. Significant improvements were noted in neurological disability, as determined by EDSS score (p<0·0001), neurological rating scale score (p=0·0001), paced auditory serial addition test (p=0·014), 25-foot walk (p<0·0001), and quality of life, as measured with the short form-36 (SF-36) questionnaire (p<0·0001).

Interpretation

Non-myeloablative autologous haemopoietic stem cell transplantation in patients with relapsing-remitting MS reverses neurological deficits, but these results need to be confirmed in a randomised trial.

Funding

Division of Immunotherapy, Northwestern University.

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Photo: In multiple sclerosis, the body’s immune system attacks myelin, the substance that covers nerves and permits them to function properly. Credit: National Multiple Sclerosis Society

Los Angeles Times, January 29, 2009, by Shari Roan — Infusing multiple sclerosis patients with their own immune stem cells appears to help the immune system “reset” itself and fight off the disease, according to a study that will be published online Friday in the Lancet Neurology.

The study, an early-phase research project involving only 21 patients, is similar to other experiments in which a patient’s own stem cells are used to treat autoimmune diseases. The treatment, called autologous non-myeloablative haematopoietic stem-cell transplantation, has also shown promising results in people with lupus and diabetes.

In the new study, Dr. Richard Burt of Northwestern University’s Feinberg School of Medicine, selected people ages 20 to 53 who had early-stage MS (they had been diagnosed an average of five years) and who had not responded to at least six months of treatment with interferon beta, the standard treatment for the disease. The patients underwent chemotherapy to destroy their immune systems and were then injected with their own immune system cells that had been removed before the chemotherapy. After an average of three years of follow-up, 17 patients (81%) had improved significantly. Some have seen their symptoms disappear.

Barry Goudy, 50, entered the trial in 2003 after the medications he was taking for MS began to falter. “I would fall out of remission and have to be admitted to the hospital,” says Goudy, who lives in Michigan. “I would get numbness in my legs; very fatigued. I couldn’t climb stairs.” Since the stem-cell transplantation, however, he has been free of symptoms. “I feel normal now,” he said in an interview with The Times. “I feel like I did prior to 1995 when I was diagnosed — young and full of energy.”

“This is the first time we have turned the tide on this disease,” Burt said in a news release. “In MS the immune system is attacking your brain. After the procedure, it doesn’t do that anymore.”

More research is needed to assess the treatment, including a large, randomized, controlled trial, said Dr. Gianluigi Mancardi, of the University of Genova, Italy, in an editorial accompanying the study. Burt has begun such a study. Previous studies showed the treatment was unsuccessful on people with late-stage multiple sclerosis.

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ScienceDaily.com, January 28, 2009 — A new study has found that transplantation of stem cells from the lining of the spinal cord, called ependymal stem cells, reverses paralysis associated with spinal cord injuries in laboratory tests. The findings show that the population of these cells after spinal cord injury was many times greater than comparable cells from healthy animal subjects. The results open a new window on spinal cord regenerative strategies.

The transplanted cells were found to proliferate after spinal cord injury and were recruited by the specific injured area. When these cells were transplanted into animals with spinal cord injury, they regenerated ten times faster while in the transplant subject than similar cells derived from healthy control animals.

Spinal cord injury is a major cause of paralysis, and the associated trauma destroys numerous cell types, including the neurons that carry messages between the brain and the rest of the body. In many spinal injuries, the cord is not actually severed, and at least some of the signal-carrying nerve cells remain intact. However, the surviving nerve cells may no longer carry messages because oligodendrocytes, which comprise the insulating sheath of the spinal cord, are lost.

The regenerative mechanism discovered was activated when a lesion formed in the injured area. After a lesion formed in the transplant subject, the stem cells were found to have a more effective ability to differentiate into oligodendrocytes and other cell types needed to restore neuronal function.

Currently, there are no effective therapies to reverse this disabling condition in humans. However, the presence of these stem cells in the adult human spinal cords suggests that stem cell-associated mechanisms might be exploited to repair human spinal cord injuries.

Given the serious social and health problems presented by diseases and accidents that destroy neuronal function, there is an ever-increasing interest in determining whether adult stem cells might be utilized as a basis of regenerative therapies.

“The human body contains the tools to repair damaged spinal cords. Our work clearly demonstrates that we need both adult and embryonic stem cells to understand our body and apply this knowledge in regenerative medicine,” says Miodrag Stojkovic, co-author of the study. “There are mechanisms in our body which need to be studied in more detail since they could be mobilized to cure spinal cord injuries.”