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.
News from the University of Pennsylvania reports a new method that involves the use of fat to help heal skin without the formation of scar tissue. This work comes from the Perelman School of Medicine at the University of Pennsylvania, and it is the result of a large-scale, multi-year study that collaborated with the Plikus Laboratory for Developmental and Regenerative Biology at the University of California, Irvine. Their findings were published online in the journal Science on January 5th, 2017.
A fancy name for fat is “adipose tissue.” Adipose tissue is actually a rather complicated pastiche of different cell types. Specialized cells in adipose tissue that stores fat are called “adipocytes,” but they are more colloquially called fat cells. Fat cells are normally found in the skin, but when wounds in the skin heal and form, those underlying population of fat cells are lost. In skin tissue that is undergoing the process of healing, the most common cell types are known as “myofibroblasts.” Myofibroblasts are large cells with ruffled membranes, that are kind of a cross between smooth muscle cells and fibroblasts. They have the ability to contract like smooth muscle cells when exposed to molecules that induce smooth muscle to contract, such as angiotensin II or epinephrine. Fibroblasts, which are numerous throughout the skin and other organs, can readily differentiate into myofibroblasts, as can stellate cells found in liver or the pancreas, some smooth muscle cells, progenitor cells in stromal tissue, epithelial cells, or circulating progenitor cells (see B. Hinz, et al, The myofibroblast: one function, multiple origins, Am J Pathol. 2007 Jun;170(6):1807-16). Once it forms, scar tissue also does not properly form any hair follicles and this can give it a rather odd appearance relative to the rest of the skin. The Perelman researchers designed a new strategy to limit scar formation during healing by converting wound-based myofibroblasts into fat cells, which prevents the formation of scarring.
“Essentially, we can manipulate wound healing so that it leads to skin regeneration rather than scarring,” said George Cotsarelis, MD, the chair of the Department of Dermatology and the Milton Bixler Hartzell Professor of Dermatology at Penn, and the principal investigator of this project. “The secret is to regenerate hair follicles first. After that, the fat will regenerate in response to the signals from those follicles.”
Cotsarelis and his colleagues showed that the formation of fat in the skin and hair follicles are separate developmental events, but they are, nevertheless, linked. Hair follicles form first, and the factors required to induce hair follicle formation that are produced by the regenerating hair follicle can also convert surrounding myofibroblasts into fat cells instead of a scar. This underlying fat does not form without the formation of these new hair follicles. These new fat cells are indistinguishable from pre-existing skin-based fat cells that give the healed wound a natural look instead of leaving a scar. Cotsarelis and his gang discovered that a factor secreted by hair follicles called Bone Morphogenetic Protein (BMP) instructs the myofibroblasts to become fat. This single finding represents a tectonic shift on our understanding of myofibroblasts.
“Typically, myofibroblasts were thought to be incapable of becoming a different type of cell,” Cotsarelis said. “But our work shows we have the ability to influence these cells, and that they can be efficiently and stably converted into adipocytes.” This was shown in both the mouse and in human keloid cells grown in culture.
“The findings show we have a window of opportunity after wounding to influence the tissue to regenerate rather than scar,” said the study’s lead author Maksim Plikus, PhD, an assistant professor of Developmental and Cell Biology at the University of California, Irvine. Plikus began this research as a postdoctoral fellow in the Cotsarelis Laboratory at Penn, and the two institutions have continued to collaborate.
These new findings might very well revolutionize dematological wound treatments. These data might be useful for developing therapies that drive myofibroblasts to differentiate into adipocytes that can help wounds heal without scarring.
As Cotsarelis put it: “It’s highly desirable from a clinical standpoint, but right now it’s an unmet need.”
However, wound treatments are not the only use for this work. Fat cell loss is a common complication of other clinical conditions. HIV treatments, cancer, scleroderma, are just a few of the diseases that can cause wasting and drastic weight loss. Also, because fat cells are also lost naturally because of the aging process, especially in the face, which leads to permanent, deep wrinkles, something that available anti-aging treatments cannot satisfactorily address.
“Our findings can potentially move us toward a new strategy to regenerate adipocytes in wrinkled skin, which could lead us to brand new anti-aging treatments,” Cotsarelis said.
The Cotsarelis Lab is now examining how hair follicle regeneration can promote skin regeneration. The Plikus Laboratory would like to know more about the role of BMP in wound healing and are conducting further studies with using human cells and human scar tissue.
Severe wounds are typically treated with full thickness skin grafts. Some new work by researchers from Michigan Tech and the First Affiliated Hospital of Sun Yat Sen University in Guangzhou, China might provide a way to use a patient’s own stem cells to make split thickness skin grafts (STSG). If this technique pans out, it would eliminate the needs for donors and could work well for large or complicated injury sites.
This work made new engineered tissues were able to capitalize on the body’s natural healing power. Dr. Feng Zhao at Michigan Tech and her Chinese colleagues used specially engineered skin that was “prevascularized, which is to say that Zhao and other designed it so that it could grow its own veins, capillaries and lymphatic channels.
This innovation is a very important one because on of the main reasons grafted tissues or implanted fabricated tissues fail to integrate into the recipient’s body is that the grafted tissue lacks proper vascular support. This leads to a condition called graft ischemia. Therefore, getting the skin to form its own vasculature is vital for the success of STSG.
STSG is a rather versatile procedure that can be used under unfavorable conditions, as in the case of patients who have a wound that has been infected, or in cases where the graft site possess less vasculature, where the chances of a full thickness skin graft successfully integrating would be rather low. Unfortunately, STSGs are more fragile than full thickness skin grafts and can contract significantly during the healing process.
In order to solve the problem of graft contraction and poor vascularization, Zhao and others grew sheets of human mesenchymal stem cells (MSCs) and mixed in with those MSCs, human umbilical cord vascular endothelial cells or HUVECs. HUVECs readily form blood vessels when induced, and growing mesenchymal stem cells tend to synthesize the right cocktail of factors to induce HUVECs to form blood vessels. Therefore this type of skin is truly poised to form its own vasculature and is rightly designated as “prevascularized” tissue.
Zhao and others tested their MSC/HUVEC sheets on the tails of mice that had lost some of their skin because of burns. The prevascularized MSC/HUVEC sheets significantly outperformed MSC-only sheets when it came to repairing the skin of these laboratory mice.
When implanted, the MSC/HUVEC sheets produced less contracted and puckered skin, lower amounts of inflammation, a thinner outer skin (epidermal) thickness along with more robust blood microcirculation in the skin tissue. And if that wasn’t enough, the MSC/HUVEC sheets also preserved skin-specific features like hair follicles and oil glands.
The success of the mixed MSC/HUVEC cell sheets was almost certainly due to the elevated levels of growth factors and small, signaling proteins called cytokines in the prevascularized stem cell sheets that stimulated significant healing in surrounding tissue. The greatest challenge regarding this method is that both STSG and the stem cell sheets are fragile and difficult to harvest.
An important next step in this research is to improve the mechanical properties of the cell sheets and devise new techniques to harvest these cells more easily.
According to Dr. Zhao: “The engineered stem cell sheet will overcome the limitation of current treatments for extensive and severe wounds, such as for acute burn injuries, and significantly improve the quality of life for patients suffering from burns.”
This paper can be found here: Lei Chen et al., “Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Re-pair,” Theranostics, October 2016 DOI: 10.7150/ thno.17031.
As we age, the capacity of our stem cells to heal and replace damaged cells and tissues decline. This age-associated decrease in adult stem cell function seems to be a major contributor to the physiological decline during aging. A new paper, by Efstathios Gonos and his colleagues at the National Hellenic Research Foundation in Athens, Greece gives one possible technique that might improve the function of stem cells in an aging body.
Cells contain a multiprotein complex called the “proteasome” that degrades unneeded or defective proteins. The proteasome controls protein half-lives, function, and the protein composition of the cell. Functional failure of the proteasome has been linked to various biological phenomena including senescence and aging. The role of the proteasome in stem cells aging, however has received little attention to date.
Gonos and his coworkers used mesenchymal stem cells from umbilical cord Wharton’s Jelly and human fat. Because they were able to compare the proteasome activity in very young and aged stem cells, Gonos and others discovered a significant age-related decline in proteasome content and activity between these two types of stem cells. The proteasome from Warton’s Jelly mesenchymal stem cells were consistently more active and displayed more normal function and activity than those from human fat. In fact, not only were the protease activities of the proteasomes from the aging stem cells decreased, but they also displayed structural alterations.
These differences in proteasomal activity were not only reproducible, but when the proteasome of young stem cells were compromised, the “stemness,” or capacity of the stem cells to act as undifferentiated cells, was negatively affected.
Even more surprisingly, once after mesenchymal stem cells from human donors lost their ability to proliferate and act as stem cells (their stemness, that is) their decline could be counteracted by artificially activating their proteasomes. Activating the proteasome seems to help the cell “clean house,” get rid of junk proteins, and rejuvenate themselves.
Gonos and his team found that the stem cell-specific protein, Oct4, binds to the promoter region of the genes that encode the β2 and β5 proteasome subunits. Oct4 might very well regulate the expression of these proteasome-specific genes.
From this paper, it seems that a better understanding the mechanisms regulating protein turnover in stem cells might bring forth a way to stem cell-based interventions that can improve health during old age and lifespan.
Producing dopamine-making neurons from stem cells for transplantation into Parkinson’s disease patients remains challenging. Differentiating stem cells into dopaminergic neurons is not as efficient a process as we would like it to be. While several laboratories have managed to make pretty good batches of dopaminergic neurons, reliably producing large and pure batches of dopamine-making neurons from pluripotent stem cells is still somewhat problematic. Secondly, transplanting dopamine-making neurons into either the midbrain or the striatum of the brain represents another patch of problems because the production of too much dopamine can cause unwanted, uncontrollable movements. Preclinical assessments of stem cell-derived dopamine neurons in laboratory animals have produced positive, but highly varied results, even though the transplanted cells are very similar at the time of transplantation.
“This has been frustrating and puzzling, and has significantly delayed the establishment of clinical cell production protocols,” said Malin Parmar, who led the study at Lund University.
To address this issue, Parmar and his colleagues used modern global gene expression studies to gain a better understand the molecular changes that drive the differentiation of stem cells into dopamine-making neurons. Parmar conducted these experiments in collaboration with a team of scientists at Karolinska Institute. In their paper, which appeared in the journal Cell Stem Cell, Parmar and his colleagues used single-cell RNA seq to construct the neuronal development of dopaminergic neurons.
These neurons are characterized by the expression of a gene called LMX1a. However, it turns out that LMX1a-expressing neurons includes not only midbrain dopaminergic neurons (see below at the substantia nigra), but also subthalamic nuclear neurons.
These findings reveal that markers used to identify midbrain dopaminergic neurons do not specifically isolate midbrain dopaminergic neurons, but isolate a mixture of cells. Is there a way to separate these two populations?
Indeed, there is. Parmar and his colleagues in the laboratory of Thomas Perlmann showed that although dopaminergic neurons from the midbrain and subthalamic nuclear neurons are related, they do express a distinct profile of genes that are specific to the two cell types. The authors argue that the application of these distinct marker genes can help optimize those protocols that differentiate dopaminergic neurons from pluripotent stem cells.
See Nigel Kee and others, “Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages,” Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.10.003.
Dr. Ronald J. Buckanovich, professor of hematology/oncology and gynecologic oncology at the University of Michigan Medical School, and his colleagues have identified a protein that help ovarian cancer cells multiply and spread to other organs. When he and his coworkers inhibited this protein with an antibody they were able to stop the spread of ovarian cancer cold.
The EGFL6 or epidermal growth factor like 6 precursor protein, which is also known as MAEG, maps to human Xp22 chromosome. The EGFL6 protein is expressed primarily in fetal tissues and during early development (see Yeung G., et al., (1999) Genomics 62, 304–307; and Buchner G., et al., (2000) Genomics 65, 16–23). The expression of MAEG has also been detected in several tissues, including the dermis of the trunk, hair follicles, and the mesenchyme of the cranio-facial region (see Buchner G., and others, (2000) Mech. Dev. 98, 179–182). EGFL6 protein has been proposed as a possible biomarker in ovarian cancer (Buckanovich R. J., and others, (2007) J. Clin. Oncol. 25, 852–861).
In this paper, which appeared in Cancer Research, Buckanovich and others amplified the expression of EGFL6 in ovarian cancer cells. Increased EGFL6 expression stimulated cancer growth some two-three times. This effect was observed in cultured ovarian cancer cells and in a mouse model of ovarian cancer. Conversely, elimination of EGFL6 greatly reduced ovarian cancer growth, decreasing the rate of growth some four-fold.
EGFL6 specifically acts in cancer stem cells. To review, in tumors, not all cancer cells are the same. Inside malignant tumors or even among circulating cancerous cells (as in the case of leukemia) there are usually a variety of different types of cancer cells. The stem cell theory of cancer proposes that among cancerous cells, a small population among them act as stem cells that reproduce themselves and sustain the cancer. Cancer stem cells, therefore, are like normal stem cells that renew and sustain our organs and tissues. Therefore, cancer cells that are not stem cells can certainly adversely affect health, but they cannot sustain the cancer long-term. Therefore, cancer stem cells fuel the growth and spread of cancers and also are often resistant to chemotherapy and radiation treatments.
Further experiments by Buckanovich and his colleagues showed that EGFL6 cause cancer stem cells to divide asymmetrically so that the one of the daughter cells remains a cancer stem cell while the other daughter cell is a cancer cell that can affect the patient but cannot sustain the cancer. This asymmetric cell division also generates a good deal of diversity among cancer cells.
Buckanovich noted: “What this means is that the stem cell population remains stable. But the daughter cells, which can have a burst of growth, multiply, and allow the cancer to grow.”.
EGFL6 does more than just promote cancer cell proliferation. EGFL6 is also a promoter of cancer stem cell migration. When blood vessels were engineered to express EGFL6, tumor metastasis (spread) was even more robust.
Treatment of tumor-afflicted mice with an antibody that specifically binds to EGFL6 and inactivates it caused a 35% reduction in cancer stem cells and significantly reduced overall tumor growth. Additionally, the antibody also prevented tumor metastasis.
Buckanovich thinks that targeting EGFL6 might be a potential therapy for women with stage 3 ovarian cancer. Such a treatment might control the growth and spread of ovarian cancers.
Dr. Buckanovich added: “The bigger implication is for women at high risk of ovarian cancer. These patients could be treated before cancer develops, potentially blocking cancer from developing or preventing it from spreading. If cancer did develop, it could be diagnosed at an early stage, which would improve patient outcomes.”.
The next step for Buckanovich and his colleagues is developing an antibody that can properly work in human cancer patients.