Jared Ehrhart from the University of South Florida, who also serves as the Director of Research and Development at Saneron CCEL Therapeutics Inc, and his coworkers have shown that cells from umbilical cord blood can not only improve the health of mice that have an experimental form of Alzheimer’s disease (AD), but these can also be administered intravenously, which is safer and easier than other more invasive procedures.
Laboratory mice can be engineered to harbor mutations that can cause a neurodegenerative disease that greatly resembles human AD. One such mouse is the PSAPP mouse that harbors two mutations that are known to cause an inherited, early-onset form of AD in humans. By placing both mutations in the same mouse, the animal forms the characteristic protein plaques more rapidly and shows significant AD symptoms and brain pathology.
Ehrhart used PSAPP mice to test the ability of human umbilical cord blood to ameliorate the symptoms of AD. He injected one million Human Umbilical Cord Blood Cells (HUCBCs) into the tail veins of PSAPP mice and 2.2 million into the tail veins of Sprague-Dawley rats. Then he harvested their tissues at 24 hours, 7 days, and 30 days after injection. Then Ehrhart and his team used a variety of techniques to detect the presence of the HUCBCs.
Interestingly, the HUCBCs were able to cross the blood-brain barrier and take up residence in the brain. The cells remained in the brain and survived there for up to 30 days and did not promote the growth of any tumors.
Several studies have shown that the administration of HUCBCs to mice with a laboratory form of AD can improve the cognitive abilities of those mice (see Darlington D, et al., Cell Transplant. 2015;24(11):2237-50; Banik A, et al., Behav Brain Res. 2015 Sep 15;291:46-59; Darlington D, et al., Stem Cells Dev. 2013 Feb 1;22(3):412-21). However, in such cases it is essential to establish that the administered cells actually found their way to the site of damage and exerted a regenerative response.
Even though Ehrhart and his troop found that the intravenously administered HUCBCs were widely distributed throughout the bodies of the animals, they persisted in the central nervous system for up to one month after they were injected. In the words of this publication, which appeared in Cell Transplantation, the HUCBCs were “broadly detected in both in the brain and several peripheral organs, including the liver, kidneys, and bone marrow.”. The fact that such a minimally invasive procedure like intravenous injection can effectively introduce these cells into the bodies of the PSAPP mice and still produce a significant therapeutic effect is a significant discovery.
Ehrhart and his colleagues concluded that HUCBCs might provide therapeutic effects by modulating the inflammation that tends to accompany the onset of AD. Furthermore, these cells do not need to be delivered by means of an invasive procedure like intracerebroventricular injection. Furthermore, even though HUCBCs were detected in other organs, their numbers in those places was not excessive and the ability of the HUCBCs to cross the blood-brain barrier suggests that these cells might serve as safe, effective therapeutic agents for AD patients some day.
If something lodges in the blood vessels that feed the brain – say a blood clot, piece of bone marrow after a bone has been broken, or tissue debris from damaged tissue – the brain undergoes a loss of blood flow. Since the brain received its oxygen and nutrients from the bloodstream, blockage of the vessels that feed the brain can lead to the death of brain cells.
Such a phenomenon is called a stroke or a Trans-Ischemic Attack. However, if the heart stops, blood flow to the brain ceases; not because of blockage of the blood vessels that feed the brain, but because the pump that propels through the bloodstream has stopped and, therefore, blood flow stops. Such a condition is known as global cerebral ischemia or GCI.
GCI is one of the most challenging clinical issues encountered during cardiac arrest and, unfortunately, typically indicates a poor prognosis. Severe neurological damage develops in 33%–50% of GCI patients who have survived a cardiac arrest that was documented by a medical professional. In those rare cases of survival after cardiac arrest that was not documented by a medical professional, the percentage of neurological defects is 100%. I hope this convinces you that CGI is a problem.
In order to treat GCI, physicians usually induce hypothermia, which lowers and maintains the core body temperature at 32°C–34°C. Presently, this is the only treatment regime that has been demonstrated to improve neurological recovery. Unfortunately, there are many technical difficulties in the application of this therapy. Special equipment is required, and complications such as blood clots and infection are perennial problems. Is there a better way to treat GCI?
Sang Won Suh from the Hallym University College of Medicine in South Korea and his colleagues have used fat-based mesenchymal stem cells to treat laboratory animals that have suffered GCI. The results of their study are encouraging.
Suh and his coworkers used Sprague-Dawley rats for this study. They anesthetized the rats and then clamped their carotid arteries to reduce blood flow to the brain for seven minutes. This effectively simulates GCI in these laboratory animals. After the clamps were removed, some animals were given one million fat-based mesenchymal stem cells, and others were simply restored by means of unclamping the carotid arteries plus fluid reconstitution. The rats were subjected to behavioral tests three days before the procedure and seven days after it. These tests consisted of placing adhesive tape the forepaws of the animals and then measuring the day it tool for the animals to remove to adhesive tape. After the seventh day post-procedure, the rats were put down and their brains were examined for cell death, structure, blood vessel densities, and degree of inflammation.
When the brains of these animals were examined, it was clear that the animals that had received fat-based mesenchymal stem cells suffered much less cell death than the untreated animals.
In the figure above you can see a Fluoro-Jade B staining of these brains. FJB stains detect dying cells. As you can see, the brain from the rats that experienced GCI without any stem cell treatments had lots of dying cells in their brains. The “sham” operated rats – rats that were operated, but their carotid arteries were not clamped – had no cell death in their brains. The animals that had their carotid arteries clamped, but were given fat-based mesenchymal stem cells had a little cell death. The graph above shows the vast differences between the stem cell-treated and the non-stem cell-treated groups. Truly these are significant results. Other experiments that detected Now this is no a surprise, since Ohtaki and others showed a very similar result in 2008 (OhtakiH, et al. Proc Natl Acad Sci USA 105:14638–14643). Suh, and his group, however, took these experiments further to determine why these cells prevented cell death in the brain after GCI.
When Suh and his team examined the leakage of large proteins into the brain, they saw something quite remarkable; the mesenchymal stem cell-treated rats only leaked a little protein into their brains compared to the non-stem cell-treated rats.
The presence of the brown color indicated the presence of a protein in the brain that normally does not find its way to the brain unless the integrity of the blood-brain barrier is compromised. As you can see, the non-treated animals have a truckload of this protein in their brains, which indicates that their blood-brain barriers are very leaky. On the contrary, the stem cell-treated brains are not nearly as leaky and the sham operated brains are not leaky at all.
These results suggest that the stem cells help maintain the structural integrity of the blood-brain barrier in GCI patients and this prevents nasty things from the bloodstream, such as immune cells and so on from accessing the brain and ravaging it. To test this hypothesis, Suh and others examined the brains for the presence of neutrophils, which are white blood cells that show up when inflammation occurs. These cells are not found in the brain unless the blood-brain barrier is damaged. Sure enough, brains from the sham-operated rats showed no signs of neutrophils, brains from the non-stem cell-treated rats were chock full of neutrophils, and the brains from the stem cell-treated rats only had a few neutrophils.
A conclusion from this paper states: “Administration of MSCs decreased the delayed neuronal damage in a transient global cerebral ischemia model by prevention of BBB disruption, endothelial damage, and neutrophil infiltration.”
Clearly this merits more work. Larger animal models will need to be examined, and also it would be nice to know if administration of exosomes from mesenchymal stem cells can elicit a similar biological response. However his is a very hopeful beginning to what might become a fruitful bit of clinical research.
Glymphatics is a new subdiscipline in neuroscience that was essentially discovered by a Danish neuroscientist named Maiken Nedergaard. Dr. Nedergaard gave a fine seminar on this subject on Sunday.
Glymphatics consists of the system that removes waste products from the brain. Dr. Nedergaard showed movies that showed how the cerebrospinal fluid that bathes the periphery of the brain pulsates as it moves over the brain. When die molecules are injected into the cerebrospinal fluid, these dyes wend up in the blood system. How does this happen?
Nedergaard reasoned that diffusion of the fluid was far too slow for the dye to get to the blood system as fast as it does. Instead, she suspected that fluid moves by means of a “convection current.” How does this work? The blood vessels that feed the brain are surrounded by cells known as astrocytes. These astrocytes prevent molecules from entering the brain unless they can properly negotiate their way across these astrocytes, and this forms the basis for the blood-brain barrier. Cerebrospinal fluid moves across the cells of the brain and is removed by the astrocyte-surrounded vessels. This sink for the cerebrospinal fluid essentially pulls the cerebrospinal fluid across the brain cells and serves as the means by which the brain is cleansed of waste products.
This system, however, is subject to regulation, since the flow of fluid from the cerebrospinal fluid depends on the size of the spaces between brain cells. As it turns out, the spaces between brain cells in larger during sleep than when we are awake. Therefore, sleep seems to be the means by which our bodies clear the rubbish from our brains.
The molecule that controls the space between brain cells is norepinephrine. How it does that remains uncertain, but this is the molecule that is released during sleep to help clear out the garbage in the brain.
Since Alzheimer’s disease, Parkinson’s disease, other neurodegenerative diseases include the accumulation of protein aggregates in the brain, the removal of waste products in the brain would seem to be a rather important process. Also, when there is a head injury, surgeons sometimes leave the skull cap open while the brain heals. This, however, hamstrings the glymphatic system and surgeons should replace the skull cap so that the glymphatic system can do its job. Secondly, if norepinephrine can regulate this system, then this might be a way to increase clearance of waste products from the brain to reduce or delay the accumulation of protein aggregates in the brain.
I am in Washington DC at the Society for Neuroscience 2014 meeting. There is some incredible science here. Let me just share a few of the things I saw today:
The Thompson laboratory from UC Irvine (my alma mater – go anteaters!) made a model system for the blood-brain barrier from induced pluripotent stem cells. These scientists made iPSCs that had similar genetic defects to those observed in patients with Huntington’s disease. These iPSCs were then differentiated into blood-brain barrier cells and showed that these cells showed defects similar to those seen in patients with Huntington’s disease. The barrier leaked, which makes this a good model to study blood brain barrier defects in patients with neurological diseases.
Another poster described the use of vesicles from human fat-based stem cells to treat laboratory animals with a type of Huntington’s disease. These vesicles attenuated Huntington’s disease pathology and delayed its onset.
There were several other brilliant posters, and tomorrow, there will be even more. I will blog about those as time permits.
Irinotecan is an anticancer drug that was approved for use in 1996. It is a modified version of the plant alkaloid camptothecin, and even though it shows significant activity against brain tumors in culture, but in a living body, this drug poorly penetrates the blood-brain barrier. Therefore irinotecan usually does not accumulate to appreciable levels in the brain and is typically not used to treat brain tumors.
That could change, however, if a new strategy published in paper by Marianne Metz and her colleagues from the laboratory of Karen Aboody at the Beckman Research Institute at the City of Hope in Duarte, California, in collaboration with colleagues from several other laboratories.
In this paper, Metz and her co-workers genetically engineered neural stem cells to express enzymes called “carboxylesterases.” These carboxyesterase enzymes convert irinotecan, which is an inactive metabolite, to the active form, which is known as “SN-38.” The efficient conversion of irinotecan to SN-38 in the brain greatly accelerates the therapeutic activity of this drug in the brain. Also, the constant conversion of irinotecan to another molecule accelerates the transport of irinotecan past the blood brain barrier.
To test this strategy. Metz and others grew the engineered neural stem cells in culture and measured their ability to make carboxylesterases in culture, and their ability to convert irinotecan into SN-38 in culture. In both cases, the engineered neural stem cells made a boat-load of carboxylesterase and converted irinotecan into SN-38 in spades. More importantly, the genetically engineered neural stem cells behaved exactly as they did before, which shows that the genetic manipulation of these cells did not change their properties.
Next, Metz others tested the ability of the engineered neural stem cells to kill human brain tumor cells in culture in the presence of irinotecan. Once again, the genetically engineered neural stem cells effectively killed human brain tumor cells in culture in a irinotecan-concentration-dependent manner. When these genetically engineered neural stem cells were injected into the brains of mice with brain tumors, intravenous administration of irinotecan produced high levels of SN-38 in the brain. This shows that these cells have the capacity to increase the production of SN-38 in the brain.
This strategy is similar to other strategies that been used in various clinical trials, but because neural stem cells have a tendency to move into brain tumor tissue and surround it, they represent an efficient and effective way to deliver anticancer drugs to brain tumors. Also, since the particular neural stem cell line used in this experiment (HB1.F3.CD) does not cause tumors and is also not recognized as foreign by the immune system, it is a particularly attractive stem cell line for such an anti-tumor strategy.
Italian researchers have derived stem cells from skin cells that can reduce the damage to the nervous system cause by a mouse version of multiple sclerosis. This experiment provides further evidence that stem cells from patients might be a feasible source of material to treat their own maladies.
The principal investigators in this work, Cecilia Laterza and Gianvito Martino, are from the San Raffaele Scientific Institute, Milan and the University of Milan, respectively.
Because multiple sclerosis results from the immune system attacking the myelin sheath that surrounds nerves, most treatments for this disease consist of agents that suppress the immune response against the patient’s own nerves. Unfortunately, these treatments have pronounced side effects, and are not effective in the progressive phases of the disease when damage to the myelin sheath might be widespread.
The symptoms of loss of the myelin sheath might one or more of the following: problems with touch or other such things, muscle cramping and muscle spasms, bladder, bowel, and sexual dysfunction, difficulty saying words because of problems with the muscles that help you talk (dysarthria), lack of voluntary coordination of muscle movements (ataxia), and shaking (tremors), facial weakness or irregular twitching of the facial muscles, double vision, heat intolerance, fatigue and dizziness; exertional exhaustion due to disability, pain, or poor attention span, concentration, memory, and judgment.
Clinically, multiple sclerosis is divided into the following categories on the basis of the frequency of clinical relapses, time to disease progression, and size of the lesions observed on MRI. These classifications are:
A) Relapsing-remitting MS (RRMS): Approximately 85% of cases and there are two types – Clinically isolated syndrome (CIS): A single episode of neurologic symptoms, and Benign MS or MS with almost complete remission between relapses and little if any accumulation of physical disability over time.
B) Secondary progressive MS (SPMS)
C) Primary progressive MS (PPMS)
D) Progressive-relapsing MS (PRMS)
The treatment of MS has 2 aspects: immunomodulatory therapy (IMT) for the underlying immune disorder and therapies to relieve or modify symptoms.
To treat acute relapses:
A) Methylprednisolone (Solu-Medrol) can hasten recovery from an acute exacerbation of MS.
B) Plasma exchange (plasmapheresis) for severe attacks if steroids are contraindicated or ineffective (short-term only).
C) Dexamethasone is commonly used for acute transverse myelitis and acute disseminated encephalitis.
For relapsing forms of MS, the US Food and Drug Administration (FDA) include the following:
A) Interferon beta-1a (Avonex, Rebif)
B) Interferon beta-1b (Betaseron, Extavia)
C) Glatiramer acetate (Copaxone)
D) Natalizumab (Tysabri)
F) Fingolimod (Gilenya)
G) Teriflunomide (Aubagio)
H) Dimethyl fumarate (Tecfidera)
For aggressive MS:
A) High-dose cyclophosphamide (Cytoxan).
In order to treat multiple sclerosis, restoring the damaged myelin sheath is essential for returning patients to their former wholeness.
In this study, this research team reprogrammed mouse skin cells into induced pluripotent skin cells (iPSCs), and then differentiated them into neural stem cells. Neural stem cells can differentiate into any cell type in the central nervous system.
Next, Laterza and her colleagues administered these neural stem/progenitor cells “intrathecally,” which simply means that they were injected into the spinal cord underneath the meninges that cover the brain and spinal cord to mice that had a rodent version of multiples sclerosis called EAE or experimental autoimmune encephalomyelitis.
EAE mice are made by injecting them with an extract of myelin sheath. The mouse immune system mounts and immune response against this injected material and attacks the myelin sheath that surrounds the nerves. EAE does not exactly mirror multiple sclerosis in humans, but it comes pretty close. While multiple sclerosis does not usually kill its patients, EAE either kills animals or leaves them with permanent disabilities. Animals with EAE also suffer severe nerve inflammation, which is distinct from multiple sclerosis in humans in which some nerves suffer inflammation and others do not. Finally, the time course of EAE is entirely different from multiple sclerosis. However, both conditions are caused by an immune response against the myelin sheath that strips the myelin sheath from the nerves.
The transplanted neural stem cells reduced the inflammation within the central nervous system. Also, they promoted healing and the production of new myelin. However, most of the new myelin was not made by the injected stem cells. Instead the injected stem cells secreted a compound called “leukemia inhibitory factor” that promotes the survival, differentiation and the remyelination capacity of both internal oligodendrocyte precursors and mature oligodendrocytes (these are the cells that make the myelin sheath). The early preservation of tissue integrity in the spinal cord limited the damage to the blood–brain barrier damage. Damage to the blood-brain barrier allows immune cells to infiltrate the central nervous system and destroy nerves. By preserving the integrity of the blood-brain barrier, the injected neural stem cells prevented infiltration of blood-borne of the white blood cells that are ultimately responsible for demyelination and axonal damage.
“Our discovery opens new therapeutic possibilities for multiple sclerosis patients because it might target the damage to myelin and nerves itself,” said Martino.
Timothy Coetzee, chief research officer of the National Multiple Sclerosis Society, said of this work: “This is an important step for stem cell therapeutics. The hope is that skin or other cells from individuals with MS could one day be used as a source for reparative stem cells, which could then be transplanted back into the patient without the complications of graft rejection.”
Obviously, more work is needed, but this type of research demonstrates the safety and feasibility of regenerative treatments that might help restore lost function.
Martino added, “There is still a long way to go before reaching clinical applications but we are getting there. We hope that our work will contribute to widen the therapeutic opportunities stem cells can offer to patients with multiple sclerosis.”
See Cecilia Laterza, et al. iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF. NATURE COMMUNICATIONS 4, 2597: doi:10.1038/ncomms3597.
Mesenchymal stem cells (MSCs) are found in multiple tissues and locations throughout our bodies, and they have the ability to differentiate into bone, fat, cartilage, and smooth muscle. MSCs also have the ability to suppress unwanted immune responses and inflammation. Therefore, MSCs are prime candidates for regenerative medical treatments.
MSCs have been used to experimentally treat traumatic brain injury (for example, Galindo LT et al., Neurol Res Int 2011;2011:564089). One of the main concerns after traumatic brain injury is damage to the blood brain barrier (BBB). BBB damage allows inflammatory cells to access the brain and further damage it. Therefore, healing the damage to the BBB or protecting the BBB after a traumatic brain injury is vital to the brain after a traumatic brain injury.
After a traumatic brain injury, the vascular system suffers damage and begins to leak. When blood leaks into tissues, it tends to irritate the tissues and damage them. MSCs release a soluble factor known as TIMP3 (tissue metalloproteinase-3) that degrades blood-based proteins known to cause damage to tissues when blood vessels leak. TIMP3 production by MSCs can also protect the BBB from degradation after a traumatic brain injury.
Researchers from the University of Texas Health Sciences Center, UC San Francisco, and two biotechnology companies have examined the protective role of MSCs and one particular protein secreted by MSCs in protecting the BBB after traumatic brain injury.
Shibani Pati, from UC San Francisco, and his collaborators from the University of Texas, Houston, MD Anderson Cancer Center, Amgen, and Blood Systems Research Institute (San Francisco) used MSCs to staunch the increased permeability the BBB after a traumatic brain injury.
They used a mouse model in these experiments and induced traumatic brain injuries in these mice. Then they gave MSCs to some, and soluble TIMP3 to others, and buffer to another group as a control. They discovered that the MSCs mitigated BBB damage after a traumatic brain injury. However, they also found that soluble TIMP3 could also protect the BBB approximately as well as MSCs. This suggested that the TIMP3 secretion by MSCs is the main mechanism by which MSCs protect the BBB after a traumatic brain injury.
To test this hypothesis, Pati and his colleagues administered MSCs to mice that had experienced traumatic brain injury, but they also co-administered a soluble inhibitor to TIMP3. They discovered that this inhibitor completely abolished the ability of MSCs to protect the BBB after a traumatic brain injury. They also found that the main target of TIMP3 was vascular endothelial growth factor. Apparently after a traumatic brain injury, massive release of vascular endothelial growth factor causes the breakdown of BBB structures. TIMP3 degrades vascular endothelial growth factor, which prevents BBB breakdown.
These findings suggest that administration of recombinant proteins such as TIMP3 after a traumatic brain injury can protect the BBB and decrease brain damage. Clinical trial anyone?