Stem Cell Factor Delivery into Heart Muscle After Heart Attack May Enhance Cardiac Repair and Reverse Injury


Stem Cell Factor or SCF is a small peptide that circulates throughout the bloodstream and eventually finds its way to the bone marrow where it summons bone marrow-based stem cells to the sight of injury for tissue repair purposes. Unfortunately, it takes injured tissues time to express SCF at high enough levels to recruit bone marrow stem cells to come and accelerate tissue healing. This is particularly the case in the heart after a heart attack. For this reason, scientists are trying to find new and better ways to increase SCF production in the damaged heart.

To that end, cardiologists at the Icahn School of Medicine at Mount Sinai have discovered that delivering SCF directly to damaged heart muscle after a heart attack seems to augment heart muscle repair and regenerate injured tissue.

“Our discoveries offer insight into the power of stem cells to regenerate damaged muscle after a heart attack,” said lead study author Kenneth Fish, Director of the Cardiology Laboratory for Translational Research, Cardiovascular Research Center, Mount Sinai Heart, Icahn School of Medicine at Mount Sinai.

In this study, Fish and his colleagues used gene transfer to administer SCF to the heart shortly after inducing heart attacks in a pig model system in order to test its regenerative repair response. Fish and his coworkers developed a novel SCF gene transfer delivery system that stimulated the recruitment and expansion of adult cardiac stem cells directly to injury sites that reversed heart attack damage. In addition, the gene therapy improved cardiac function, decreased the death of heart muscle cells, increased regeneration of heart tissue blood vessels, and reduced the formation of heart tissue scarring.

“It is clear that the expression of the stem cell factor gene results in the generation of specific signals to neighboring cells in the damaged heart resulting in improved outcomes at the molecular, cellular, and organ level,” says Roger J. Haijar, senior study author and Director of the Cardiovascular Research Center at Mount Sinai. “Thus, while still in the early stages of investigation, there is evidence that recruiting this small group of stem cells to the heart could be the basis of novel therapies for halting the clinical deterioration in patients with advanced heart failure.”

The cell surface receptor for SCF is the c-Kit protein, and cells that possess the c-Kit protein are called c-Kit+ cells. c-Kit+ cells not only respond to SCF, but serve as resident cardiac stem cells that naturally increase in numbers after a heart attack and through cell proliferation are directly involved in cardiac repair.

To date, there is a great need for new interventional strategies for cardiomyopathy to prevent the progression of this disease to heart failure. Heart disease is the number one cause of death in the United States, with cardiomyopathy or an enlarged heart from heart attack or poor blood supply due to clogged arteries being the most common cause of the condition. Cardiomyopathy also causes a loss of heart muscle cells and changes in heart shape, which lead to the heart’s decreased pumping efficiency.

“Our study shows our SCF gene transfer strategy can mobilize a promising adult stem cell type to the damaged region of the heart to improve cardiac pumping function and reduce myocardial infarction sizes resulting in improved cardiac performance and potentially increase long-term survival and improve quality of life in patients at risk of progressing to heart failure,” says Dr. Fish.

“This study adds to the emerging evidence that a small population of adult stem cells can be recruited to the damaged areas of the heart and improve clinical outcomes,” says Dr. Hajjar.

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.

The Nooks and Crannies in Bone Marrow that Nurture Stem Cells


Stems cells in our bodies often require a specific environment to maximize their survival and efficiency. These specialized locations that nurture stem cells is called a stem cell niche. Finding the right niche for a stem cell population can go a long way toward growing more stem cells in culture and increasing their potency.

To that end, a recent discovery has identified the distinct niches that exist in bone marrow for hematopoietic stem cells (HSCs), which form the blood cells in our bodies.

A research team from Washington university School of Medicine in St. Louis has shown that stem niches in bone marrow can be targeted, which may potentially improve bone marrow transplants and cancer chemotherapy. Drugs that support particular niches could encourage stem cells to establish themselves in the bone marrow, which would greatly increase the success rate of bone marrow transplants. Alternatively, tumor cells are known to hide in stem cell niches, and if drugs could disrupt such niches, then the tumor cells would be driven from the niches and become more susceptible to chemotherapeutic agents.

Daniel Link, the Alan A. and Edith L. Wolff Professor of Medicine at Washington University, said, “Our results offer hope for targeting these niches to treat specific cancers or to impress the success of stem cell transplants. Already, we and others are leading clinical trials to evaluate whether it is possible to disrupt these niches in patients with leukemia or multiple myeloma.”

Working in mice, Link and his colleagues deleted a gene called CXCL12, only in “candidate niche stromal cell populations.” CXCL12 which encodes a receptor protein known to be crucial for maintaining HSC function, including retaining HSCs in the bone marrow, controlling  HSCs activity, and repopulating the bone marrow with HSCs after injury.

CXCL12 crystal structure
CXCL12 crystal structure

CXCL12 signaling pathways

In bone marrow, HSCs are surrounded by a whole host of cells, and it is difficult to precisely identify which type of cells serve as the niche cells. These bone marrow cells are known collectively as “stroma,” but there are several different types of cells in stroma. Cells that have been implicated in the HSC niche include endosteal osteoblasts (osteoblasts are bone-making cells and the endosteum in the layer of connective tissue that lines the inner cavity of the bone), perivascular stromal cells (cells that hang out around blood vessels), CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin–positive mesenchymal progenitors. Basically, there are a lot of cells in the stroma and figuring out which one is the HSC niche is a big deal.

bone marrow stromal cells

When HSCs divide, they form two cells, one of which replaces the HSC that just divided and a new cells called a hematopoietic progenitor cell (HPC), which can divide and differentiate into either a lymphoid progenitor or a myeloid progenitor. The lymphoid progenitor differentiates into either a B or T lymphocyte and the myeloid progenitor differentiates into a red blood cell, or other types of white blood cells (neutrophil, basophil, macrophage, platelet or eosinophil). As the cells become more differentiated, they lose their capacity to divide.

HSC differentiation

Deleting CXCL12 from mineralizing osteoblasts (bone making cells) did nothing to the HSCs or those cells that form lymphocytes (lymphoid progenitors). Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, causes mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the bloodstream, and loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from blood vessel cells causes a modest loss of long-term repopulating activity. Deletion of Cxcl12 from nestin-negative mesenchymal progenitors causes a marked loss of HSCs, long-term repopulating activity, and lymphoid progenitors. All of these data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow. Also, the expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Link summarized his results this way: “What we found was rather surprising. There’s not just one niche for developing blood cells in the bone marrow. There’s a distinct niche for stem cells, which have the ability to become any blood cell in the body, and a separate niche for infection-fighting cells that are destined to become T cells and B cells.”

These data provide the foundation for future investigations whether disrupting these niches can improve the effectiveness of cancer chemotherapy.

In a phase 2 study at Washington University, led by oncologist Geoffrey Uy, assistant professor of medicine, Link and his team are evaluating whether the drug G-CSF (granulocyte colony stimulating growth factor) can alter the stem cell niche in patients with acute lymphoblastic leukemia and whose disease is resistant to chemotherapy or has recurred. The FDA approved this drug more than 20 years ago to stimulate the production of white blood cells in patients undergoing chemotherapy, who have often weakened immune systems and are prone to infections.

Uy and his colleagues want to evaluate G-CSF if it is given prior to chemotherapy. Patients enrolled at the Siteman Cancer Center will receive G-CSF for five days before starting chemotherapy, and the investigators will determine whether it can disrupt the protective environment of the bone marrow and make cancer cells more sensitive to chemotherapy.

This trial is ongoing, and the results are not yet in, but Link’s work has received a welcome corroboration of his work. A companion paper was published in the same issue of Nature by Sean Morrison, the director of the Children’s Medical Center Research Institute at the University of Texas Southwestern Medical Center in Dallas. Morrison and his team used similar methods as Link and his colleagues and came to very similar conclusions.

Link said, “There’s a lot of interest right now in trying to understand these niches. Both of these studies add new information that will be important as we move forward. Next, we hope to understand how stem cells niches can be manipulated to help patients undergoing stem cell transplants.”

Rejuvenating Aged Stem Cells With a Fountain-Of-Youth Cocktail


Stem cell researchers from the laboratory of Ren-Ke Li at the University of Toronto have discovered a cocktail that can kick old, lagging stem cells in the backside and renew their regenerative capacities.

Donated bone marrow stem cells are transplanted into patients with leukemia, or diseases that compromise bone marrow function. Unfortunately, even though such therapies save hundreds to thousands of lives every year, some of these patients die or become horribly ill because the patient rejects some of the cells in the donated bone marrow. To reduce the risk of bone marrow rejection, stem cells treatments have used stem cells from the patient’s own body. Unfortunately, such a strategy is unusable in older patients, since their stem cell function has been vitiated by the ravages of age. If there is a way to beef up the stem cell function of an older patient, why then, this protocol would definitely be preferred.

Ren-Ke Li, professor in the Division of Cardiovascular Surgery and a member of the Institute for Biomaterials and Biomedical Engineering at the University of Toronto, Canada and his colleague Milica Radisic, an associate professor of chemical engineering have designed a unique micro-environment that allows heart tissue to grow from stem cells donated by elderly patients.

This micro-environment utilizes a porous scaffold made of collagen (the protein found in scar tissue), and embedded in this scaffold are two growth factors (vascular endothelial growth factor and basic fibroblast growth factor). Radisic and Li and their co-worked seeded these scaffolds with stem cells taken from younger (~50 years old) and older donors (~75 years old) and then used them to repair the left ventricles of rats with damaged hearts.

The scaffolds without growth factors and seeded with stem cells from older donors did not repair the hearts very well, but those scaffolds without growth factors and seeded with stem cells from younger donors did a good job of repairing the hearts. When the scaffolds impregnated with growth factors were seeded with stem cells from older donors, the patches did a much better job of repairing the hearts; they did as good a job of facilitating heart repair and those scaffolds seeded with stem cells from younger patients.

Patch Morphology 28 Days After Implantation In Vivo(A) Representative images of rat hearts show the outer border of the patches depicted by the yellow dotted line. (B) Patch area was quantified using computerized planimetry. The patch area increased in all groups from the original size of 39 mm2(red dotted line) at the time of SVR. Patch area in the Old group was significantly larger after implantation than that in the other groups. The addition of cytokines significantly prevented patch expansion. (C) Representative images of heart slices stained with Masson's trichrome. Arrows indicate patch thickness. (D) Patch thickness was quantified using computerized planimetry. The patches in the Old group were significantly thinner than patches in the Young and Young + GF groups. Cytokine enhancement did not significantly increase patch thickness for old or young cells. *p < 0.05, **p < 0.01 vs. Old; Old n = 5, Young n = 8, Old + GF n = 6, Young + GF n = 8. GF = growth factor.
Patch Morphology 28 Days After Implantation In Vivo(A) Representative images of rat hearts show the outer border of the patches depicted by the yellow dotted line. (B) Patch area was quantified using computerized planimetry. The patch area increased in all groups from the original size of 39 mm2(red dotted line) at the time of SVR. Patch area in the Old group was significantly larger after implantation than that in the other groups. The addition of cytokines significantly prevented patch expansion. (C) Representative images of heart slices stained with Masson’s trichrome. Arrows indicate patch thickness. (D) Patch thickness was quantified using computerized planimetry. The patches in the Old group were significantly thinner than patches in the Young and Young + GF groups. Cytokine enhancement did not significantly increase patch thickness for old or young cells. *p < 0.05, **p < 0.01 vs. Old; Old n = 5, Young n = 8, Old + GF n = 6, Young + GF n = 8. GF = growth factor.

When Li and his team tracked the molecular changes in the stem cells grown on the scaffolds, they found that these cells acted like younger stem cells. In Li’ words: “We saw certain aging factors turned off.” The levels of two molecules in particular, p16 and RGN were reduced in the older stem cells grown on the growth factor-containing scaffolds, which turned back the clock in these cells and returned them to a more robust and healthy state.

Li and Radisic hope to experiment with their micro-environment in order to make it as effective as possible. According to Li, “We can create much better tissues which can then be used to repair defects such as aneurysms.” Li also thinks that these cells could be used to repair the heart after a heart attack.

See Kai Kang, et al., Aged Human Cells Rejuvenated by Cytokine Enhancement of Biomaterials for Surgical Ventricular Restoration,” Journal of the American College of Cardiology 2012 60(21): 2237 DOI: 10.1016/j.jacc.2012.08.985.