Using CXCR4 to Make Stem Cells Stay Put: Regenerating Intervertebral Discs


The migration of several different types of stem cells is regulated by a receptor called “CXCR4” and the molecule that binds to this receptor, SDF-1. SDF-1 is a powerful summoner of white blood cells. During early development, SDF-1 mediates the migration of hematopoietic cells from fetal liver to bone marrow and plays a role in the formation of large blood vessels. During adult hood, SDF-1 plays an important role in making new blood vessels by recruiting endothelial progenitor cells (EPCs) from the bone marrow. Consequently, SDF-1 has a role in tumor metastasis where cancer cells that express the receptor CXCR4 are attracted to metastasis target tissues that release SDF-1. SDF-1 also attracts mesenchymal stem cells and helps them suppress the breakdown of bone.

Hopefully, I have convinced you that SDF-1 and its receptor CXC4 are important molecules. Can overexpression the CXCR4 receptor improve the retention of stem cells within an injured tissue?

Xiao-Tao Wu and Feng Wang from Zhongda Hospital in Nanjing, China and their colleagues have used this CXCR4 receptor/SDF-1 system to test this question in the damaged spinal cord.  This work was published in the journal DNA and Cell Biology (doi:10.1089/dna.2015.3118).

Isolated MSCs were treated with genetically engineered viruses to so that would overexpress the CXCR4 receptor. In order to track these cells under medical imaging scans, the MSCs were also labeled with superparamagnetic iron oxide (SPIO). Next, rabbits that had suffered injuries to their intervertebral discs that lie between the vertebrae were given infusions of these labeled, genetically engineered MSCs. Images of the spine were taken at 0, 8, and 16 weeks after the surgery. The degeneration of the damaged intervertebral discs were also evaluated by disc height (damaged, degenerating intervertebral discs tend to shrink and lose height).

The SPIO-labeled CXCR4-MSC could be detected within the intervertebral discs by MRI 16 weeks post-transplantation. The MSCs that had been engineered to overexpress CXCR4 showed better retention within the discs, relative to implanted MSCs that had not been engineered to overexpress CXCR4.

Did the implanted MSCs affect the integrity of the intervertebral discs? Indeed they did. Compared to the control group, loss of disc height was slowed in the animals that received the CXCR4-overexpressing MSCs. Also, the genetically engineered MSCs seemed to make more cartilage-specific materials, like the giant molecule aggrecan and type II collagen. There is a caveat here, since there is no indication that measured protein directly; only mRNAs. Until the quantities of these molecules can be directly shown to increase in the disc, the increases in these cartilage-building molecules can be said to be presumptive, but not proven.

From these experiments, it seems reasonable to conclude that CXCR4 overexpression promoted MSC retention within the damaged intervertebral discs and the increased stem cell retention enhanced stem cell-based disc regeneration. Therefore this SDF-1/CXCR4 signaling pathway might be a way to drive stem cell migration and infiltration within degenerated intervertebral discs.

Intravenous Administration of Lipitor-treated Stem Cells on the Heart


Hao Zhang and colleagues from the Chinese Academy of Medical Sciences and Peking Union Medical College have published a rather unusual experiment in the American Journal of Translational Research. This experiment, however, could have implications for stem cell therapy in heart attack patients.

When heart attack patients are treated with stem cells, they are either injected directly into the heart muscle or released into the heart through the coronary arteries by means of angioplasty. Injecting stem cells directly into the heart requires special equipment and training. Releasing cells into the coronary arteries causes most cells to end up in the lungs or other organs, and the retention of the stem cells is poor. Introducing cells by means intravenous administration would be supremely simple, but in animal experiments, intravenously administered stem cells almost never get to their target organ.

When the heart undergoes a heart attack, the damaged heart cells release a molecule called SDF1 or stromal cell-derived factor 1. SDF1 summons stem cells to the damaged areas by binding to the surfaces of stem cells and drawing them to the higher concentrations of SDF1. SDF1 binds to a receptor on the surfaces of stem cells called CXCR4. Unfortunately, when stem cells are administered intravenously to animals that have just experienced a heart attack, the stem cells do not have enough CXCR4 on their surfaces to properly respond to the SDF1 being secreted by the damaged heart.

Zhang and his colleagues capitalized on an observation made several years ago. When stem cells are exposed to statin drugs that are normally used to lower serum cholesterol levels, the stem cells increase the number of CXCR4 molecules on their surfaces. Statins have also been shown to increase stem cell survival once the cells get to the heart, but Zhang and his team wanted to know if pre-treating stem cells with statins could increase their migration to the damaged heart.

The Zhang group isolated mesenchymal stem cells from rat bone marrow and treated these cells with increasing concentrations of the drug Lipitor (atorvastatin). Indeed, increasing amounts of Lipitor increased the number of CXCR4 molecules on the surfaces of the mesenchymal stem cells (MSCs), This increase in CXCR4 molecules peaked at 24 hours, after which the number of receptors declined. These Lipitor-treated MSCs also migrated much more robustly in culture when treated with SDF1.

Next, Zhang’s group pre-treated MSCs with Lipitor and labeled them with an innocuous tracking molecule. 24 hours after giving some laboratory rats heart attacks, these MSCs were administered to the rats in their tail veins. Two other groups of similarly treated rats were given either MSCs that had not been pre-treated with Lipitor, or just buffer.

The Lipitor-treated MSCs were found in significantly higher quantities in the hearts of laboratory animals, relative to the other animals. Secondly, these Lipitor pre-treated MSCs cut the size of the heart scar in half, and there was also substantially less inflammation in hearts from animals treated with Lipitor pre-treated MSCs than the other groups. Heart function was also increased in the pre-treated group.

Live MSCs were observed in the hearts of the animals given Lipitor pre-treated MSCs. This is a remarkable finding, because most experiments have shown that MSCs administered to the heart after a heart attack ad usually dead within 21 day after administration. However the Lipitor pre-treated MSCs survive and flourish in the damaged heart, which suggests that SDF1 not only attracts stem cells but also increases their rates of survival.

This is a somewhat off-beat experiment at first glance, but if MSCs could be pre-treated with a drug like Lipitor and then administered to heart patients intravenously, they would survive in the heart, convey greater benefits, and their administration would be safer, and not require special equipment or training. With a little luck, this idea will reach human clinical trials in a few years; provided that further animal and cell culture studies confirm these results, elucidate the mechanism of SDF1-mediated survival, and show that such augmentation of function is also observed in human MSCs.

Bone Marrow Stem Cells Treat Chronic Pain


Nerve damage as a result of type 2 diabetes, surgical amputation, chemotherapy and other conditions can lead to chronic pain. Such chronic pain can resist painkiller medications and other treatments and is debilitating.

New studies from scientists at Duke University with mice have shown that injections of bone marrow-derived stem cells might be able to relieve this type of chronic, neuropathic pain. This study was recently published in the Journal of Clinical Investigation and might be the springboard for advanced cell-based therapies to treat chronic pain conditions, lower back pain and spinal cord injuries.

Ru-Rong Ji, professor of anesthesiology and neurobiology at the Duke School of Medicine and his team used bone marrow stromal cells (BMSCs) that were isolated from bone marrow aspirations. BMSCs have been shown in a variety of clinical trials and basic research experiments to produce an array of healing factors and can differentiate into many cell types of cells in the body. BMSCs are being tested in small-scale clinical studies with people who suffer from inflammatory bowel disease, heart damage and stroke. BMSCs might also be useful for treating pain, but it’s not clear how they work.

“Based on these new results, we have the know-how and we can further engineer and improve the cells to maximize their beneficial effects,” said Professor Ji. In his team’s study, stromal cells were used to treat mice with pain caused by nerve damage. The cells were delivered by means of lumbar puncture, which infused the BMSCs into the cerebrospinal fluid (CSF) that bathes the spinal cord.

cerebrospinal-fluid-brain-flow

Mice treated with the bone marrow stromal cells were much less sensitive to painful stimuli after their nerve injury in comparison with untreated mice.

“This analgesic effect was amazing,” Ji said. “Normally, if you give an analgesic, you see pain relief for a few hours, at most a few days. But with bone marrow stem cells, after a single injection we saw pain relief over four to five weeks.”

When the spinal cords of the treated animals were examined in detail, Ji and others observed that the injected stem cells had clustered together along the nerve cells in the spinal cord.

To understand how the stem cells alleviated pain, Ji and his coworkers measured levels of anti-inflammatory molecules that have been linked to pain suppression. One of these molecules in particular, TGF-β1, was present in higher amounts in the CSF of the stem cell-treated animals compared with the untreated animals.

Immune cells typically secrete TGF-β1, which is a small protein, and it is found at low concentrations throughout the body. According to Professor Ji, people with chronic pain have been shown to possess too little TGF-β1.

In the new study, when Ji and others chemically neutralized TGF-β1 in the stem cell-treated animals, the pain-killing benefit of the infused BMSCs was reversed. This suggests that the secretion of this protein by BMSCs was a major reason these are able to abate neuropathic pain. When Ji and his crew directly injected TGF-β1 into the CSF, it provides significant pain relief, but only for a few hours, according to Ji.

However, infused BMSCs, remain at the site of infusion for as long as three months after their administration. This is just the right length of time for the cells to persist, according to Ji, because if the stem cells permanently persisted in the CSF, they have an increased risk of becoming cancerous.

Even more significantly, infused BMSCs also migrate to the site of injury. The ability of these cells to migrate to the site of injury depends on a molecule secreted by the injured nerve cells called CXCL12 (which, incidentally, has also previously been linked to neuropathic pain). CXCL12 (also known as stromal cell-derived factor-1) acts as a homing signal, since BMSCs have on their cell surfaces, a receptor for CXCL12 called CXCR4, CXCL12 acts as a kind of stem cell attractant.

In the next set of experiments, Ji and his colleagues would like to find a way to make the stromal cells more efficient. “If we know TGF-β1 is important, we can find a way to produce more of it,” Ji said. Additionally, the cells may produce other pain-relieving molecules, and Ji’s group is working to identify those.

Remote Ischemic Conditioning Enhances Stem Cell Retention in the Heart


Stem cell administration to the heart after a heart attack is a difficult venture. Direct injection into the heart muscle is definitely the most sure-fire way to get stem cells into the heart tissue. However, direct injection requires that the physician crack the patient’s chest (thoracotomy), which is exquisitely unpleasant for the patient. Alternatively, there are devices that an deliver stem cell injections into the heart through the large veins in the legs, but these procedures require special equipment and lots of skills that your average cardiologist does not have. Another way is to administer stem cells through angioplasty. Using the same procedure as stent implantation, a delivery device is replaced at the site of heart damage through over-the-wire angioplasty technology, and the stem cells are delivered slowly and gradually through the coronary blood vessels. This does not require fancy equipment, and your average cardiologist could perform this technique pretty easily.

Problems exist with both procedures. Direct injection places cells and fluid into the heart wall and there is a risk of rupture. Likewise, with over-the-wire delivery of stem cells, there is the risk of clogging the coronary artery.

With both techniques, many stem cells end up in places other than the heart. In fact, the majority of the stem cells end up somewhere else – the lungs and liver mostly. Is there a better way?

Intravenous administration would be sweet, but that has been tried and the bottom line is that it bombed (Barbash et al., Circulation. 2003 19;108(7):863-8; Freyman et al., Eur Heart J. 2006 May;27(9):1114-22).

Well, some very enterprising scientists from China had an idea to get the intravenously administered stem cells to go to the heart and stay there. Bone marrow stem cells respond to a molecule called SDF1alpha (stromal cell derived factor-1alpha). On their cell surfaces, bone marrow cells have a receptor called CXCR4 which binds the SDF1alpha and bone marrow cells move towards higher and higher concentrations of SDF1alpha. Therefore, can you get the heart to make more SDF1alpha?

Sure. You can genetically engineer it to make more SDF1alpha. If you do that, the stem cells will pour out of the bone marrow and go to the heart and help fix it (Sundararaman S et al., Gene Ther. 2011 18(9):867-73). However, is there another way to get more SDF1alpha in the heart?

Yes there is. Let me introduce Remote Ischemic Conditioning or RIC. RIC increases the protection against injury that results from loss of blood flow to an organ. The way RIC works is that the blood supply to another organ is clamped so that this other organ is deprived of oxygen long enough to sound the alarm, but not long enough to do it serious damage. This deprivation of oxygen induces a flash of SDF1alpha production, which brings stem cells from bone marrow to the bloodstream and to the damaged organ.

Qin Jiang and colleagues from the Peking Union Medical College in Beijing, China used RIC in animals that had undergone a heart attack to determine if RIC could recruit more stem cells to the heart. Also, they administered bone marrow stem cells intravenously to see if RIC increased stem cell retention in the heart.

Jiang and others broke their laboratory rats into three groups (it gets a little complicated).

The first group was given heart attacks and then split into two subgroups. One subgroup received RIC and the second subgroup received surgery but no RIC.

The second group was given a heart attack and then split into six subgroups. Once subgroup was given RIC and intravenous bone marrow mesenchymal stem cells. the second received bone marrow mesenchymal stem cells by no RIC, only the incision, the third subgroup only received intravenous mesenchymal stem cells, the fourth group received RIC and intravenous saline, the fifth subgroup received no RIC, only an incision and intravenous saline, and the sixth subgroup received only intravenous saline.

The third group was given heart attacks and then split into two groups, one of which received RIC, intravenous mesenchymal stem cells and intravenous antibodies against CXCR4, and the other of which received RIC, mesenchymal stem cells and an antibody against nothing in particular.

The results showed that RIC GREATLY increased the amount of SDF1alpha in the heart. There was simply no getting around this. At 1 hour after RIC, SDF1alpha and VEGF (vascular endothelial growth factor) levels were up, but these levels decreased by 3 hours and back to normal by 6 hours after RIC.

Did these increased SDF1alpha levels increase stem cell retention? Oh yes!! The RIC-treated rats had almost twice the number of stem cells in their hearts than the animals that did not have RIC. Did this make a functional difference? Again, yes! The RIC-treated animals had hearts that functioned more normally (relatively speaking) than hearts from the non-RIC-treated animals.

The third experiment was even more informative, since the co-administration of the CXCR4 antibody abrogated the response induced by RIC. This demonstrates that effects of RIC are mediated by the SDF1alpha/CXCR4 axis and blocking this signaling axis prevented any benefits from RIC.

This paper is short, but very informative. It suggests that a relatively simple procedure like RIC could potentially improve the clinical efficacy of stem cell treatments. If this can be shown to work in larger animals, then clinical trials might be warranted. In fact clinical trials are presently underway in which SDF1alpha is being engineered into the heart to treat heart attack patients (see Hajjar RJ, Hulot JS. Circ Res. 2013 Mar 1;112(5):746-7).

Engineered Mesenchymal Stem Cells Make Blood Vessels that Help Heal Ailing Hearts


Another term for a heart attack is a myocardial infarction (MI). A heart attack or an MI occurs when the blood supply to the heart that flows through coronary blood vessels is interrupted. The interruption of blood flow deprives the heart of nourishment and oxygen, and the downstream blood vessels and heart muscle die as a result. The decrease in blood vessel density after a MI can increase cell death, which increases the amount of cell death and the size of the heart scar. Therefore, growing more blood vessels in the heart after a heart attack, which is known as therapeutic angiogenesis, is a potentially strategy in treating an MI (see Ziebart T, et al., (2008) Circ Res 103: 1327–1334)..

To this end, a few clinical trials have attempted to used stem cells that can make blood vessels to reverse heart damage caused by an MI (see Ripa RS, et al. (2007) Circulation 116: I24–I30 and Schachinger V, et al. (2006) N Engl J Med 355: 1210–21).

Among those therapeutic agents for heart attack patients, mesenchymal stem cells (MSCs) are considered excellent candidates. MSCs have the ability to differentiate into smooth muscle, or blood vessels, which means that they can help revascularize the heart after a MI. The problem with MSCs is their tendency to die off rapidly after transplantation into the heart after a heart attack (see Ziegelhoeffer T, et al. (2004) Circ Res 94: 230–38 & O’Neill TT, et al., Circ Res 97: 1027–35; & Perry TE, et al. (2009) Cardiovasc Res 84: 317–25).

To fix this problem, MSCs can be either preconditioned before implantation (see previous posts) or genetically engineered to withstand the hostile conditions inside the heart after a heart attack.

Previously, Muhammad Ashraf and Yigang Wang from the University of Cincinnati genetically engineered MSCs to express a surface protein called CXCR4.  CXC4R is the receptor for a chemokine known as CXCL12/SDF-1.  SDF-1 is a rather potent stem cell recruitment molecule.

When transplanted into the hearts of rodents that had just experienced a heart attack, MSCs that expressed CXCR4 showed increased mobilization and engraftment into the damaged areas of the heart. Also, the pumping abilities of the heart regions into which the MSC-CXCR4s were infused increased, and the MSC-CXCR4 cells cranked up their secretion of blood vessel-inducing growth factors (vascular endothelial growth factor-A or VEGF-A), This led to increased formation of new blood vessels and a decrease in the early signs of left ventricular remodeling (see Zhang D, et al. (2010) Am J Physiol Heart Circ Physiol 299: H1339– H1347; Huang W, et al. (2010) J Mol Cell Cardiol 48: 702–712; &.Zhang D, et al. (2008) J Mol Cell Cardiol 44: 281–292). While these papers show truly stunning results, it was still, even after all this work, unclear if the MSCs were actually differentiating into blood vessel cells and making blood vessels.

To nail this down, Wang and his group used a clever little technique. They engineered MSCs to express CXCR4 and the viral TK gene. TK stands for “thymidine kinase,” which is an enzyme involved in nucleotide synthesis from a virus. The TK enzyme is not found in human cells, and is therefore a target for antiviral drugs. If treated with antiviral drugs that target the TK enzyme, only cells with the TK gene will be killed.

When Wang and his group used their CXCR4-engineered MSCs to treat the heart of mice that had recently suffered a heart attack, they found that their hearts improved and that these same heart were covered with new blood vessels. However, when this experiment was repeated with CXCR4-MSCs that also had the TK gene, Wang his co-workers fed the mice a drug called ganciclovir, which kills only those cells that possess the TK gene. In these mice, their heart failed to improve and also were completely devoid of the new blood vessels.

This paper nicely shows that without viable MSCs, no new blood vessels were made. This strongly suggests that the engineered MSCs are differentiating into blood vessel cells and making new blood vessels, which helps the heart recover from the heart attack and shrinks the size of the dead area of the heart.

What are the implications for human clinical trial\? This is difficult to say. Before clinical trials with genetically engineered cells are approved those cells will need to go through piles of safety tests before they can be used in clinical trials. Once that hurdle is passed, then they can be used in human clinical trials, and they will certainly prove efficacious for human patients.