Dosing Recent Heart Attack Patients with G-CSF Doesn’t Seem To Work


Granulocyte-Colony Stimulating Factor (G-CSF)is a small protein that stimulates the bone marrow to produce more of a particular class of white blood cells called granulocytes and release them into the bloodstream. A commercially available version of G-CSF called Filgrastim (Neupogen) is used to boost the immune system of cancer patients whose immune systems have taken a beating from chemotherapy.

Because several clinical trials have shown that implanting bone marrow mononuclear fractions into the hearts of heart attack patients can improve the heart health of some heart attack patients, clinicians have supposed that injecting heart attack patients with drugs like filgrastim, which moves many bone marrow-derived cells into the bloodstream might also provide some relief for heart attack patients.

Nice idea, but it does not seem to work. Two clinical trials, STEMMI and REVIVAL-2, have given G-CSF to heart attack patients at different times after their heart attacks. Unfortunately both studies have failed to show a difference from the placebo.

In the REVIVAL-2 study, 114 patients were enrolled, and 56 received 10 micrograms per kilogram body weight G-CSF for five days, and the remaining patients received a placebo treatment.  G-CSF and the placebo were administered to patients five days after the hearts were successfully reperfused by percutaneous coronary intervention (this is a fancy way of saying stenting).  This study was double-blinded, placebo-controlled and well designed.  Unfortunately, when patients were studied seven years after treatment, there were no statistically significant differences between the treatment and the placebo groups when it came to the number of deaths, heart attacks, and strokes.  Thus, the authors conclude that G-CSF administration did not improve clinical outcomes for patients who had a heart attack (see Birgit Steppich, et al, Atherosclerosis and Ischemic Disease 115.4, 2016).

A second clinical trial, the STEMMI trial, was a prospective trial in which G-CSF treatment was begun 10-65 hours after reperfusion.  Here again, there were no structural differences between the placebo group and the G-CSF-treated group six months after treatment and a five-year follow-up analysis of 74 patients revealed no differences in the occurrence of major cardiovascular incidents between the two treatment groups (R.S. Ripa, and others, Circulation 2006; 113: 1983-1992).

The STEM-AMI clinical trial also showed no differences in clinical outcomes after G-CSF treatment as compared to placebo in 60 patients after three years (F. Achilli, and others, Heart 2014, 100: 574-581).

Why does this technique fail?  It is possible that the white blood cells that are mobilized by G-CSF are low-quality and do not express particular genes.  A study in rats has shown that G-CSF infusion increases the number of progenitor cells in the bloodstream, but fails to increase the number of progenitor cells in the heart after a heart attack (D. Sato, and others, Experimental Clinical Cardiology, 2012; 17:83-88).  In order for cells to home to the infarcted heart, they must express particular proteins on their surfaces.  For example, the cell surface protein CXCR4 is known to play an integral role in progenitor cell homing, along with several other proteins (see Taghavi and George, American Journal of Translational Research 2013; 5:404-411; Shah and Shalia, Stem Cells International 2011;2011:536758; Zaruba and Franz, Expert Opinion in Biological Therapy 2010; 10:321-335).  Indeed, Stein and others have shown that progenitor cells mobilized with G-CSF in human patients lack CXCR4 and other cell adhesion proteins thought to play a role in homing to the infarcted heart (Thromb Haemost 2010;103:638-643).

Therefore, even though all of these studies have not uncovered a risk in G-CSF treatment, the consensus of the data seems to be there no clinical benefit is conferred by treating heart attack patients with G-CSF.

Stem Cells from Bone Marrow Help Heal Hard-to-Heal Bone Fractures


A new study that has appeared in the journal STEM CELLS Translational Medicine demonstrates the potential of a subset of stem cells called CD34+ in treating stubborn bone fractures that prove hard to heal.

The body has mechanisms for the repair of broken bones. Consequently, most patients recover from broken bones with little or no complication. However, up to 10 percent of all fracture patients experience fractures that refuse to heal. Such heard to heal fractures can lead to several debilitating side effects that include infection and bone loss, and the healing of hard to heal fractures often requires extensive treatment that includes multiple operations and prolonged hospitalization as well as long-term disability.

Regenerating broken bones with stem cells could offer an answer to this medical conundrum. Adult human peripheral blood CD34+ cells have been shown to contain a robust population of endothelial progenitor cells (EPCs) and hematopoietic stem cells, which give rise to all types of blood cells. These two types of stem cells might be good candidates for this therapy.

However, while other types of stem cells have been tested for their bone regeneration potential, the ability of CD34+ stem cells to facilitate bone healing has not been examined; that is until now. A phase I/II clinical study that evaluated the capacity of CD34+ to stimulate bone regeneration was published in the current edition of STEM CELLS Translational Medicine. This study was conducted by researchers at Kobe University Graduate School of Medicine, led by Tomoyuki Matsumoto, M.D., and Ryosuke Kuroda, M.D., members of the university’s department of orthopedic surgery and its Institute of Biomedical Research and Innovation (IBRI).

Matsumoto’s and Kuroda’s study was designed to evaluate the safety, feasibility and efficacy of autologous and G-CSF-mobilized CD34+cells in patients with non-healing leg bone breaks that had not healed in nine months. Seven patients were treated with CD34+ stem cells after receiving bone grafts.

In case you were wondering, G-CSF is a drug that releases stem cells from the bone marrow into the blood. It is given by injection or intravenously, and works rather well to mobilize bone marrow stem cells into the peripheral circulation.  It has clinical uses for patients recovering from chemotherapy.  Filgrastim (Neupogen) and PEG-filgrastim (Neulasta) are two commercially-available forms of recombinant G-CSF.

“Bone union was successfully achieved in every case, confirmed as early as 16.4 weeks on average after treatment,” Dr. Kuroda said.

Dr. Matsumoto added, “Neither deaths nor life-threatening adverse events were observed during the one year follow-up after the cell therapy. These results suggest feasibility, safety and potential effectiveness of CD34+ cell therapy in patients with nonunion.”

Atsuhiko Kawamoto, MD, Ph.D., a collaborator in IBRI, said, “Our team has been conducting translational research of CD34+ cell-based vascular regeneration therapy mainly in cardiovascular diseases. This promising outcome in bone fracture opens a new gate of the bone marrow-derived stem cell application to other fields of medicine.”

Although the study documents a relatively small number of patients, the results suggest the feasibility, safety and potential effectiveness of CD34+ cell therapy in patients with non-healing breaks,” said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine.

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.”