Engineered Stem Cells from Human Umbilical Cord Blood Eradicates Pancreatic Tumor


Tissue-specific stem cells called mesenchymal stem cells (MSCs) are a very efficient way to delivery new drugs to cancer sites. One of the reasons these cells do such a good job with cancers in that MSCs have a liking for tumors, and once MSCs are injected into a patient or laboratory animal with tumors, the MSCs make a “B-line” for the tumor and get cozy with it.

Interleukin-15 (IL-15) is a small protein synthesized by white blood cells in our bodies, and IL-15 has a demonstrated ability to stop tumors in their tracks. Unfortunately, IL-15 is broken down quickly once it is injected into the body and consequently, has to be given in very high quantities for it to work. At such high concentrations, IL-15 causes severe side effects, and therefore, it has not been pursued as an anti-tumor agent to the degree that it deserves.

To get around this problem, a Chinese group led by Kexing Fan from the International Joint Cancer Institute in Shanghai, China, genetically engineered MSCs isolated from human umbilical cord blood so that they expressed IL-15. When these engineered MSCs that expressed a mouse version of IL-15 were subjected to experimental verification, the expressed IL-15 activated white blood cells to divide just like native IL-15.

Next, Fan’s group used these souped-up cells to treat In mice afflicted with pancreatic tumors. Pancreatic cancer is an indiscriminate killer, since by the time it causes any symptoms, it is usually so advanced, that there is little to be done in order to treat it. Thus new strategies to treat this yep of cancer are eagerly being sought. Systemic administration of IL-15-expressing MSCs significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice. The tumors of these mice showed extensive cell death, and other types of immune cells known to fight tumor cells (NK and T cells) had also accumulated around the tumor. Other experiments confirmed that the injected MSCs did indeed migrate toward the tumors and secrete IL-15 at the site of the tumors.

Interestingly, those mice that were cured from the pancreatic tumors, appeared to have a kind of resistance of these tumors. Namely, when Fan and his colleagues tried to reintroduce the same tumor cells back into the cured mice, the tumor cells would not grow. Thus the engineered MSCs not only tuned the immune system against the tumor, but they effectively vaccinated the mice against it as well.

Overall, these data seem to support the use of IL-15-producing MSCs as an innovative strategy for the treatment of pancreatic tumors.

A Molecular Switch that Causes Stem Cell Aging


A study from the Cincinnati Children’s Hospital Medical Center, in collaboration with the University of Ulm in Germany has discovered a molecular switch that causes the aging of blood stem cells. This same work suggests a therapeutic strategy to delay stem cell aging.

Hematopoietic stem cells (HSCs) reside in the bone marrow and make all the red and white blood cells that populate the bloodstream. Proper HSC function is absolutely vital to the ongoing production of different types of blood cells that allow the immune system to fight infections and organs to receive adequate quantities of oxygen.

Hartmut Geiger from the Cincinnati Children’s Hospital Medical Center and the University of Ulm was the senior researcher on this project. Dr. Geiger said, “Although there is a large amount of data showing that blood stem cell function declines during aging, the molecular processes that cause this remain largely unknown. This prevents rational approaches to attenuate stem cell aging. This study puts us significantly closer to that goal through novel findings that show a distinct switch in a molecular pathway is very critical to the aging process.”

The pathway to which Dr. Geiger referred is the Wnt signaling pathway, which plays a foundational role in animal development, cell-cell communication, tissue generation, and is also involved in the pathology of various diseases.

Crystal structure of XWnt8
Crystal structure of XWnt8

Analysis of mouse models and cultured HSCs showed that under normal conditions, Wnt signaling in HSCs occurred through the so-called “canonical” Wnt signaling pathway. The canonical Wnt signaling pathway utilizes the typical components of Wnt signaling that were first identified in the fruit fly and then isolated and characterized in vertebrates (shown below).

Canonical Wnt signaling

However, Wnt proteins can also signaling through other, distinct signal transduction pathways, and these types of pathways are collectively known as “noncanonical” Wnt signaling pathway. In aging HSCs, a switch from canonical Wnt signaling to noncanonical Wnt signaling marked the onset of HSC aging.  See below for one example of non-canonical Wnt signaling.

Non-canonical Wnt signaling

To test this observation, Geiger’s group overexpressed Wnt5 in HSCs (a Wnt protein known to induced signaling through noncanonical Wnt signaling pathways), and immediately, the HSCs began to show the signs of aging.

One of the targets of Wnt5 signaling is a protein called Cdc42, which influences the cytoskeleton of cells.  Therefore, Geiger and his crew asked if Cdc42 was activated in those HSCs that overexpressed Wnt5.  The answer to this question was a clear “yes.”  Then they treated cultured HSCs with a molecule that inhibited Cdc42 activity.  This treatment reversed the aging process in HSCs.

To test their hypothesis in a living animal, Geiger and others removed a copy of the Wnt5 gene from HSCs in laboratory mice.  Mice that lacked functional Wnt5 protein in HSCs, showed rejuvenation of the aged HSCs.  Mice that lacked both copies of the Wnt5 gene showed a delayed aging process in their HSCs.

Even though this study has definitely made an important contribution to understanding HSC aging, more work is needed before a therapeutic strategy is in place.

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.

Treating Crohn’s Disease Fistulas with Fat Stem Cells


All of us have probably heard of Crohn’s disease or have probably known someone with Crohn’s disease. While the severity of this disease varies from patient to patient, some people with Crohn’s disease simply cannot get a break.

Crohn’s disease is one of a group of diseases known as IBDs or “Inflammatory Bowel Diseases.” IBDs include Crohn;s disease, which can affect either the small or large intestine and rarely the esophagus and mouth, ulcerative colitis, which is restricted to the large intestine, and other rarer types of IBDs known that include Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behçet’s disease, and Indeterminate colitis.

Crohn’s disease (CD) involves the patient’s immune system attacking the tissues of the gastrointestinal tract, which leads to chronic inflammation within the bowel. While the exact mechanism by which this disease works is still not completely understood and robustly debated, Crohn’s disease was originally thought to be an autoimmune disease in which the immune system recognizes some kind of surface protein in the gastrointestinal tract as foreign and then attacks it. However, genetic studies of CD, linked with clinical and immunological studies have shown that this is not the case. Instead, CD seems to be due to a poor innate immunity so that the bowel has an accumulation of intestinal contents that breach the lining of the gastrointestinal tract, resulting in chronic inflammation. A seminal paper by Daniel Marks and others in the Lancet in 2006 provided hard evidence that this is the case. When Marks and others tested the white blood cells from CD patients and their ability to react to foreign invaders, those cells were sluggish and relatively ineffective. Therefore, Crohn’s seems to be an overactivity of the acquired immunity to make up for poor innate immunity.

Given all that, one of the biggest, most painful consequences of CD are anal fistulas. If those sound painful it’s because they are. A fistula is a connection between to linings in your body that should not normally be connected. In CD patients, the anus and the attached rectum get kicked about by excessive inflammation and tears occur. These tears heal, but the healing can cause connections between linings that previously did not exist. Therefore fecal material not comes out of the body in more than one place. Sounds disgusting? It gets worse. Those areas that leak feces are not subject to extensive pus formation and they must be fixed surgically. But how do you fix something that is constantly inflamed? It’s an ongoing problem in medicine.

Enter stem cells to the rescue, maybe. In Spain, a multicenter clinical study has just been published that shows that fat-derived mesenchymal stem cells might provide a better way to treat these fistulas in CD patients. Mesenchymal stem cells have the ability to suppress inflammation, and for that reason, they are excellent candidates to accelerate healing in cases such as these.

Galindo and his group took 24 CD patients who had at least one draining fistula (yes, some have more than one) and gave them 20 million fat-derived mesenchymal stem cells. These cells were extracted from someone else, which is an important fact, since liposuction procedures on these patients might have added to their already surfeit of inflammation.

For this treatment, the cells were administered directly on the lesion, which is almost certainly important. If the closing of the fistula was incomplete after 12 weeks, then the patients were given another dose of 40 million fat-derived mesenchymal stem cells right on the lesion. All these patients were followed until week 24 after the initial stem cell administration.

The results were very hopeful. There were no major adverse effects six months after the stem cell treatment. This is a result seen over and over with mesenchymal stem cells – they are pretty safe when administered properly. Secondly, full analysis the data showed that at week 24 69.2% of the patients showed a reduction in the number of draining fistulas. Even more remarkably, 56.3% of the patients achieved complete closure of the treated fistula. That is just over half. Also, 30% of the cases showed complete closure of all existing fistulas. These results are exciting when you consider the criteria they used for complete closure: absence of draining pus through its former opening. complete “re-epithelization” of the tissue, which means that the lining of the tissue is healed, looks normal and is properly attached to the proper neighbors, and magnetic resonance image (MRI) scans of the region must look normal. For these patients, the MRI “Score of Severity,” which is a measure of the structural abnormality of the anal region, showed statistically significant reductions at week 12 with a marked reduction at week 24. Folks that’s good news.

Galindo interprets his results cautiously and notes that this is a small study, which is true. He also states that the goal of this study was to ascertain the safety of this technique, and when it comes to safety, this technique is certainly safe. When it comes to efficacy, another larger study is required that specifically examined the efficacy of this technique. Galindo is, of course, quite correct, but this is certainly a very exciting result, and hopefully these cells will get further chances to “strut their therapeutic stuff.”

See de la Portilla F, et al Expanded allogeneic adipose-derived stem cells (eASCs) for the treatment of complex perianal fistula in Crohn’s disease: results from a multicenter phase I/IIa clinical trial.  Int J Colorectal Dis. 2013 Mar;28(3):313-23. doi: 10.1007/s00384-012-1581-9. Epub 2012 Sep 29.

Directly Programming Skin Cells to Become Blood-Making Stem Cells


Within our bones lies a spongy, ribbon-like material called bone marrow.  Bone marrow is home to several different populations of stem cells, but the star of the stem cell show in the bone marrow are the hematopoietic stem cells or blood-making stem cells.   When a patient receives a bone marrow transplant these are the stem cells that are transferred, take up residence in the new bone marrow, and begin making new red and white blood cells for the patient.  Because bone marrow is such a precious commodity from a clinical standpoint, finding a way to make more of it is essential.

Hematopoiesis from Pluripotent Stem Cell

A new report from scientists at Mt Sinai Hospital in New York suggest that the transfer of specific genes into skin fibroblasts can reprogram mature, adult cells into hematopoietic stem cells that look and function exactly like the ones normally found within our bone marrow.

A research team at the Icahn School of Medicine at Mount Sinai led by Kateri Moore screen a panel of 18 different genes for their ability to induce blood-forming activity when transfected into fibroblasts. Kateri and others discovered that a combination four different genes (GATA2, GFI1B, cFOS, and ETV6) is sufficient to generate blood vessel precursors with the subsequent appearance of hematopoietic stem cells. These cells expressed several known hematopoietic stem cell surface proteins (CD34, Sca1 and Prominin1/CD133).

Reprogramming of fibroblasts to HSCs

“The cells that we grew in a Petri dish are identical in gene expression to those found in the mouse embryo and could eventually generate colonies of mature blood cells,” said Carlos Filipe Pereira, first author of this paper and a postdoctoral research fellow in Moore’s laboratory.

The combination of gene factors that we used was not composed of the most obvious or expected proteins,” said Ihor Lemischka, a colleague of Dr. Moore at Mt. Sinai Hospital.  “Many investigators have been trying to grow hematopoietic stem cells from embryonic stem cells, but this process has been problematic.  Instead, we used mature mouse fibroblasts, pick the right combination of proteins, and it worked.”

According to Pereira, there is a rather critical shortage of suitable donors for blood stem cells transplants.  Bone marrow donors are currently necessary to meet the needs of patients suffering from blood diseases such as leukemia, aplastic anemia, lymphomas, multiple myeloma and immune deficiency disorders.  “Programming of hematopoietic stem cells represents an exciting alternative,” said Pereira.

“Dr. Lemischka and I have been working together for over 20 years in the fields of hematopoiesis and stem cell biology,” said Kateri Moore.  “It is truly exciting to be able to grow these blood forming cells in a culture dish and learn so much from them.  We have already started applying this new approach to human cells and anticipate similar success.”