Turning Stem Cells into Drug Factories


Wouldn’t it be nice to have cells that express the right molecules at the right place and the right time to augment or even initiate healing?

Researchers at the Brigham and Women’s Hospital and Harvard Stem Cell Institute have inserted modified messenger RNA to induce mesenchymal stem cells to produce adhesive proteins  (PSGL-1)and secrete interleukin-10, a molecule that suppresses inflammation. When injected into the bloodstream of mice, these modified stem cells home to the right location, stick to that site, and secrete interleukin-10 (IL-10) to suppress inflammation.

Improving MSC therapeutic potential viamRNA transfection with homing ligands and immunomodulatory factors. Illustration of (A) mRNA-engineered MSCs that express a combination of homing ligands (PSGL-1 and SLeX) and an immunomodulatory factor (IL-10), and (B) targeting mRNA-engineered MSCs to site of inflammation.
Improving MSC therapeutic potential viamRNA transfection with homing ligands and immunomodulatory factors. Illustration of (A) mRNA-engineered MSCs that express a combination of homing ligands (PSGL-1 and SLeX) and an immunomodulatory factor (IL-10), and (B) targeting mRNA-engineered MSCs to site of inflammation.

Jeffrey Karp, Harvard Stem Cell Institute principal faculty member and leader of this research, said this about this work: “If you think of a cell as a drug factory, what we’re doing is targeting cell-based, drug factories to damaged tissues, where the cells can produce drugs at high enough levels to have a therapeutic effect.”

Karp’s paper reports a proof-of-principle study has piqued the interest of several biotechnology companies, since it has the potential to target biological drug to disease sites. While ranked as the top sellers in the drug industry, biological drugs are still challenging to use. Karp’s approach might improve the clinical applications of biological drugs and improve the somewhat mixed results of clinical trials with mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have emerged as one of the favorite sources for stem cell therapies. The attractiveness of MSCs largely lies with their availability, since they are found in bone marrow, fat, liver, muscle, and many other places. Secondly, MSCs can be grown in culture for a limited period of time without a great deal of difficulty. Third, MSCs tend to be ignored by the immune system when injected. For these reasons, MSCs have been used in many clinical trials, and they appear to be quite safe to use.

To genetically modify MSCs, Karp and his co-workers made chemically modified messenger RNAs (mRNAs) whose bases differed slightly from natural mRNAs. These chemical modifications did not affect the recognition of the mRNA by the protein synthesis machinery of the MSCs, but did affect the recognition of these mRNAs by those enzymes that degrade mRNAs. Therefore, these synthetic mRNAs are very long-lived and the transfected cells end up making the proteins encoded by these mRNAs for a very long time. RNA transfection does not modify the genome of the host cells, and this makes it a very safe procedure, since the engineered cells will express the desired protein for some time, but not indefinitely.

The mRNAs introduced into the cultured MSCs included mRNAs that encode the IL-10 protein, which is cytokine that suppresses inflammation, the PSGL-1 protein, a cell-surface protein that sticks to the P-and E-selectin receptors, and the Fut7 gene product.  FUT7 encodes an enzyme called fucosyltransferase 7, which adds a sugar called “fucose” to the PSGL-1 protein and without this sugar, PSGL-1 cannot bind to the selectins.  Selectins are stored by cells and during inflammation, they are sent to the cell surface where they can bind cells and keep them there to mediate inflammation.  By expressing PSGL-1 in the MSCs, Karp and his group hoped to that the engineered MSCs would bind to the surfaces of blood vessels and not be washed out.

e-selectin_binding

Oren Levy, lead author of this paper, said, “This opens the door to thinking of messenger RNA transfection of cell populations as next generation therapeutics in the clinic, as they get around some of the delivery challenges that have been encountered with biological agents.”

A problem that constantly troubles clinical trials that use MSCs is that they are rapidly cleared from the bloodstream within a few hours or days after they are introduced. The Harvard team showed that rapid targeting of MSCs to inflamed tissue produced a therapeutic effect despite rapid clearance of the MSCs.

Karp and his colleagues would like to extend the lifespan of these cells in the bloodstream and they are presently experimenting with new synthetic mRNAs that encode pro-survival factors.

“We’ve interested to explore the platform nature of this approach and see what potential limitations it may have or how far we can actually push it. Potentially we can simultaneously deliver proteins that have synergistic therapeutic impacts,” said Weian Zhao, another author of this paper.

Keeping Implanted Stem Cells in the Heart


Globally, thousands of heart patients have been treated with stem cells from bone marrow and other sources. While many of these patients have been helped by these treatments, the results have been inconsistent, and most patients only show a modest improvement in heart function.

The reason for these sometimes underwhelming results seems to result from the fact that implanted stem cells either die soon after they are delivered to the heart or washed out. Since the heart is a pump, it is constantly contracting and having fluid (blood) wash through it. Therefore, it is one of the last places in the body we should expect implanted stem cells to stay put.

To that end, cardiology researchers a Emory University in Atlanta, Georgia have packaged stem cells into small capsules made of alginate (a molecule from seaweed) to keep them in the heart once they are implanted there.

alginate_formel

W. Robert Taylor, professor of medicine and director of the cardiology division at Emory University School of Medicine, and his group encapsulated mesenchymal stem cells in alginate and used them to male a “patch” that was applied to the hearts of rats after a heart attack. Taylor’s group compared the recovery of these animals to those rats that had suffered heart attacks, but were treated with non-encapsulated cells, or no cells at all. The rats treated with encapsulated cells not only showed a more robust recovery, but they had larger numbers of stem cells in their hearts and showed better survival.

Histological appearance of encapsulated human mesenchymal stem cells (hMSCs). Light microscopic appearance of encapsulated hMSCs at the time of implantation with approximately 200 cells within each 250 μm capsule. (Scale bar=100 μm)
Histological appearance of encapsulated human mesenchymal stem cells (hMSCs). Light microscopic appearance of encapsulated hMSCs at the time of implantation with approximately 200 cells within each 250 μm capsule. (Scale bar=100 μm)

Of this work, Taylor said, “This approach appears to be an effective way to increase cell retention and survival in the context of cardiac cell therapy. It may be a strategy applicable to many cell types for regenerative therapy in cardiovascular medicine.

Readers of this blog might remember that I have detailed before the inhospitable environment inside the heart after a heart attack. Oxygen levels are low because blood vessels have died, and roving white blood cells are gobbling up cell debris and releasing toxic molecules while they do it. Also the dying cells have released a toxic cocktail of molecules that make the infarcted area very inhospitable. Injecting stem cells into this region is an invitation for more cells to die. Previous experiments have shown that preconditioning stem cells either by genetically engineering them to withstand high stress levels of by growing them in high-stress conditions prior to implantation can increase their survival in the heart.

Taylor also pointed out that the mechanical forces of the contracting heart can squeeze them and displace them from the heart, much like pinching a watermelon seed between your fingers causes it to slip out. “These cells are social creatures and like to be together,” said Taylor. “From some studies of cell therapy after myocardial infarction, one can estimate that more than 90 percent of the cells are lost in the first hour. With numbers like that, it’s easy to make the case that retention is the first place to look to boost effectiveness.”

Encapsulation keeps the mesenchymal stem cells together in the heart and “keeps them happy.” Encapsulation, however, does not completely cut off the cells from their environment. They can still sense the cardiac milieu and release growth factors and cytokines while they are protected from marauding white blood cells and antibodies that might damage, destroy, or displace them.

Alginate already has an impressive medical pedigree as a biomaterial. It is completely non-toxic, and chefs use it to make edible molds to encase other types of tasty morsels. Dentists use alginate to take impressions of a patient’s teeth and it is also used a component of wound dressings. One of Taylor’s co-authors, an Emory University colleague named Collin Weber has used alginate to encapsulate insulin-producing islet-cells that are being tested in clinical trials with diabetics.

Encasing cells in alginate prevents them from replacing dead cells, but mesenchymal stem cells tend to do the majority of their healing by means of “paracrine” mechanisms; that is to say, mesenchymal stem cells tend to secrete growth factors, cytokines and other healing molecules rather than differentiating into heart cells. Mesenchymal stem cells can be isolated from bone marrow or fat.

One month after suffering from a heart attack, those rats that had suffered a heart attack saw their ejection fractions (a measure of how much volume the heart pumps out with every beat) fell from an average of 72% to 34%. However, rats treated with encapsulated mesenchymal stem cells saw an increase in their ejection fractions from 34% to 56%. Those treated with unencapsulated mesenchymal stem cells saw their ejection fractions rise to 39%.

Detailed cardiac functional analysis by cardiac magnetic resonance imaging (CMR) and transthoracic echocardiography (TTE) showed improvement in animals treated with encapsulated human mesenchymal stem cells (hMSCs). A, Representative short axis CMR at end systole of animals treated with encapsulated hMSCs or controls. Myocardial thinning and chamber dilation, delineated by traced endocardium (red) and epicardium (green) was reduced in the encapsulated hMSC group (arrow). Quantification of end systolic volume (B) and ejection fraction (C) by CMR at day 28 showed improved contractile function in the encapsulated hMSC treated group (n=4 per group). D, TTE comparison of untreated animals (n=9) to animals treated with encapsulated hMSCs (n=7) or hMSCs delivered by direct injection (n=7) into the infarcted myocardium showed greater benefit of treatment with encapsulated cells. Data represent mean±SEM. *P<0.05 by Dunnett's test of multiple comparisons; #P<0.05 by analysis of variance (ANOVA). LVESV indicates left ventricular end systolic volume; MI, myocardial infarction.
Detailed cardiac functional analysis by cardiac magnetic resonance imaging (CMR) and transthoracic echocardiography (TTE) showed improvement in animals treated with encapsulated human mesenchymal stem cells (hMSCs). A, Representative short axis CMR at end systole of animals treated with encapsulated hMSCs or controls. Myocardial thinning and chamber dilation, delineated by traced endocardium (red) and epicardium (green) was reduced in the encapsulated hMSC group (arrow). Quantification of end systolic volume (B) and ejection fraction (C) by CMR at day 28 showed improved contractile function in the encapsulated hMSC treated group (n=4 per group). D, TTE comparison of untreated animals (n=9) to animals treated with encapsulated hMSCs (n=7) or hMSCs delivered by direct injection (n=7) into the infarcted myocardium showed greater benefit of treatment with encapsulated cells. Data represent mean±SEM. *P

One of the main effects of implanted stem cells is the promotion of the growth of new blood vessels.  In capsule-treated rats, the damaged area of the heart had a blood vessel density that was several times that of the hearts of control animals.  Also, the area of cell death was much lower in the hearts treated with encapsulated MSCs.

Treatment of hearts with encapsulated human mesenchymal stem cells (hMSC) post myocardial infarction reduced myocardial scarring at 28 days. A, Representative sections of infarcted hearts stained with Masson's Trichrome and treated with encapsulated hMSCs or control gels. Blue indicates fibrotic scar. ×15, scale bar=1 mm. B, Animals treated with encapsulated hMSCs showed reduced scar area (7±1%; n=6) at 28 days compared to control treated hearts (MI: 12±1%, n=8; MI+Gel: 14±2%, n=7; MI+Gel+hMSC: 14±1%, n=7; MI+Gel+Empty Caps: 12±2%, n=5). Data represent mean±SEM. *P<0.05. MI indicates myocardial infarction.
Treatment of hearts with encapsulated human mesenchymal stem cells (hMSC) post myocardial infarction reduced myocardial scarring at 28 days. A, Representative sections of infarcted hearts stained with Masson’s Trichrome and treated with encapsulated hMSCs or control gels. Blue indicates fibrotic scar. ×15, scale bar=1 mm. B, Animals treated with encapsulated hMSCs showed reduced scar area (7±1%; n=6) at 28 days compared to control treated hearts (MI: 12±1%, n=8; MI+Gel: 14±2%, n=7; MI+Gel+hMSC: 14±1%, n=7; MI+Gel+Empty Caps: 12±2%, n=5). Data represent mean±SEM. *P

The encapsulated stem cells seem to stay in the heart for just over ten days, which is the time is takes for the alginate hydrogels to break down.  Taylor said that he and his lab would like to test several different materials to determine how long these capsules remain bound to the patch.

The goal is to use a patient’ own stem cells as a source for stem cell therapy.  Whatever the source of stem cells, a patient’s own stem cells must be grown outside the body for several days in a stem cell laboratory, much like Emory Personalized Immunotherapy Center in order to have enough material for a therapeutic effect.

Foregut Stem Cells


Scientists from Cambridge University have designed a new protocol that will convert pluripotent stem cells into primitive gut stem cells that have the capacity to differentiate into liver, pancreas, or some other gastrointestinal structure.

Nicholas Hannan and his colleagues at the University of Cambridge Welcome Trust MRC Stem Cell Institute have developed a technique that allows researchers to grow a pure, self-renewing population of stem cells that are specific to the human foregut, which is the upper section of the human digestive system. These types of stem cells are known as “foregut stem cells” and they can be used to make liver, pancreas, stomach, esophagus, or even parts of the small intestine. Making these types of gastrointestinal tissues can provide material for research into gastrointestinal abnormalities, but might also serve as a source of material to treat type 1 diabetes, liver disease, esophageal and stomach cancer, and other types of severe gastrointestinal diseases.

“We have developed a cell culture system which allows us to specifically isolate foregut stem cells in the lab,” said Hannan. “These cells have huge implications for regenerative medicine, because they are the precursors to the thyroid upper airways, lungs, liver, pancreas, stomach, and biliary systems.”

Hannan did this work in the laboratory of Ludovic Vallier, and they think that their technique will provide the means to analyze the precise embryonic development of the foregut in greater detail. “We now have a platform from which we can study the early patterning events that occur during human development to produce intestines, liver, lungs, and pancreas,” said Hannan.

To make foregut stem cells, Hannan begins with a pluripotent stem cell line; either an embryonic stem cell line or an induced pluripotent stem cell line. Then he differentiated them into definitive endoderm by treating them with CDM-PVA and activin-A (100 ng/ml), BMP4 (10 ng/ml), bFGF (20 ng/ml), and LY294002 (10 mM) for 3 days. Once they differentiated into endoderm, the endodermal cells were grown in RPMI+B27 medium with activin-A (50 ng/ml) for 3-4 days in order to generate foregut stem cells.

(A) GFP-expressing hPSCs were differentiated into hFSCs. (B) Single GFP-positive hFSCs were seeded onto a layer of non-GFP hFSCs and then expanded for five passages. The resulting population was then split into culture conditions inductive for liver or pancreatic differentiation. (C and D) GFP-hFSCs differentiated for 25 days were found to respectively generate cells expressing liver markers (ALB, LDL-uptake) and pancreatic markers (PDX1, C-peptide) from both hESC-derived (C) and hIPSC-derived (D) hFSCs.
(A) GFP-expressing hPSCs were differentiated into hFSCs.  (B) Single GFP-positive hFSCs were seeded onto a layer of non-GFP hFSCs and then expanded for five passages. The resulting population was then split into culture conditions inductive for liver or pancreatic differentiation.  (C and D) GFP-hFSCs differentiated for 25 days were found to respectively generate cells expressing liver markers (ALB, LDL-uptake) and pancreatic markers (PDX1, C-peptide) from both hESC-derived (C) and hIPSC-derived (D) hFSCs.

These foregut stem cells (FSCs) can self-renew, and can also differentiate into any part of the foregut. Thus, FSCs can grow robustly in culture, and they can also differentiate into foregut derivatives. However, these cells also do not form tumors. When injected into mice, they failed to form tumors.

(A) Large cystic hFSC outgrowth under the kidney capsule of a NOD-SCID mouse. (B) Cryosection of a hFSC outgrowth showing large cystic structures lined with epithelial cells. (C) Immunocytochemistry showing foregut outgrowths expressing EpCAM, PDX1, AFP, and NKX2.1. Scale bars, 100 μm or 50 μm as shown. See also Figure S4.
(A) Large cystic hFSC outgrowth under the kidney capsule of a NOD-SCID mouse.  (B) Cryosection of a hFSC outgrowth showing large cystic structures lined with epithelial cells.  (C) Immunocytochemistry showing foregut outgrowths expressing EpCAM, PDX1, AFP, and NKX2.1.  Scale bars, 100 μm or 50 μm as shown. See also Figure S4.

What are the advantages to FSCs as opposed to making pancreatic cells or liver cells from pluripotent stem cells? These types of experiments always create cultures that are impure. Such cultures are difficult to use because not all the cells have the same growth requirements and they would be dangerous for therapeutic purposes because they might contain undifferentiated cells that might grow uncontrollably and cause a tumor. Therefore, FSCs provide a better starting point to make pure cultures of pancreatic tissues, liver tissues, stomach tissues and so on.

Ludovic Vallier, the senior author of this paper said this of his FSCs, “What we have now is a better starting point – a sustainable platform for producing liver and pancreatic cells. It will improve the quality of the cells that we produce and it will allow us to produce the large number of uncontaminated cells we need for the clinical applications of stem cell therapy.”

Vallier’s groups is presently examining the mechanisms that govern the differentiation of FSCs into specific gastrointestinal cell types in order to improve the production of these cells for regenerative medicine.

Human Umbilical Cord Mesenchymal Stem Cells and Rheumatoid Arthritis


A collaborative study between physicians at the Hospital of Chinese People’s Liberation Army and the University of Oklahoma Health Sciences Center has examined the efficacy of umbilical cord mesenchymal stem cell treatments in combination with drugs in patients with active rheumatoid arthritis (RA).

RA may exist in 0.5-1.0% of the general population. In 2005, an estimated 1.5 million US adults aged ≥ 18 (0.6%) had RA. RA is characterized by chronic inflammation of the joints that causes cartilage and bone damage and deformity. It occurs in women two to three times more often than men.

Treatment of RA requires the administration of disease-modifying antirheumatic drugs (DMARDs), Unfortunately, these drugs have sizable side effects, and less debilitating treatments would be a welcome addition to the treatment options for RA patients.

A paper by Liming Wang and colleagues that was published in Stem Cells and Development examines the efficacy of combining DMARDs with infusions of umbilical cord mesenchymal stem cells (MSCs). Since MSCs have the ability to suppress an overactive immune response, such treatments might provide relief from the symptoms of RA and decrease the dependence on DMARDs.

In this study, Wang and others enrolled 172 RA patients and divided them into two groups: 36 of them were treated with DMARDs alone and 136 were treated with DMARDs plus umbilical cord MSCs (UC-MSCs). Of these 136 patients, 76 were treated for 3 months, 45 for 6 months, and 15 for 8 months. Each of these groups consisted of patients who could and who could not tolerate DMARDs. All patients in the second group received 4 x 10[7] UC-MSCs in 40 milliliters of liquid, but the first group received stem cell “solvent” (whatever that is) without UC-MSCs.

The results clearly showed that UC-MSCs treatments are safe. Patients blood work-ups before and after treatment show no significant differences. Secondly, the DMARD-only group did not show any improvements, but they did not get worse either. The DMARD + UC-MSC group showed quantifiable improvements. These patients reported feeling better in health assessment questionnaires, their serum levels of C-reactive protein and rheumatoid factor went down and their numbers of regulatory T-cells went up. The joint evaluations of these patients also improved (the so-called DAS28 score). All of these are measures of the severity of RA, and in the DMARD + UC-MSC groups, all the these markers improved.

Other markers of RA severity such as IL-6 and TNF-alpha also decreased in the DMARD + UC-MSC patients.

From these data, Wang and others conclude that “UC-MSCs are suitable pllications in the clinic and provide an additional choice to many RA patients.”

The data in this paper are rather clear. The benefits of a single UC-MSC treatment are significant. For this reason, umbilical cord MSCs should be regarded as a potential adjuvant treatment for RA patients.

Inhibition of a Heart-Specific Enzyme After a Heart Attack Decreases Heart Damage and Prevents Remodeling


Cardiac Troponin I-interacting Kinase or TNNI3K is an enzyme that was initially identified in fetal and adult heart tissue, but was undetectable in other tissues. The function of this enzyme remains unknown, but Chinese scientists showed that overexpression of TNNI3K in cultured heart muscle cells causes them to blow up and get large (hypertrophy). Earlier this year, a research team from Peking Union Medical College showed that overexpression of TNNI3K in mice caused enlargement of the heart (Tang H., et al., J Mol Cell Cardiol 54 (2013): 101-111). These results suggested that TNNI3K is a potential therapeutic target for heart attack patients.

To that end, Ronald Vagnozzi and his colleagues in the laboratory of Thomas Force at Temple University School of Medicine and their collaborators designed small molecules that can inhibit TNNI3K activity, and these small molecules decrease cardiac remodeling after a heart attack in rodents. Large animal trials are planned next.

In the first experiments of this paper, Vagnozzi and others showed that the levels of TNNI3K in the heart increase after a heart attack. Measurements of TNNI3K protein levels failed to detect it in all tissue other than the heart. Furthermore, it was present throughout the heart, and mainly in heart muscle and not in blood vessels, fibroblasts, and other types of non-muscle heart tissues.

Next, Vagnozzi and others measured TNNI3K protein levels in heart transplant patients. The heart tissues of these patients, who had badly dysfunctional hearts showed higher than usual levels of TNNI3K protein. Thus, TNNI3K is associated with heart tissue and is up-regulated in response to heart dysfunction.

The next experiment examined the effects of overexpressing the human TNNI3K gene in mice. While the overexpression of TNNI3K did not affect heart function of structure under normal circumstances, under pathological conditions, however, this is not he case. If mice that overexpressed TNNI3K where given heart attacks and then “reperfused,” means that the blood vessel that was tied off to cause the heart attack was opened and blood flowed back into the infarcted area. In these cases, mice that overexpressed TNNI3K had a larger area of cell death in their hearts than their counterparts that did not overexpress TNNI3K. The reason for this increased cell death had to do with the compartment in the cell that generated most of the energy – the mitochondrion. TNNI3K causes the mitochondria in heart muscle cells to go haywire and kick out all kinds of reactive oxygen-containing molecules that damage cells.

Cell damage as a result of reactive oxygen-containing molecules (known as reactive oxygen species or ROS) activates a pathway in heart cells called the “p38” pathway, which leads to programmed cell death.

p38 signaling

Once Vagnozzi and his colleagues nailed down the function of TNNI3K in heart muscle cells after a heart attack, they deleted the gene that encodes TNNI3K and gave those TNNI3K-deficient mice heart attacks. Interestingly enough, after a heart attack, TNNI3K-deficient mice showed much small dead areas than normal mice. Also, the levels of the other mediators of TNNI3K-induced cell death (e.g., oxygen-containing molecules, p38, ect.) were quite low. This confirms the earlier observations that TNNI3K mediates the death of heart muscle cells after a heart attack, and inhibiting TNNI3K activity decreases the deleterious effects of a heart attack.

And now for the pièce de résistance – Vagnozzi and his crew synthesized small molecules that inhibited TNNI3K in the test tube. Then they gave mice heart attacks and injected these molecules into the bellies of the mice. Not only were the infarcts, or areas of dead heart muscle cells small in the mice injected with these TNNI3K inhibitors, but the heart of these same mice did not undergo remodeling and did not enlarge, showed reduced scarring, and better ventricular function. This is a proof-of-principle that inhibiting TNNI3K can reduce the pathological effects of a heart attack.

This strategy must be tested in large animals before it can move to human trials, but the strategy seems sound at this point, and it may revolutionize the treatment of heart attack patients.

Primed Fat-Based Stem Cells Enhance Heart Muscle Proliferation


A Dutch group from the University of Groningen has shown that fat-based stem cells can enhance the proliferation of cultured heart muscle cells. The stem cells used in these experiments were preconditioned and this pretreatment greatly enhanced their ability to activate heart muscle cells.

This paper, by Ewa Przybyt, Guido Krenning, Marja Brinker, and Martin Harmsen was published in the Journal of Translational Medicine. To begin, Przybyt and others extracted human adipose derived stromal cells (ADSC) from fat tissue extracted from human liposuction surgeries. To do this, they digested the fat with enzymes, centrifuged and washed it, and then grew the remaining cells in culture.

Then they used rat neonatal heart muscle cells and infected them with viruses that causes them to glow when certain types of light was shined on them. Then Przybyt and others co-cultured these rat heart cells with human ADSCs.

In the first experiment, the ADSCs were treated with drugs to prevent them from dividing and then they were cultured with rat heart cells in a one-to-one ratio. The heart muscle cells grew faster with the ADSCs than they did without them. To determine if cell-cell contact was required for this stimulation, they used the culture medium from ADSCs and grew the heart cell on this culture medium. Once again, the heart cells grew faster with the ADSC culture medium than without it. These results suggest that the ADSCs stimulate heart cell proliferation by secreting factors that activate heart cell division.

Another experiment subjected the cultured heart cells to the types of conditions they might experience inside the heart after a heart attack. For example, heart cells were subjected to low oxygen tensions (2% oxygen), and inflammation – two conditions found within the heart after a heart attack. These treatments slowed heart cell growth, but this heart cell growth was restored by adding the growth medium of ADSCs. Even more remarkably, when ADSCs were grown in low-oxygen conditions or treated with inflammatory molecules (tumor necrosis factor-alpha or interleukin-1beta), the culture medium increased the fractions of cells that grew. Therefore, ADSCs secrete molecules that increase heart muscle cell proliferation, and increase proliferation even more after the ADSCs are preconditioned by either low oxygen tensions or inflammation.

In the next experiment, Przybyt and others examined the molecules secreted by ADSCs under normal or low-oxygen tensions to ascertain what secreted molecules stimulated heart cell growth. It was clear that the production of a small protein called interleukin-6 was greatly upregulated.

Could interleukin-6 account for the increased proliferation of heart cells? Another experiment showed that the answer was yes. Cultured heart cells treated with interleukin-6 showed increased proliferation, and when antibodies against interleukin-6 were used to prevent interleukin-6 from binding to the heart cells, these antibodies abrogated the effects of interleukin-6.

Przybyt and others then took these results one step further. Since the signaling pathways used by interleukin-6 are well-known, they examined these pathways. Now interleukin-6 signals through pathways, once of which enhances cell survival, and another pathway that stimulated cell proliferation. The cell proliferation pathway uses a protein called “STAT3” and the survival function uses a protein called “Akt.” Both pathways were activated by interleukin-6. Also, the culture medium of ADSCs that were treated with interleukin-6 induced the interleukin-6 receptor proteins (gp80 and gp130) in cultured heart muscle cells. This gives heart muscle cells a greater capacity to respond secreted interleukin-6.

This paper shows that stromal stem cells from fat has the capacity, in culture, to activate the growth of cultured heart muscle cells. Also, if these cells were preconditioned with low oxygen tensions or pro-inflammatory molecules, those fat-based stem cells secreted interleukin-6, which enhanced heart muscle cell survival, and proliferation, even if those heart muscle cells are exposed to low-oxygen tensions or inflammatory molecules.

This suggests that preconditioned stem cells from fat might be able to protect heart muscle cells and augment heart healing after a heart attack. Alternatively, cardiac administration of interleukin-6 after a heart attack might prove even more effective to protect heart muscle cells and stimulate heart muscle cell proliferation. Human trials anyone?

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

“Noncontroversial” Embryonic Stem Cells?


An article from Bioscience Technology, a working scientist’s rag, has argued that everyone can have their lifetime supply of embryonic stem cells. Below is a summary of the article, after which I will comment on it.

Susan Fisher is the director of the UCSF Human Embryonic Stem Cell program. Last week, her lab reported that they have efficiently created embryonic stem cell lines from the cells removed from early embryos for Preimplantation Genetic Diagnosis (PGD) clinics. PGD takes a single cell from an early embryo that was created by means of in vitro fertilization, and subjects that single cell to genetic analyses to determine if the embryo carries a genetic disease. Because early human embryos have the ability to “regulate,” the removal of a single simply spurs the cells of the embryo to undergo extra cell divisions. The embryos subjected to PGD are then either destroyed, if they harbor a genetic disease, or implanted into the mother’s womb and gestated.

However, these cells removed from embryos could also be used to make an embryonic stem cell culture, since they could be seeded in culture to make an embryonic stem (ES) cell line. Therefore, in theory, cells could now be routinely removed from in vitro fertilization (IVF) clinic embryos, to provide them with a lifetime supply of their own embryonic stem cells. Because these cells were made without destroying embryos, they would be uncontroversial.

“Back in the mid-2000’s, when California was trying to decide whether to fund ES cell research, thousands of interested people would come out to hear us speak about topics like this,” says Fisher, interviewed after her report to the New York Stem Cell Foundation conference last week. “It is possible this particular, refined approach will generate that kind of interest now.”

ES cells have the greatest potency of any human stem cells and they can potentially form every cell type in the adult human body. Because such cells were recently harvested, they would not possess any of the mutations that ES cultures can acquire when they are grown for long periods of time in culture.

Traditionally, ES cell lines have been derived from stored, spare embryos from IVF clinics that were donated by other patients. Therefore, they are not immunologically identical to patients who potentially need them. Patients who receive non-matching tissues must take harsh immunosuppressive drugs for years to avoid rejecting the cells, and even then, over time the immune eventually wins the fight in some cases.

In recent years, scientists have turned to induced Pluripotential Stem Cells (IPSCs). IPSCs are made by genetically engineering adult cells to express four genes that de-differentiate the cells so that they are embryonic-like cells. IPSCs have been a boon to research, since scientists hace used them to make “disease in a dish” models on which to try drugs. But IPSCs are often riddled with mutations, as they come from adults. They have not yet hit the clinic as a result (although trials are upcoming).

However, Fisher, following on the heels of very preliminary work published in the journal Nature by the biotechnology company ACT, has refined the ability to create possibly uncontroversial stem cells—that are immunological matches to patients. By removing one cell from a very young human embryo, Fisher thinks that scientist might be able to produce a veritably unlimited supply of ES cells that are immunologically identical to the embyros from which they came. And as the embryos aren’t destroyed, but implanted into the mothers’ uteruses, the derivation of these tailor-made ES cells should be uncontroversial. “We will see how this is received,” Fisher says.

The process, she reported, is robust, if still not easy to pull off. This procedure, however, is labor-intensive and required a great deal of skill to pull off. In Fisher’s lab at UCSF, they derived ten human ES cell lines from four eight-cell embryos and one 12-cell embryo from a single couple.

When compared to standard ES cells, the UCSF lines were healthy and “formed derivatives of the three germ layers” like standard ES cells. Furthermore, these cells could form trophoblasts (placental cells), and Fisher’s team used them to create the first human trophoblast stem cell line. This is something that standard ES cells cannot do and this could make the UCSF cells useful in the clinic for diseases affecting the placenta.

Will patients begin turning to such cells? A few companies in the mid-2000s started offering designer ES cells like these, but that practice ended due to lack of interest or understanding, Fisher says. Additionally, some technical problems—later fully rectified—associated with the earlier Nature ACT paper may have cast a pall on enthusiasm for the approach, others in the field note.

“It remains to be seen if a place will be found for both iPS and ES cells,” Fisher concludes.

Now follows my comments:

Human embryos are very young human beings.  They do not have the right to vote, own property, or get a driver’s license, but they at least have the right not to be harmed.  By withdrawing cells from the embryo, you are potentially harming it.  “But wait,” proponents will tell you, “there are hundreds or even thousands of children who have been born who grew from embryos that were subjected to PGD and their rates of birth defects are no higher than everyone else’s.”  So their rates of birth defects are lower, but have we followed them for the rest of their lives to establish that removing a blastomere during early development does no harm?

“Oh come on,” you say.  But there are studies in mice that show that removing blastomere from early embryos does not cause higher rates of birth defects, but it does cause higher rates of neurological defects that manifest later in life.  Yu and others found that “mice generated after blastomere biopsy showed weight increase and some memory decline compared with the control group. Further protein expression profiles in adult brains were analyzed by a proteomics approach. A total of 36 proteins were identified with significant differences between the biopsied and control groups, and the alterations in expression of most of these proteins have been associated with neurodegenerative diseases. Furthermore hypomyelination of the nerve fibers was observed in the brains of mice in the biopsied group. This study suggested that the nervous system may be sensitive to blastomere biopsy procedures and indicated an increased relative risk of neurodegenerative disorders in the offspring generated following blastomere biopsy.”  In another paper, Yang and others showed that “blastomere biopsy, increases the rate of embryo death at 4.5-7.5 dpc, but does not affect the development of surviving 7.5 dpc embryos.”  In human embryos, time-lapse photography of biopsied embryos by Kirkegaard K, Hindkjaer JJ and Ingerslev HJ showed that “blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.”  Finally, Sugawara and others showed that “The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.”

There is reason to believe that this procedure potentially hurts the embryo.  Also, not all blastomeres in the early embryo are equally competent to make ES lines (see Lorthongpanich et al., Reproduction. 2008 Jun;135(6):805-1).  Therefore, if more than one blastomere must be taken from the embryo, the risks to it definitely increases (see Groossens et al., Hum. Reprod. (2008) 23 (3): 481-492).  The embryo has a basic right not to be harmed, but PGD potentially harms it without its consent.  This is barbaric.  With any other procedure we would say so, but this seems to be alright because we are dealing with embryos and they are too small and young.  This is ageism and size discrimination.  These are not “uncontroversial stem cells.”  They are anything but.  

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.

Tiny, Poorly-Controlled Study Shows No Benefit for Stem Cell Treatment in Children with Optic Nerve Hypoplasia


Optic nerve hypoplasia (ONH), an underdevelopment of optic nerves that occurs during fetal development, can appear as an isolated condition or as a part of a group of disorders characterized by brain anomalies, developmental delay, and endocrine abnormalities. ONH is a leading cause of blindness in children in North America and Europe and is the only cause of childhood blindness that shows increasing prevalence. No treatments have been shown to improve vision in these children.

RetinaRetina ONH

Because stem cells heal or even regenerate some tissues, some have considered stem cell treatments as an option for this condition.  However, a very small clinical study at Children’s Hospital Los Angeles found no evidence that stem cell therapies improve vision for children with optic nerve hypoplasia (ONH). Their results are reported in the Journal of the American Association for Pediatric Ophthalmology and Strabismus (AAPOS).

Families with a child that has ONH are traveling to China to undergo stem cell treatments that would be illegal in the United States. Because there are presently no viable treatment options available to improve vision in ONH children, such trips are often an act of desperation. The American Association for Pediatric Ophthalmology and Strabismus has also expressed its concern about these procedures, which are usually rather expensive, and have a dubious safety record.

Pediatric neuro-ophthalmologist Mark Borchert, MD, director of both the Eye Birth Defects and Eye Technology Institutes in The Vision Center at Children’s Hospital Los Angeles, realized that a controlled trial of sufficient size was needed to evaluate whether stem cell therapy is effective as a treatment for children with ONH. He agreed to conduct an independent study at the behest of Beike Biotech, which is based in Shenzhen, China and offers a stem cell treatment for ONH. This treatment uses donor umbilical cord stem cells and injects these cells into the cerebrospinal fluid.

Beike Biotech identified 10 children with bilateral ONH (ages 7 to 17 years) who had volunteered to travel to China for stem cell therapy. These patients gave their consent to participate in the study and Children’s Hospital found matched controls from their clinic. However, only two case-controlled pairs were evaluated because Beike Biotech was only able to recruit two patients.

Treatments consisted of six infusions over a 16-day period of umbilical cord-derived mesenchymal stem cells and daily infusions of growth factors. Visual acuity, optic nerve size, and sensitivity to light were to be evaluated one month before stem cell therapy and three and nine months after treatment.

Unfortunately no therapeutic effect was found in the two case-control pairs that were enrolled. “The results of this study show that children greater than 7 years of age with ONH may have spontaneous improvement in vision from one examination to the next. This improvement occurs equally in children regardless of whether or not they received treatment. Other aspects of the eye examination included pupil responses to light and optic nerve size; these did not change following treatment. The results of this research do not support the use of stem cells in the treatment of ONH at this time,” said lead author Cassandra Fink, MPH, program administrator at The Vision Center, Children’s Hospital Los Angeles.

However, confounding factors affect the interpretation of these results because the test subjects received additional alternative therapies (acupuncture, functional electrical stimulation and exercise) while receiving stem cell treatments. They were not supposed to receive such treatments. Additionally, the investigators could not determine the effect of these additional therapies on the subjects’ eyes.

“This study underscores the importance of scientifically testing these procedures to validate them and ensure their safety. Parents of afflicted children should be aware that the science behind the use of stem cell technology is unclear. This study takes a step toward testing this technology and finds no beneficial effect,” said William V. Good, MD, senior associate editor, Journal of AAPOS and Clinical Professor of Ophthalmology and Senior Scientist at the Smith-Kettlewell Eye Research Institute.

Basically, we have an incredibly small study that is also poorly controlled. Because the optic nerve forms during embryonic, fetal and postnatal development, using stem cells to make new nerves seems like a long shot as a treatment.  I better treatment strategy might be to increase the myelination of the optic nerve with neural stem cells, oligodendrocyte precursor cells (OPCs), or Schwann cells.  In general, this study does little to establish the lack of efficacy of such a stem cell treatment.

Faulty Stem Cell Regulation Contributes to Down Syndrome Deficits


People who have three copies of chromosome 21 have a genetic condition known as Down Syndrome (DS). In particular, patients who have an extra copy of a small portion of chromosome 21 (q22.13–q22.2) known as the Down Syndrome Critical Region or DSCR have the symptoms of DS. The DSCR contains at least 30 genes or so and some of them tightly correlate to the pathology of DS. For example, the APP (amyloid protein precursor) gene accounts for the accumulation of amyloid protein in the brains of DS patients. DS patients develop Alzheimer disease-like pathology by the fourth decade of life, and the APP protein is overexpressed in the adult Down syndrome brain. Another gene found in the DSCR called DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) encodes a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and this protein participates in various cellular processes. Overproduction of DYRK1A seems to cause the abnormal brain development observed in DS babies.

Another gene found in the DSCR is called USP16 and this gene encodes a protein that removes small peptides called ubiquitin from other proteins. Ubiquitin attachment marks a protein for degradation, but it can also mark a protein to do a specific job. USP16 removes ubiquitin an either stops the protein from acting or prevents the proteins from being degraded. Overexpression of UPS16 occurs in DS patients, and too much UPS16 protein affects stem cell function.

Michael Clarke, professor of cancer biology at the Stanford University School of Medicine, said, “There appear to be defects in the stem cells in all the tissues we tested, including the brain.” Clarke continued, “We believe USP16 overexpression is a major contributor to the neurological deficits seen in Down Syndrome.” Clarke’s laboratory conducted their experiments in mouse and human cells.

Additional work by Clarke and his colleagues showed that downregulation of USP16 partially rescues the stem cell proliferation defects found in DS patients.

Clarke’s study suggests that drugs that reduce the activity of USP16 could reduce the some of the most profound deficits in DS patients.

This paper also details some of the pathological mechanisms of DS. DS patients age faster and exhibit early Alzheimer’s disease. The reason for this seems to rely on the overexpression of UPS16, which accelerates the rate at which stem cells are used during early development. This accelerated rate of stem cell use burns out and exhausts the stem cell reserves and, consequently, the brains age faster and are susceptible to the early onset of neurodegenerative diseases.

After examining laboratory mice that had a rodent form of DS, Clarke and his coworkers turned their attention to USP16 overexpression in human cells. Clarke collaborated with a Stanford University neurosurgeon named Samuel Cheshier and their study showed that skin cells from normal volunteers grew much more slowly when the Usp16 gene was overexpressed. Furthermore, neural stem cells, which normally clump into little balls of cells called neurospheres, no longer formed these structures when Usp16 was overexpressed in them.

a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.
a, Proliferation analysis, as well as SA-βgal and p16Ink4a staining, of three control and four Down’s syndrome (DS) human fibroblast cultures show growth impairment and senescence of Down’s syndrome cells. b, c, Lentiviral-induced overexpression of USP16 decreases the proliferation of two different control fibroblast lines (b), whereas downregulation of USP16 in Down’s syndrome fibroblasts promotes proliferation (c). d, Overexpression of USP16 reduces the formation of neurospheres derived from human adult SVZ cells. The right panel quantifies the number of spheres in the first and second passages. P < 0.0001. All the experiments were replicated at least twice. Luc, luciferase.

Conversely, when cultured cells from DS patients had their USP16 activity levels knocked down, their proliferation defects disappeared. In Clarke’s words, “This gene is clearly regulating processes that are central to aging in mice and humans, and stem cells are severely compromised. Reducing Usp16 expression gives an unambiguous rescue at the stem cell level. The fact that it’s also involved in this human disorder highlights how critical stem cells are to our well-being.”

Isolation of Pancreatic Stem Cells


There has been a robust debate as to whether or not the pancreas has a stem cell population. Several studies suggested that the pancreatic duct cells could differentiate into hormone-secreting pancreatic cells. Unfortunately, when the cells of the pancreatic duct are marked, they clearly never contribute to regeneration of the pancreas. According to an article that appeared in the journal Developmental Cell by Oren Ziv, Benjamin Glaser, and Yuval Dor entitled “The Plastic Pancreas,” tying off the pancreatic duct kills off the acinar cells, but it leads to a large increase in the number of hormone-secreting beta cells. Something seems to be contributing cells to the adult pancreas. However when lineage studies tried to confirm that the pancreatic duct cells formed the new cells, it failed to find any connection between the new cells in the pancreas and the duct.

Pancreas

Recent experiments from Chris Wright’s lab suggest that the acinar cells are a population of progenitor cells that divide and differentiate into different kinds of pancreatic cell types after injury to the pancreas. A similar result was observed in work by Desai and others. If that’s not odd enough for you, another set of experiments from Pedro Herrera research group has shown once all the insulin-secreting beta cells are killed off, the adjacent glucagon-secreting cells transdifferentiate into insulin-secreting beta cells. Therefore, something interesting is afoot in the pancreas.

All these experiments were done with rodents. Whether or not they are transferable to human remains uncertain. Nevertheless, a fascinating paper in EMBO Journal from Hans Clevers lab at the Hubrecht Institute, Utrecht, Netherlands haws succeeded in culturing pancreatic precursor cells.

Here’s how they did it. Clevers and his crew took the pancreatic duct of mice and partially tied it off. In order to stem cells from the digestive tract to grow, they must upregulate a signaling pathway called the “Wnt” pathway. The Wnt pathway is quiet in the pancreas, but one the pancreas is injured, the Wnt pathway swings into gear and the cells begin to divide.

When Clevers and company dropped pancreatic duct tissue into culture, Wnt signaling activity soared and the cells grew into a mini-organ (organoid) that resembled and tiny pancreas in a culture dish. In fact, a single cell taken from the pancreatic duct could be cultured into an organoid.

Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding. (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.
Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding.  (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.

This experiment shows that there are techniques for growing unlimited quantities of pancreatic cells.  The therapeutic possibilities of this technology is tremendous.  In Clever’s own words, “We have found a way to activate the Wnt pathway to produce an unlimited expansion of pancreatic stem cells isolated from mice.  By changing the growth conditions we can select two different fates for the stem cells and generate large numbers of either hormone-producing beta cells or pancreatic duct cells.”

Can this work with human pancreatic duct cells?  That is the $64,000 question.   Clevers and his groups will almost certainly try to answer this questions next.  If Clevers and his crew can get this to work, then the possibilities are vast indeed.

Mesenchymal Stem Cell Transplantation to Heal Mother’s Childbirth Injury


Occasionally. vaginal birth can lead to injury in the mother. Some of these injuries are relatively light and the mother heals rather quickly, but others can be more severe. Stress urinary incontinence (SUI) affects 4-35% of women who have given birth via vaginal delivery. SUI causes unintentional leakage from the bladder during heavy exercise, laughter, coughing, sneezing, heavy lifting, or jumping. SUI can cause discomfort, embarrassment, and some degree of social isolation. Unfortunately the treatments for SUI range from surgery to physiotherapy and they do little good.

In order to provide better options for mothers, researchers at the Cleveland Clinic’s Department of Biomedical Engineering have used female rats with birth-induced injuries as a model system. In this model system, injections of mesenchymal stem cells improved recovery from childbirth-induced injuries.

Previous work by this research group showed that injected mesenchymal stem cells tended to move into the spleen. However, if the urethra and vagina were damaged by childbirth trauma prior to mesenchymal stem cell injections, the cells targeted the damaged tissues and secreted trophic factors, which stimulated the differentiation and survival of remaining cells, and also induced the mesenchymal stem cells to engraft into the smooth muscles around the urethra and vagina. These activities accelerated and improved recovery of the animals from SUI.

Margot S. Damaser from the Cleveland Clinic said, “Stem cell-based therapy has recently gained attention as a promising treatment for SUI. Stem cell therapies may be more feasible and less invasive than current therapies.”

Other kinds of stem cells have been used to experimentally treat SUI in laboratory animals. Autologous or self-donated muscle stem cells have been used to treat SUI in animals and in human clinical trials. Fat-based stem cells have also been used, but only in animal models.

Damaser believes that mesenchymal stem cells have the added advantage of not being recognized by the immune system and therefore the possibility to implanting stem cells from an unrelated donor is a possibility for older patients.

“Since rat MSCs were used in this study, the results can only be applied to rat models of injury-treated rats,: said Damaser. “Human adult stem cells need to be investigated in future studies to see if these findings also apply to humans.”

Other researchers think that this procedure might serve as a treatment for SUI in older women. “This study provides evidence that mesenchymal stem cell transplantation could favorably impact a side effect of delivery and aging by releasing factors that can influence the urethra and vagina to treat stress urinary incontinence,” said Amit N Patel, director of cardiovascular regenerative medicine at the University of Utah. “Further studies are required to confirm that this animal study translates to humans.”

Induced Pluripotent Stem Cells Do Not Cause Immune Rejection


A paper appeared in the journal PLoS One by Liu and others that showed that heart muscle cells made from induced pluripotent stem cells were rejected by the immune system of mice. The way induced pluripotent stem cells (iPSCs) are made introduces mutations, many of which are harmless. However, mutations that alter the cell surface proteins of iPSC derivatives can cause the immune system of the host to attack and destroy any transplanted cells.

Are adult cells made from iPSC recognized by the immune system? Are the mouse experiments merely an anomaly of the mouse system?

Dr. Jun Takahashi of Kyoto University’s Center for iPS Cell Research and Application and his research group have examined how monkeys respond to implanted derivatives of iPSCs. They made iPSCs from monkey cells taken from the inside of the mouth. Then Takahashi and his group made midbrain-specific neurons from them and transplanted them back into the monkeys. Only a minimal immune response against these cells was observed. However if a monkey received midbrain neurons made from another animal’s cells, then a robust immune response followed.

Therefore, in non-human primates, iPSC derivatives are not rejected by the immune system of the host.

Takahashi said of this experiment, “These findings give a rationale to start autologous transplantation – at least of neural cells – in clinical situations.”  Takahashi’s last statement is critically important – “At least of neural cells.” The brain is an immunologically privileged organ that normally does not have immune cells lurking in its midst. The heart, however, is constantly under immunological surveillance. Therefore, even though this experiment shows that IPSC derivatives are not rejected in non-human primates under these circumstances, there might be circumstances under which they are rejected.

Since there are ways to screen iPSCs and their derivatives for mutations that might sensitize the immune system to the host, such screenings could almost certainly decrease the rate of immunological rejection. Such screening were not done in either this experiment or in the experiments of Liu and others.

New Treatment for Mesothelioma Attacks Cancer Stem Cells


The term mesothelium refers to a membrane that forms the lining of several body cavities such as the pleural cavity that contains the lungs or the peritoneum that contains the gastrointestinal tract. Inhalation of asbestos fibers repeatedly injures the mesothelium, and the cycle of injury and regeneration selects for cells that grow faster and faster. Such conditions predispose people to cancer and cancer of the mesothelium or mesothelioma is a consequence of repeated exposure to asbestos fibers.

Mesothelium

In the United Kingdom (UK), asbestos was banned in 1985, but the number of asbestos-related deaths has climbed from 153 in 1968 to 2,321 in 2009 and epidemiologists have estimated that the number of asbestos-related deaths will continue to rise over the next 20 years, peaking in 2020.

Mesothelioma treatments do not provide a terribly good prognosis. The drug cisplatin alone or in combination with pemetrexed (brand name Alimta) or cisplatin in combination with raltitrexed, but raltitrexed is no longer commercially available for this type of treatment regime. Cisplatin has also been used in combination with gemcitabine or vinorelbine. If cisplatin cannot be used then carboplatin can be substituted. In all cases, survival rates are rather underwhelming.

The importance of this clinical issue to stem cell biology is that mesothelioma is a type of cancer driven by rogue stem cells that grow uncontrollably. To that end, Dean Fennell and his team from Leicester University, UK have conducted a clinical trial to test a new treatment for mesothelioma.

The Meso2 study, conducted by Synta Pharmaceuticals, examined the efficacy of a drug called ganetspib as a treatment for mesothelioma. The trial is being led by Fennell and his research team, and will enroll about 140 patients.

Ganetespib is a unique drug in that it targets a protein called HSP90 (heat shock protein 90). Heat shock proteins help proteins fold and help unfolded proteins refold. They are called heat shock proteins because their expression increases when temperatures are raised. Since cancer cells grow quickly and make large quantities of protein, hamstringing those proteins that fold other proteins can gum up the internal workings of the cell and cause them to die.

HSP90

Fennell said, “We think this is a new way to being able to target mesothelioma. Laboratory tests show ganetespib is extremely active in mesothelioma, and combined with chemotherapy, this treatment could shrink cancers down and improve symptoms for patients.”

There is also a second clinical trial called COMMAND (Control of Mesothelioma with MAintenance Defactinib) is being sponsored by a Verastem, a Cambridge, Massachusetts-based pharmaceutical company and it will test a new drug called defactinib.

Defactinib inhibits a protein called FAK (focal adhesion kinase), which is also crucial for cancer stem cell function and for the conversion of cancer stem cells into tumors.  FAK acts as an adapter between the cell adhesion molecules on the surface of the cell, and the internal skeleton proteins of the cell.  Therefore when the cell attaches to another cell or a substratum, FAK and the proteins associated with it transmits a message to the rest of the cell that the cell has attached to another cell.  For a great website about FAK, see here.

FAK

Defactinib inhibits FAK and prevents the cell from adapting to its environment and since FAK is involved with spread of the cell over its substratum, proliferation of the cell and migration of the cell.  Inhibition of FAK prevents the cell from properly responding to surface stimuli, and the cell stops growing.

The COMMAND trial will enroll some 350-400 people and Fennell’s lab is involved with starting this trial.

Cancer stem cells can cause cancer to return to return after chemotherapy because most chermotherapeutic strategies attack the progeny of cancer stem cells and not the cancer stem cells themselves.  Inhibiting the FAK protein takes away something cancer stem cells crucially need and Fennell hopes that treatments like defactinib or ganetespib will positively help mesothelioma patients.

Stem Cell Behavior in Three-Dimensional Matrices


Scientists from Case Western Reserve in Cleveland, Ohio have used hydrogels (jello-like materials) to make three-dimensional structures that direct stem cell behavior.

Physical and biochemical signals guide stem cell behavior and directs them to differentiate and make tissues like muscle, blood vessels, or bone. The exact recipes to produce each particular tissue remains unknown, but the Case Western Reserve team has provided a way to discover these recipes.

Ultimately, scientists would like to manipulate stem cells in order to repair or replace damaged tissues. They would also like to engineer new tissues and organs.

Eben Alsberg. associate professor of biomedical engineering and orthopedic surgery at Case Western Reserve, who was also the senior author on this research said, “If we can control the spatial preservation of signals, we have be able to have more control over cell behavior and enhance the rate and quality of tissue formation. Many tissues form during development and healing processes at least in part due to gradients of signals: gradients of growth factors, gradients of physical triggers.”

Alsberg and his colleagues have tested their system on mesenchymal stem cells, and in doing so have turned them into bone or cartilage cells. Regulating the presentation of certain signals in three-dimensional space may be a key to engineering complex tissues; such tissues as bone and cartilage. For example, if we want to convert cartilage-making cells into bone-making cells or visa-verse, several different signals are required to induce the stem cells to change into different cell types in order to form the tissues you need.

To test their ideas, Alsberg and coworkers two different growth factors directed the stem cells to differentiate into either bone or cartilage.  One of these growth factors, transforming growth factor-beta (TGF-beta) promotes cartilage formation while a different growth factor, bone morphogen protein-2 (BMP-2).  Alsberg and his crew placed mesenchymal stem cells into an alginate hydrogel with varying concentrations of these growth factors.  Alginate comes from seaweed and when you hit it with ultraviolet light, it crosslinks to form a jello-like material called a hydrogel.   To create gradients of these growth factors, Alsberg developed a very inventive method in which they loaded a syringes with these growth factors and hooked them to a computer controlled pump that released lots of BMP-2 and a little TGF-1beta and tapered the levels of BMP-2 and then gradually increased the levels of TGF-1beta (see panel A below).  

 Fabrication of microparticle-based gradient alginate hydrogels. (A) Photograph of gradient making system. (B) Flow rates of two syringes to pump a linear gradient for a 5 cm length × 2 mm diameter alginate hydrogel. After linear gradient pumping for 3 min, an additional 50 μL of alginate solution, which is the volume from the Y point to the beginning of quartz tube, was further pumped into a spiral mixer for 1 min. (C) Photomicrographs of microparticles in cross-sections of gradient alginate hydrogel segments. Segments 1-10 represent sequential segments of the gel. (D) Quantification of microparticles in each segment of gradient alginate hydrogels.
Fabrication of microparticle-based gradient alginate hydrogels. (A) Photograph of gradient making system. (B) Flow rates of two syringes to pump a linear gradient for a 5 cm length × 2 mm diameter alginate hydrogel. After linear gradient pumping for 3 min, an additional 50 μL of alginate solution, which is the volume from the Y point to the beginning of quartz tube, was further pumped into a spiral mixer for 1 min. (C) Photomicrographs of microparticles in cross-sections of gradient alginate hydrogel segments. Segments 1-10 represent sequential segments of the gel. (D) Quantification of microparticles in each segment of gradient alginate hydrogels.

The result has an alginate hydrogen with mesenchymal stem cell embedded in it that had a high concentration of BMP-2 at one end and a high concentration of TGF-1beta at the other end.  Alsberg also modified the hydrogel by attached RGD peptides to it so that the stem cells would bind the hydrogel.  The peptide RGD (arginine-glycine-aspartic acid) binds to the integrin receptors, which happen to be one of the main cell adhesion protein on the surfaces of these cells.  This modification increases the exposure of the mesenchymal stem cells to the growth factors.  After culturing mesenchymal stem cells in the hydrogel, they discovered that the majority of the cells were in the areas of the hydrogel that had the highest concentration of RDG peptides.  

In another other experiment Alsberg and others varied the crosslinks in the hydrogel.  They used hydrogels with few crosslinks that were more flexible and hydrogels that have quite a few crosslinks and were stiffer.  The stem cells clearly preferred the more flexible hydrogels.  Alsberg thinks that the more flexible hydrogels might show better diffusion of the growth factors and better waste removal.  

“This is exciting,” gushed Alsberg.  “We can look at this work as a proof of principle.  Using this approach, you can use any growth factor or any adhesion ligand that influences cell behavior and study the role of gradient presentation.  We can also examine multiple different parameters in one system to investigate the role of these gradients in combination on cell behavior.”  

This technology might also be a platform for testing different recipes that would direct stem cells to become fat, cartilage, bone, or other tissues.  Also, since this hydrogel is also biodegradable, stem cells grown in the hydrogel could be implanted into patients.  Since the cells would be in the process of forming the desired tissue, their implantation might restore function and promote healing.  Clearly Alsberg is on to something.  

New Drug Prevents Viral Infections in Stem Cell Transplant Patients


Because bone marrow transplant patients have had their bone marrows wiped out with radiation or rather severe drugs, their immune systems tend to be kaput until the transplanted bone marrow stem cells start making new immune cells to reconstitute the immune system. Consequently, bone marrow transplant patients can contract a whole host of truly diabolical diseases.

One disease that shows up with some frequency in bone marrow transplant patients is cytomegalovirus (CMV) infections. CMV can cause pneumonia, diarrhea, digestive tract ulcers, and other problems. Some antiviral drugs do exist (ganciclovir, or its prodrug valganciclovir, foscarnet, and cidofovir), but they can cause kidney dysfunction or bone marrow suppression. Neither of these are desirable side effects. Clearly new drugs are needed (see Ahmed, A. Infect Disord Drug Targets. 2011 Oct;11(5):475-503).

A new clinical trial by researchers at Dana-Farber Cancer Institute and Brigham and Women’s Hospital has tested a drug called CMX001. When bone marrow transplant patients took it shortly after transplant, they were much less likely to contract CMV infections that those who did not take the drug.

The study’s lead author, Francisco Marty from Dana-Farber and Brigham and Women’s said: “With current agents, between 3 and 5 percent of allogeneic transplant patients develop CMV disease within six months of transplantation, and a small number of them die of it. There is clearly a need for better treatments with fewer adverse effects. This clinical trial examined whether the disease can be prevented, rather than waiting for blood tests to show that treatment is needed.”

By the time we become adults, most of use have been infected by CMV. However in most cases our immune systems hold it in check. In stem cell transplant patients, however, the immune system is replaced with those of a donor after receiving sizable doses of chemotherapy. During this period, long-dormant viruses, such as CMV, can reactivate and cause CMV disease. CMV is a type of herpes virus. Herpes viruses do a very good job of keeping a low profile and hiding in various types of cells. Only by treating with an effective anti-viral drug can CMV disease be thwarted.

In this Phase 2 clinical trial, 230 stem cell transplant recipients at 27 different centers across the United States were randomly assigned to either the oral CMX001 group to the placebo group. All patients took the drugs or placebos after their bone marrow transplant procedure and the drugs or placebos were taken for 9-11 weeks.

Those patients that took 100 milligrams of CMX001 twice a week, 10% had a CMV event in which CMV was detectable in the blood and the symptoms of CMV disease appeared. However, 37% of those patients who took the placebo had a CMV event. The most common side effect was diarrhea, which is no surprise given the fragile state of these patients.

“The results show the effectiveness of CMX001 in preventing CMV infections in this group of patients,” said Marty. “Because CMX001 is known to be active against other herpes viruses and against adenoviruses that sometimes affect transplant patients, it may be useful as a preventative or treatmentagent for those infections as well.”

See New England Journal of Medicine, 2013; 369(13): 1227.

Increasing Engraftment Rates of Umbilical Cord Blood Transplantations


Harvard Stem Cell Institute (HSCI) researchers have published initial results of a Phase Ib human clinical trial of a therapeutic that has the potential to improve the success of blood stem cell transplantation. This publication marks a success for the HSCI and their ability to carry a discovery from the lab bench to the clinic. This was actually the mandate for the HSCI when it was founded.

This Phase 1b safety study was published in the journal Blood, and it included 12 adult patients who underwent umbilical cord blood transplantation for leukemia or lymphoma at the Dana Farber Cancer Institute and Massachusetts General Hospital. Each patient received two umbilical cord blood units; one of which was untreated and another that was treated with a small molecule called 16,16 dimethyl prostaglandin E2 (dmPGE2). The immune systems of all 12 patients were successfully reconstituted and their bone marrow tissues were able to make blood cells. However, 10 of the 12 patients had blood formation that was solely derived from those umbilical cord blood cells that had been treated with dmPGE2.

This clinical test is now entering Phase II, during which the HSCI scientists will determine the efficacy of this treatment in 60 patients at 8 different medical centers. They expect results from this trial within 18-24 months.

The success of the HSCI depended on collaborations with scientists at different Harvard-affiliated institutions. These collaborations included 1) Leonard Zon, chair of the HSCI Executive Committee and Professor of Stem Cell and Regenerative Biology at Harvard, and his colleagues, 2) Dana-Farber Cancer Institute and Massachusetts General Hospital, led by hematologic oncologist and HSCI Affiliated Faculty member Corey Cutler, and 3) Fate Therapeutics, Inc., a San Diego-based biopharmaceutical company of which Zon is a founder, sponsored the Investigational New Drug application, under which the clinical program was conducted, and translated the research findings from the laboratory into the clinical setting.

“The exciting part of this was the laboratory, industry, and clinical collaboration, because one would not expect that much close interplay in a very exploratory trial,” Cutler said. “The fact that we were able to translate someone’s scientific discovery from down the hall into a patient just a few hundred yards away is the beauty of working here.”

Gastroenterologists have been interested in dmPGE2 for decades, because it has the ability to protect the intestinal lining from stress. However, its ability to amplify stem cell populations was identified in 2005 during a chemical screen exposing 5,000 known drugs to zebrafish embryos. Wolfram Goessling, MD, PhD, and Trista North, PhD former Zon postdoctoral fellows, were involved in that work.

“We were interested in finding a chemical that could amplify blood stem cells and we realized looking at zebrafish embryos that you could actually see blood stem cells budding from the animal’s aorta,” Zon said. “So, we elected to add chemicals to the water of fish embryos, and when we took them out and stained the aortas for blood stem cells, there was one of the chemicals, which is this 16,16 dimethyl prostaglandin E2, that gave an incredible expansion of stem cells—about a 300 to 400 percent increase.”

The dramatic effects of this molecule on blood stem cells causes Zon, who practices as a pediatric hematologist, consider how this prostaglandin could be applied to bone marrow transplantation. Bone marrow transplantations are often used to treat blood cancers, including leukemia and lymphoma. Bone marrow contains the body’s most plentiful reservoir of blood stem cells, and so patients with these conditions may be given bone marrow transplants to reconstitute their immune systems after their cancer-ravaged bone marrow has been wiped out with chemotherapy and radiation.

Zon designed a preclinical experiment, similar to the one later done with cord blood patients, in which mice undergoing bone marrow transplants received two sets of competing bone marrow stem cells, one set treated with dmPGE2 and a second untreated set.

“What we found was the bone marrow stem cells that were treated with prostaglandin, even for just two hours, had a four times better chance of engrafting in the recipient’s marrow after transplant,” he said. “I was very excited to move this into the clinic because I knew it was an interesting molecule.”

Zon and his team’s then visited the Dana Farber Cancer Institute (DFCI). There, they presented the mouse research at bone marrow transplant rounds and found physicians interested in giving the prostaglandin to patients.

“We basically sat down in a room and we brainstormed a clinical trial based on their scientific discovery, right then and there,” said Farber oncologist Corey Cutler. “They knew that it was something they could bring to the clinic, but they just didn’t know where it would fit. We said, if this molecule does what you say it does, significant utility would lie in umbilical cord blood transplants.”

A cord blood transplant is similar to a bone marrow transplant, but the blood stem cells are not from an adult donor but from the umbilical cord blood of a newborn. The degree of tissue matching is less in an umbilical cord blood transplant than in a bone marrow transplant. The umbilical cord stem cells are young and incipient and the immune system simply does not recognize them as readily as adult cells. Therefore, potentially fatal graft-versus-host disease is less common with umbilical cord blood transplants. About 10-20 percent of stem cell transplantation procedures now use umbilical cord blood. However the main disadvantage of umbilical cord blood transplantations is that the cord blood contains uses smaller amounts of cells, which makes engraftment is more difficult.

Umbilical cord blood transplants fail about 10 percent of the time. Therefore, increasing the procedure’s success would significantly help patients who do not have adult bone marrow donors, including a disproportionate number of non-Caucasian patients in North America. Increasing the engraftment rate would also allow the use of smaller umbilical cord blood units that are potentially better matches to their recipients, increasing the number of donations that go on to help patients.

Fate Therapeutics received the first green light from the US Food and Drug Administration, and the DFCI Institutional Review Board for this clinical trial. Umbilical cord blood processing was done by Dana-Farber’s Cell Manipulation Core Facility, directed by HSCI Executive Committee member Jerome Ritz, MD. There was a stumbling block in that once the human trial was underway with the first nine patients in that the protocol in use, which was developed in mice, did not translate to improved engraftment in humans.

“The initial results were very disappointing,” Cutler said. “We went back to the drawing board and tried to figure out why, and it turned out some of the laboratory-based conditions were simply not optimized, and that was largely because when you do something in the lab, the conditions are a little bit different than when you do it in a human.”

Fate Therapeutics discovered that the human cord blood was being handled at temperatures that were too cold (4-degrees Celsius) for the prostaglandin to biologically activate the stem cells. Therefore even after prostaglandin treatment, the umbilical cord blood did not show enhanced engraftment rates. Fate further demonstrated that performing the incubation of the hematopoietic stem cells at 37-degrees Celsius and increasing the incubation time from 1 hour to 2 hours elicited a much stronger gene and protein expression response that correlated with improved engraftment in animal models.

In running a second cohort of the Phase Ib trial, which included 12 patients, dmPGE2 appeared to enhance the engraftment properties of the blood stem cells in humans and was deemed safe to continue into Phase II. “It’s probably the most exciting thing I’ve ever done,” Zon said. “Basically, to watch something come from your laboratory and then go all the way to a clinical trial is quite remarkable and very satisfying.”