G-CSF Fails to Improve Long-Term Clinical Outcomes in REVIVAL-2 Trial


Granulocyte-Colony Stimulating Factor (G-CSF) is a glycoprotein (protein with sugars attached to it) that signals to the bone marrow to produce granulated white blood cells (specifically neutrophils), and to release stem cells and progenitor cells into the peripheral circulation.

This function of G-CSF makes it a candidate treatment for patients who have recently experienced a heart attack, since the release of stem cells from the bone marrow could, in theory, bring more stem cells to the damaged heart to heal it. Additionally, G-CSF is known to induce the proliferation and enhance the survival of heart muscle cells.

In several experiments with laboratory animals showed that G-CSF treatments after a heart attack significantly reduced mortality (Moazzami K, Roohi A, and Moazzimi B. Cochrane Database Systematic Reviews 2013; 5: CD008844. However, in a clinical trial known as the REVIVAL-2 trial, a double-blind, placebo-controlled study, G-CSG treatment failed to influence the performance of the heart six months after administration.

Now Birgit Steppich and others have published a seven-year follow-up of the subjects in the original REVIVAL-2 study to determine if G-CSF had long-term benefits that were not revealed in the short-term study. These results were published in the journal Thrombosis and Haemostasis (115.4/2016).

Of the initially enrolled 114 patients, 106 patients completed the seven-year follow-up. The results of this trial showed that G-CSF treatment for five days in successfully revascularized heart attack patients did not alter the incidence of death, recurrent heart attacks, stroke, or secondary adverse heart events during the seven-year follow-up.

These results are similar to those of the STEMMI trial, which treated patients with G-CSF for six days 10-65 hours after the reperfusion. In a five-year follow-up of 74 patients, there were no differences in the occurrence of major cardiovascular events between the G-CSF-treated group and the placebo group (Achili F, et al., Heart 2014; 100: 574-581).

Therefore, it appears that even though G-CSF worked in laboratory rodents that had suffered heart attacks, this treatment does not consistently benefit human heart attack patients. Although why it does not work will almost certainly require more insights than we presently possess.

Patient’s Own Peripheral Blood Stem Cells Benefits Stroke Patients


A study conducted in Taiwan has examined the ability of a patient’s circulating peripheral blood stem cells to benefit stroke patients.

In this study, one of two groups of stroke patients received injections of their own peripheral blood stem cells (PBSCs) directly into the brain but the other group received standard care. Those patients who received the PBSCs experienced some improvement in stroke scales and functional capabilities. Patients who received their own PBSCs also were given injections of granulocyte-colony stimulating factor (G-CSF), which seems to protect the nervous system after trauma to it.

“In this phase 2 study, we provide the first evidence that intracerebral injection of autologous (self-donated) PBSCs can improve motor function in those who have suffered a ,” said stroke and have motor deficits as a result,” said Woei-Cheng Shyu of the China Medical University, who is the corresponding author of this study. “Our study demonstrates that this therapeutic strategy was feasible and safe in stroke patients who suffered a prior stroke, but within five years from the onset of symptoms.”

Strokes, also known as trans-ischemic attacks (TIAs) result from blockage in blood vessels that feed the brain. The lack of blood flow to the brain starves it of oxygen, and the cells of the brain begin to die off. Because neuronal death as a result of stroke limits functional recovery, stem cell therapy is advancing as a potentially effective regenerative treatment for stroke.

Also, in many types of stem cell trials, PBSCs are the stem cell of choice. The ease of isolating these cells without invasive procedures makes them the stem cell choice for many clinical trials. In order to utilize PBSCs, patients must amplify their supply of PBSCs, and injections of G-CSF seems to do just that.

In this study, all patients had suffered a prior stroke as long as five years prior to being treated.

At the end of the 12-month follow-up, the group of 15 patients with neurological deficits who received the PBSC injections into the brain experienced neurological and functional improvements, according to several different clinical measurements.

On the other hand, the 15-patient control group who had neurological deficits but did not receive the PBSC injections did not experience the same beneficial results.

In the experimental group, nine of the 15 patients showed proper activation of the motor nerves after stimulating that part of the brain with a magnet. This procedure, called transcranial magnetic stimulation or TMS, places a magnet above skull, directly above the part of the brain you want to stimulate. The rapidly changing magnetic field generated by the magnet produces weak electrical currents in the brain, which stimulates nearby neurons. In this experiment, researchers targeted the precentral gyrus, which is the portion of the brain where the primary motor cortex. Because strokes sometimes kill off neurons in the primary motor cortex, stimulation of the primary motor cortex will not lead to stimulation of motor nerves, but in this experiment, 9 of 15 patients who received the PBSC injections who positive motor evoke potential or MEPs after TMS. Why this ratio was not 15 out of 15 remains unclear at this time.

primary motor cortex

One of the main conclusions of this work, is that “Despite this success, it should be noted that this was a preliminary study and, due to the small number of patients, are tentative. In the future we plan to conduct a multi-center, large-scale, double-blind, placebo-controlled randomized studies [sic] to better evaluate the effect of PBSC implantation in patients suffering from the effects of past stroke.”

Umbilical Cord Blood Cells Combined with Growth Factors Improves Traumatic Brain Injury Outcomes


Approximately 2 million Americans experience a traumatic brain injury every year. Most of these are individuals who employed in high-risk jobs such as the military, firefighting, police work and others types of essential but highly dangerous jobs. No matter how small the injury, individuals who have suffered a traumatic brain injury (TBI) can suffer from a whole host of motor, behavioral, intellectual and cognitive disabilities over the short or long-term. Unfortunately, there are few clinical treatments for TBI, and the few we have are rather ineffective.

In order to design better, more effective treatments for TBI, neuroscientists at the Center of Excellence for Aging and Brain Repair, Department of Neurosurgery in the USF Health Morsani College of Medicine, University of South Florida, have used umbilical cord stem cells in combination with growth factors to treat TBIs in mice.

This study investigated the ability of several strategies, both by themselves and in combination with other therapies, to treat rats with a laboratory form of TBI. In particular, the USF team discovered that a combination of human umbilical cord blood cells (hUBCs) and granulocyte colony stimulating factor (G-CSF), a growth factor, was more therapeutic than either administered alone, or each with saline, or saline alone.

“Chronic TBI is typically associated with major secondary molecular injuries, including chronic neuroinflammation, which not only contribute to the death of neuronal cells in the central nervous system, but also impede any natural repair mechanism,” said study lead author Cesar V. Borlongan, PhD, professor of neurosurgery and director of USF’s Center of Excellence for Aging and Brain Repair. “In our study, we used hUBCs and G-CSF alone and in combination. In previous studies, hUBCs have been shown to suppress inflammation, and G-CSF is currently being investigated as a potential therapeutic agent for patients with stroke or Alzheimer’s disease.”

In previous studies, Borlongan and his team showed that G-CSF can mobilize stem cells from bone marrow and induce them to home to and infiltrate injured tissues. While there, the cells promote neural cell self-repair. Cells from human umbilical cord blood also have the ability to suppress inflammation and promote cell growth.

“Our results showed that the combined therapy of hUBCs and G-CSF significantly reduced the TBI-induced loss of neuronal cells in the hippocampus,” said Borlongan. “Therapy with hUBCs and G-CSF alone or in combination produced beneficial results in animals with experimental TBI. G-CSF alone produced only short-lived benefits, while hUBCs alone afforded more robust and stable improvements. However, their combination offered the best motor improvement in the laboratory animals.”

“This outcome may indicate that the stem cells had more widespread biological action than the drug therapy,” said Paul R. Sanberg, distinguished professor at USF and principal investigator of the Department of Defense funded project. “Regardless, their combination had an apparent synergistic effect and resulted in the most effective amelioration of TBI-induced behavioral deficits.”

This particular study examined motor improvements or improvements in movement, but the USF group suggested that future combination therapy research should also include analysis of cognitive improvement in the laboratory animals with TBI.

In short, umbilical cord cell and growth factor treatments tested in animal models could offer hope for millions, including U.S. war veterans with traumatic brain injuries.

Post-script:  On Twitter, Alexey Bersenev made some very helpful observations about this paper.  In this paper, the authors used whole human umbilical cord blood.  They did not attempt to separate any of the different cell types from the cord blood.  Now when such whole blood is used, it is easy to assume that the stem cells in the blood that are doing the regenerative work.  However, as Alexey graciously pointed out, you cannot assume that the stem cells are responsible for the therapeutic effects for at least two main reasons:  1)  the number of stem cells in the cord blood is quite small relative to the other cells; 2) some of the non-stem cells in the blood turn out to have therapeutic effects.  See here and here.  I have seen some of these papers before, but I did not think much of them.  Therefore, until the cell populations in the umbilical cord blood are dissected out and studied, all we can say with any confidence is SOMETHING in the cord blood is conveying a therapeutic effect, but the identity of the therapeutic culprit remains unclear at this time.

A Link Between Stem Cells, Atherosclerosis, and Cholesterol


Researchers at the University of Buffalo have discovered that stem cells are involved in the inflammation that promotes atherosclerosis.

Atherosclerosis or hardening of the arteries occurs when fat, cholesterol, and other substances build up in the walls of arteries and form hard structures called plaques. With the passage of time, these plaques can grow and block the arteries, depriving tissues of oxygen and nutrition.

High serum cholesterol levels have been unequivocally linked to an increased risk of arteriosclerosis. However, the deposition of cholesterol and other molecules underneath the inner layer (intima) of arteries requires a phenomenon known as inflammation. Inflammation occurs in response to tissue damage and it involves the dilation of blood vessels, increased blood flow the damaged area, the recruitment of white blood cells to the area, and increased heart, volume, and pain at the area in question. Increased inflammation within blood vessels damages the intimal layer and allows the deposition of cholesterol and other molecules underneath it to form an atheroma or a plaque.

The stem cell link to atherosclerosis is that the bone marrow-based stem cells that make our blood cells (hematopoietic stem/progenitor cells or HSPCs) ramp up their production of white blood cells in response to increased serum cholesterol levels.

Thomas Cimato, assistant professor in the Department of Medicine in the UB School of Medicine and Biomedical Sciences, said of his publication, “Our research opens up a potential new approach to preventing heart attack and stroke, by focusing on interactions between cholesterol and the HSPCs. Cimto also suggested that these findings could lead to the development of a useful therapy in combination with statins, or a treatment in place of statins for those who cannot tolerate statins.

In Cimato’s study, high cholesterol levels were shown to cause increases in the levels of interleukin -17 (IL-17). IL-17 is a cytokine that recruits monocytes and neutrophils to the site of inflammation. IL-17 boosts levels of granulocyte colony stimulating factor (GCSF), which is a factor that induces the release of HSPCs from the bone marrow to the peripheral circulation.

Cimato also found that statin drugs reduce the number of HSPCs in circulation, but not all patients responded similarly to statins. “We’ve extrapolated to humans what other scientists previously found in mice about the interactions between LDL, cholesterol, and these HSPCs,” said Cimato.

In order to transport cholesterol through the bloodstream, cells must construct a vehicle into which the cholesterol is packaged. Cholesterol does not readily dissolve in water. Therefore, packaging cholesterol into lipoprotein particles allows for its transport around the cell. Cell use cholesterol to vary the fluidity of their membranes, and to synthesize steroid hormones. Once cholesterol is absorbed from the diet, the cells of the small intestine package cholesterol and fat into a particle known as a chylomicron.

chylomicron

Chylomicrons are released by the small intestinal cells and they travel to the liver. In the liver, chylomicrons are disassembled and the cholesterol is packaged into a particle known as a very-low density lipoprotein particle (VLDL). After its release and sojourning through the bloodstream, the VLDL looses some surface proteins and is depleted of its fat and becomes known as a low-density lipoprotein or LDL particle.  While these particles sojourn through the bloodstream, they release fat for tissues to use as an energy source.

LDL

LDL particles are gradually removed from circulation. If they build up to high concentrations, they can be taken up by a wandering white blood cell known as a macrophage. If these macrophages take up too much LDL, they can become a foam cell.  Foams cells can become lodged underneath the intimal layer of blood vessels when inflammation occurs inside blood vessels, and this is the cause of atherosclerosis.

Increased LDL levels in mice have been shown to stimulate the release of HSPCs from bone marrow and accelerate the differentiation of these cells into white blood cells (neutrophils and monocytes) that participate in inflammation.

Mice do not regulate their cholesterol levels in the same way humans do.  Cimato commented, “mice used for atherosclerosis studies have very low total cholesterol levels at baseline.  We feed then very high fat diets in order to study high cholesterol but it isn’t easy to interpret what the levels in mice will mean in humans and you don’t know if extrapolating to humans will be valid.”

Therefore, in order to properly model cholesterol regulation in their human subjects, Cimato had them take statins for a two-week period followed by one-month intervals when they were off the drugs.  “We modeled the mechanism of how LDL cholesterol affects stem cell mobilization in humans,” said Cimato.

The experiments showed that increased LDL levels tightly correlated with IL-17 levels.

IL-17 and cholesterol levels

Secondly, blood LDL levels also correlated with GCSF levels.

LDL levels and GCSF levels

Finally, increasing GCSF levels led to higher levels of circulating HSPCs.

CD34 cells and G-CSF levels

These circulating HSPCs increase the numbers of neutrophils, monocytes, and macrophages that are involved in the formation of plaque and atherosclerosis.

The next step is to determine if HSPCs, like LDL cholesterol levels are connected to stroke, cardiovascular disease and heart attacks.