A New Target for Treating Stroke: The Spleen


If the blood vessels of the brain become plugged as a result of a clot or some other obstructive event, then the brain suffers a trans-ischemic attack (TIA), which is more commonly known as a stroke. The initial stroke starves brain cells of oxygen, which causes cell death by suffocation. However, dying brain cells  often spill enormous amounts of lethal material into the surrounding area, which kills off even more brain cells. Worse still, these dead or dying called can induce inflammation in the brain, which continues to kill off brain cells.

New work, however, from the laboratory of César Borlongan at the University of Southern Florida in Tampa, indicates that the spleen may be a target for treating the stroke-induced chronic inflammation that continues to kill brain cells after the initial stroke.

At the University of Florida Center of Excellence for Aging and Brain Repair, a study found that the intravenous administration of human bone marrow stem cells to post-stroke rats reduced the inflammatory-plagued secondary cell death associated with stroke progression in the brain. The intravenously administered cells preferentially migrated to the spleen where they reduced this post-stroke inflammation.

This study answers some of the perplexing questions surrounding animal experiments that used stem cells to treat stroke. Typically, stem cell administration to animals that suffered an artificially-induced stroke causes some functional recovery, but when their brains are examined for the stem cells that were implanted into them, very few surviving cells are observed.

“Our findings suggest that even if stem cells do not enter the brain or survive there, as long as the transplanted cells survive in the spleen the anti-inflammatory effect they promote may be sufficient enough to therapeutically benefit the stroke brain,” said César Borlongan, principal investigator of this study.

Stroke is the leading cause of death and the number one cause of chronic disability in the United States, yet treatment options are limited.

Stem cell therapy has emerged as a potential treatment for ischemic stroke, but most pre-clinical studies have examined the effects of stem cells transplanted during acute stroke (one hour to three hours aster the onset of the stroke).

In the wake of an acute stroke, an initial brain lesion forms from the lack of blood flow to the brain. The blood-brain barrier is also breached and this allows the infiltration of inflammatory molecules that trigger secondary brain cell death in the weeks and months that follow. This expanded inflammation is the hallmark of chronic stroke.

In this study, Borlongan and his colleagues intravenously administered human bone marrow stem cells 60 days after the onset of a stroke. Thus these animals were well into the chronic stroke stage.

The transplanted stem cells predominantly homes to the spleen. In fact, Borlongan and his crew found 30-times more cells in the spleens of the animals than in the brain.

While in the spleen, the stem cells squelched the production of a protein called tumor necrosis factor, which is a major inflammatory signal that increases in concentration after a stroke. The reduction of the tumor necrosis factor signal prevented the macrophages and other immune cells from leaving the spleen and going to the brain. This reduced systemic inflammation and decreased the size of the lesions in the brain caused by the stroke. There was also a trend toward reduced neuronal death and smaller decreases in learning and memory in the laboratory animals.

Borlongan explained that during the chronic stage of stroke, macrophages seem to fuel inflammation. “If we can find a way to effectively block the fuel with stem cells, then we may prevent the spread of damage in the brain and ameliorate the disabling symptoms many stroke patients live with,” said Borlongan.

Borlongan and his team hope to test whether transplanting human bone marrow stem cells directly into the spleen will lead to behavioral recovery in post-stroke rats.

One drug that has been approved for the emergency treatment of stroke is tPA or tissue plasminogen activating factor, which activates the blood-based protein plasminogen to form the highly active enzyme, plasmin. Plasmin is a powerful dissolved of clots, but tPA must be administered less than 4.5 hours after the onset of ischemic stroke, and benefits only three to four percent of patients.

Even though more work needs to be done, evidence from the USF group and other neurobiology groups indicates that stem fells may provide a more effective treatment for stroke over a wider time frame.

Targeting the spleen with stem cells or the anti-inflammatory molecules they sec rate offers hope for treating chronic neurodegenerative diseases like stroke at later stages.

This study, which was published in the journal Stroke, shows that it is possible to arrest the chronic inflammation that characterizes chronic stroke 60 days after the initial stroke. If such a result can be replicated in human patients, it will indeed be a powerful thing, according the Sandra Acosta, the first author on this paper.

Stem Cells Decrease Brain Inflammation and Increase Cognitive Ability After Traumatic Brain Injury


A study at the Texas Health Science Center has shown that stem cell treatments that quash inflammation soon after traumatic brain injury (TBI) might also offer lasting cognitive gains.

TBI sometimes causes severe brain damage, and it can also lead to recurrent inflammation of the brain.  This ongoing inflammation can extend the damage to the brain.  Only a few drugs help (anti-inflammatory drugs for example).  Up to half of patients with serious TBI need surgery, but some stem cells like a sub group of mesenchymal stem cells called multipotent adult progenitor cells (MAPCs) can reduce short-term inflammation, and induce functional improvement in mice with TBI.  Unfortunately, few groups have gauged the long-term effects of MAPCs on TBI.

Differentiation of MultiStem® cells into alkaline-phosphatase-positive osteoblasts (blue) and lipid-accumulating adipocytes (red).
Differentiation of MultiStem® cells into alkaline-phosphatase-positive osteoblasts (blue) and lipid-accumulating adipocytes (red).

In an article that appeared in the journal Stem Cells Translational Medicine, a research team led by the Director of the Children’s Program in Regenerative Medicine, Charles Cox, reported the use of human MAPCs in mice that had suffered TBI.

Charles Cox, Jr., MD
Charles Cox, Jr., MD

In this study, Cox and his colleagues infused MAPCs into the bloodstream of two groups of mice 2, and 24 hours after suffering a TBI.  The first group of mice received two million cells per kilogram, and mice in the other group received an MAPC dose five times stronger.

Four months after MAPC administration, those mice that had received the stronger dose continued to experience less brain inflammation and better cognition.  Spatial learning was increased and motor deficits had decreased.

According to Cox, the intravenously administered MAPCs did not cross the blood/brain barrier.  Since immune cells can cross the blood/brain barrier for a short period of time after a TBI and cause autoimmunity, this result shows that the MAPCs are quelling inflammation through “paracrine” mechanisms (paracrine means that molecules are secreted by the cells and these secreted molecules elicit various responses from nearby cells). Cox made this clear: “We spent 18 months looking for them in the brain. There was little to no engraftment there.”

Rather than entering the brain, the MAPCs “set up shop in the spleen, a giant reservoir of T and B cells. The MAPCs change the spleen’s output to anti-inflammatory cells and cytokines, which communicate with immune cells in the brain—microglia—and change their response to injury from hyper-to-anti- inflammatory. The cells alter the innate immune response to injury. We have shown this in a sequence of papers.”

Microglia
Microglia

University of Cambridge neurologist, Stefano Pluchino, has worked with immune regulatory stem cells.  Pluchino said that Cox’s study shows a “good dose response” on disability and behavior “after hyperacute, or acute, intravenous injection of MAPCs.”  However, Pluchino noted that the description of the effects of MAPCs on microglia (white blood cells in the brain that gobble up foreign matter and cell debris) is “speculative.”  Pluchino continued: “It is not clear whether these counts have been done on the injured brain hemisphere, and whether MAPC effects were observable on the unaffected hemisphere.  The distribution and half-life of these MAPCs is not clear” and has never been demonstrated convincingly in Athersys papers (side note: Athersys is the company that isolates and grows the human MAPCs). “It is also not clear if effects in the Cox study were a ‘false positive,’ secondary to a paradoxical immune suppression the xenograft modulates.” That is, a false positive could occur because human cells in animal bodies rouse immune reactions. “It is not clear where in the body these MAPCs would work, either out or into the injured brain.” Additionally the mechanism by which these cells act does not seem to be clear, according to Pluchino.

But, Pluchino added: “Athersys is already in clinic with MAPCs in graft vs. host disease, myocardial infarction, stroke, progressing towards a phase I/II clinical trial in multiple sclerosis, and completing the pre-clinical work in traumatic brain and spinal cord injuries. Everything looks great. The company is solid. The data is convincing in terms of behavioral and pathological analyses. But the points I have raised are far from clarified.”

Cox admitted that Pluchino’s points are valid.  He pointed out that human cells were used in rodents, since the FDA wants pre-clinical studies in laboratory animals in order to first evaluate the safety and efficacy of the exact cells to be used in a proposed therapy before they head to the clinic. “As we are not seeking engraftment of these cells, and would not plan to immunosuppress a trauma patient, we have not pursued animal models that use immunosuppression. Our study was designed with translationally relevant end-points, recognizing the limitations of not having a final mechanism of action determined. The growing consensus is there are many mechanism(s) of action in cell therapies.”

Cox also agreed that the suggested effects of MAPCs on microglia, “is not truly a proof of mechanism.”  However, Cox and his co-workers have developed a protocol that can potentially more accurately quantify microglia in mice. “We ultimately plan more mechanistic studies to define endogenous microglia versus infiltrating microglia and the effects of various cell therapies. “

Additionally, Cox also said that: “We have published work showing the majority of acutely infused MSCs and MAPCs are lodged in the lung after intravenous delivery. This was an acute study in non-injured animals, but others have shown similar data.” In another study, Cox’s research group showed that the cells cluster in the spleen, which corroborates work by other research groups that have used umbilical cord cells to treat stroke.

Finally, Cox disagrees that the suppression of immune cell function in animals by human cells is appropriately characterized as “a false positive.”  Cox explained that the infused cells induce a “modulation of the innate immune response, and typically, the immune rejection of a transplant is associated with immune activation, not suppression. So it well may be a ‘true positive.’”

In order for MAPCs to make to the clinical trial stage, Cox will need to investigate the mechanisms by which MAPCs suppress inflammation and if their purported effects on microglia in the central nervous system are real.  He will also need to show that these cells work in other types of laboratory animals beside mice.  Rats will probably be next, and after that, my guess is that the FDA would allow Athersys to apply for a New Drug Application.