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


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

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

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

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

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

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

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

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

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

Injected Wnt Protein Helps With Muscular Dystrophy


Duchenne muscular dystrophy is a genetic disease that affects one of every 3,500 newborn males. Because the DMD gene is located on the X chromosome, loss-of-function mutations that cause Duchenne muscular dystrophy (DMD) tend to occur in males.

Muscular dystrophy or MS affects skeletal muscles and causes muscle weakness and muscle loss, and unfortunately, the disease often progresses to a state were the muscles are so weak and damaged that even the diaphragm, which is a voluntary muscle, becomes nonfunctional, and the patients dies from an inability to breath.

Recently, Michael Rudnicki, a MS researcher from the Ottawa Hospital Research Institute in Canada, has led a research team that discovered that injections of a protein called “WNT7a” into muscles can increase the size and strength of muscles in MS mice.

Rudnicki is the director of the Regenerative Medicine Program at Ottawa Hospital Research Institute (OHRI), Canada. The results of this work were published on the Nov. 26, 2012, in the Proceedings of the National Academy of Sciences (PNAS).

For these experiments, Rudnicki collaborated with a San Diego-based biotechnology firm known as Fate Therapeutics. Fate Therapeutics specializes in developing pharmaceuticals that are based on stem cell biology, and Rudnicki is one of the founders of this company. Rudnicki hopes to begin a clinical trial of WNT7a for DMD in the near future.

In 2009, Rudnicki and co-workers showed that WNT7a protein is able to stimulate muscle repair by increasing the available supply of a population of muscle stem cells known as “muscle satellite cells.” Muscle satellite cells are located near muscle fibers but they are dormant until they are needed for muscle repair or muscle fiber regeneration. When the muscle is stressed or damaged, the satellite cells increase in number (proliferate) and mature (differentiate).

Muscle Satellite Cells

These newly published findings build on these earlier results. Once injected into the muscles of mice afflicted with DMD, the WNT7a-injected muscles showed significant increases in fiber strength and size. However, Rudnicki and others also found that WNT7a stimulated a two-fold increase in the number of satellite cells in the injected mouse muscles.

Rudnicki was worried that WNT7a was pushing satellite cells to differentiate prematurely, which was disconcerting because such premature differentiation would deplete the muscle satellite population. However, no evidence of premature differentiation was observed. Additionally, WNT7a-injected mouse muscles showed far less contraction-related injury, suggesting that WNT7a has a kind of protective effect on the muscle.

Even though these experiments were done in a mouse model of DMD, would WNT7a also work in a similar fashion in human muscles? To answer this questions, Rudnicki and his colleagues analyzed human muscle tissue from healthy male donors that had been treated with WNT7a. The results showed that the effects of this protein in skeletal muscle are the same in humans as in mice.

To summarize from their own paper: “Our experiments provide compelling evidence that WNT7a treatment counteracts the significant hallmarks of DMD, including muscle weakness, making WNT7a a promising candidate for development as an ameliorative treatment for DMD.”

The remarkable conclusion is that increasing muscle strength by injecting WNT7a into specific, vital muscle groups, such as those involved in breathing, should be considered as a therapeutic approach for this debilitating disease.

2012 in review


The WordPress.com stats helper monkeys prepared a 2012 annual report for this blog.

Here’s an excerpt:

4,329 films were submitted to the 2012 Cannes Film Festival. This blog had 50,000 views in 2012. If each view were a film, this blog would power 12 Film Festivals

Click here to see the complete report.

My deepest thanks to all my readers and best heart-felt wishes for a happy and prosperous new year.

MB

Stem Cells from Human Placenta Repair Damaged Lungs


The placenta does more than provide yet unborn babies with oxygen from the mother’s blood supply; they are also a rich source of stem cells. Vladamir Serikov from the Children’s Hospital Oakland Research Institute in Oakland, California first isolated and characterized “chorionic mesenchymal stem cells” from human placenta in 2009 (see Exp Biol Med 2009 234:813-23), and since that time, his work has been conformed by several other research labs (Cell Stem Cell 2009 5:385-95 & Dev Biol 2009 327:24-33). Now Serikov and his research team have used his hCMSCs to repair damaged lungs in laboratory animals.

In this present publication, the Serikov team grew placenta-derived hCMSCs in culture and discovered that these grew like gangbusters. After 100 doublings, the cells showed no signs of giving up and their chromosomes show no signs of shortening, which is a symptom of aging when cells are grown in culture. Stem cells, have the ability to properly maintain the ends of their chromosomes and not show these signs of aging. Serikov’s hCMSCs have this definitive stem cell ability.

Next, the Oakland-based team tried to get these hCMSCs to differentiate into various cell types using published protocols. The hCMScs formed fat cells, bone cells, blood vessel-like cells, and liver cells in culture. When treated with a molecule called nerve growth factor, hCMSCs even sprouted nerve cell-like extensions and expressed genes common found in neurons (the cells that make a propagate nerve impulses).

To determine if these cells had the capacity to heal damaged tissue, Serikov and co-workers treated human lungs that were donated by a deceased individual but were denied for transplantation with a bacterial toxin that tends to really screw up the lungs. One lobe of the lung was treated with toxin only but the other side was treated with the toxin and five million hCMSCs. The side that received only the toxin showed damage to the lining of the lungs that was reflected in poor gas exchange and high fluid uptake by the lung tissue, but the side that received the hCMSCs was able to properly pump out the liquid and maintain the structure of the lung. When this same assay was applied to cultured lung tissue from humans, it was clear that the hCMSCs helped repair the columns of lung cells through the modicum of growth factors that they secrete. Certainly, hCMSCs have the capacity to heal the lungs after they are ravaged by a deadly bacterial toxin.

Two other experiments underscored the therapeutic capacity of these cells. When hCMSCs were infused into mice after the animals have been hit with high doses of radiation, they took up residence in multiple tissues, including the intestine, lungs, brain, and liver. Therefore, hCMSCs can not help heal tissues by means of what they secrete (so-called paracrine mechanisms), but by incorporating into tissues and becoming an integral part of it. Finally, when hCMSCs were implanted into mice and examined one year later, none of the mice showed any signs of tumors. There were also no signs of pain, heart problems, distress, fever, or weight loss. Therefore, these cells seem to be well tolerated, and do not have a high capacity for tumor formation.

These preclinical studies should give way to studies in larger animals, and if those are successful, hopefully, the first human clinical trials with these amazing stem cells that come from an abundant source, the human afterbirth.

See Igor Nazarov et al., “Multipotential Stromal Stem Cells from Human Placenta Demonstrate High Therapeutic Potential,” Stem Cells Translational Medicine 2012 1:359-72.

Stem Cells from Your Nose to Treat Parkinson’s Disease?


Parkinson’s disease (PD) is a neurodegenerative disease that is a global problem and the incidence of PD increases as the population lives longer and longer. PD results from the loss of dopamine-making neurons in the midbrain. The main treatment for PD is a drug called L-DOPA, which can cross the blood-brain barrier, but this drug decreases in effectiveness as time progresses because the neurons become less sensitive to the drug and L-DOPA does not prevent dopamine-making neurons in the midbrain from dying.

substantia nigra

Experimental stem cell treatments of PD have used embryonic stem cells and induced pluripotent stem cells that were differentiated into dopamine-making neurons and transplanted into the midbrain of rodents that suffered from drug-induced PD. Unfortunately, even though symptom relief was observed, tumors were formed in many of these animals in these experiments. Until a more sure-fire way is discovered to identify and isolated dopamine-secreting neurons from other cells types, this approach will always seem too dangerous for clinical trials. References: Embryonic stem cells – Brederlau, et al., Stem Cells 2006 24:1433-40; Sonntag KC, et al. (2007) Stem Cells 25:411–418. and Roy, et al., Nature Medicine 2006 12:1259-68. Induced Pluripotent Stem Cells – Chang, et al., Cell Transplant 2012 21:313-32.

A paper that used induced pluripotent stem cells and differentiated them into dopamine-producing neurons which were transplanted into the brains of PD rodents did not produce tumors (see Hargus, et al., Proceedings of the National Academy of Sciences USA 2010 107:15921-6). It is likely that the stringent isolation procedures employed in this paper decreased tumor incidence (48 different cell lines were generated in this paper and none of them produced detectable tumors).

These experiments show that stem cell-based treatments for PD are feasible. The key is to find the right cell. Well, an old bromide says that “your nose knows.” Maybe this is true in the case of PD treatments. In the nose resides a tissue known as the “olfactory epithelium,” (OE) which is a source of stem cells that can form neurons. OEs can be harvested with minimally invasive nasal surgery (see Winstead W, et al., American Journal of Rhinology 2005 19:83-90). In fact, more than 150 different patient-specific cell lines of “human olfactory neural progenitor” (hONPs) cells have been established from cultures of adult olfactory epithelial cells taken from cadavers (see Roisen FJ, et al., Brain Research 2001 890:11-22).

 The olfactory epithelium lines the posterodorsal nasal cavity immediately inferior to the cranial cavity. The normal epithelium is composed of a handful of cell types: sustentacular cells (Sus), microvillar cells (a supporting cell variant), olfactory sensory neurons – both mature and immature (OSNs), globose basal cells (GBCs), horizontal basal cells (HBCs), Bowman’s duct and gland cells. Deep to the basal lamina, the fascicles of the olfactory axons are ensheathed by the specialized glia of the olfactory nerve, the olfactory ensheathing cells (OECs). In addition, stromal cells (fibroblasts) of the lamina propria secrete signals that regulate epithelial assembly and turnover.
The olfactory epithelium lines the posterodorsal nasal cavity immediately inferior to the cranial cavity. The normal epithelium is composed of a handful of cell types: sustentacular cells (Sus), microvillar cells (a supporting cell variant), olfactory sensory neurons – both mature and immature (OSNs), globose basal cells (GBCs), horizontal basal cells (HBCs), Bowman’s duct and gland cells. Deep to the basal lamina, the fascicles of the olfactory axons are ensheathed by the specialized glia of the olfactory nerve, the olfactory ensheathing cells (OECs). In addition, stromal cells (fibroblasts) of the lamina propria secrete signals that regulate epithelial assembly and turnover.

Human ONPs can also be differentiated into dopamine-making neurons in culture (Zhang X., et al., Stem Cells 2006 24:434-442). Therefore, these cells should be candidate stem cells for making treatments for PD.

Fred Roisen and his cohorts from the University of Louisville, Kentucky, has used hONPs to treat rats with drug-induced PD.  In their paper, Roisen and others used cultures of hONPs and then proceeded to differentiate them into dopamine-making neurons. Then they transplanted these cells into the midbrains of rats that had been treated with 6-hydroxydopamine, which is a drug that kills off dopamine-making neurons in the midbrain and induces PD. However, it is important to understand that the dopamine-producing neurons were only destroyed on the right side of the brain, thus leaving the left side intact. When they stem cells were injected into the midbrains of these rats, they were only injected into the right side, the side that had been damaged by the drugs. Therefore, the right side of the midbrain served as a control throughout these experiments.

The behavioral tests on these PD rats determined if the transplanted hONPs helped decrease the effects of PD. In all three behavioral tests, the hONP-injected rats showed significant improvements over the untreated rats. Were these improvements due to the formation of new dopamine-making neurons? The answer is a clear yes, since postmortem analyses of the brains of these rats showed that the hONP-injected rats not only showed the presence of dopamine-making neurons on the injected side, but the levels of dopamine production in the right side of the brain as compared to the left side of the brain were higher in the hONP-injected animals, even though they were three times lower than those dopamine levels found in the left side of the midbrain.

This experiment shows that hONPs should be considered serious players in the treatment of PD. In none of the transplanted animals were tumors found. Therefore, hONPs seem to be safe, they are easily acquired, and they have the capacity to form dopamine-making neurons. The goal should be to jack up the dopamine levels in the transplanted cells.

See Meng Wang, Chengliang Lu, Fred Roisen, “Adult human olfactory epithelial-derived progenitors: A potential autologous source for cell-based treatment for Parkinson’s disease,” Stem Cells Translational Medicine 2012 1:492-502.

Cultured Human Kidney Cells Improve Chronic Kidney Injury


Chronic kidney disease (CKD) is extremely expensive to treat and also leads to additional complications, such as heart and circulatory troubles. In general, when you have CKD, your life is a drag. ~13% of the worldwide population has CKD and in the US alone, the estimated Medicare costs for the treatment of this disease is $42 million.

There are drugs that can treat CKD, but these drugs (statins, angiotensin 2 receptor blockers and angiotensin converting enzyme (ACE) inhibitors, and erythropoietin to improve anemia) must be given for some time and at high doses before their effects become apparent.

The hormone erythropoietin (EPO) is made by the kidneys, and EPO signals to the bone marrow to produce more red blood cells. Recombinant versions of EPO are given to anemia patients, and have also been used illicitly in aerobic athletics to artificially boost red blood cell production (e.g., Floyd Landis, Lance Armstrong, etc.). However, EPO has another function in CKD in that EPO administration seems to protect the kidney from damage caused by low oxygen delivery. EPO production is quite low in CKD patients and this might play a role in the problems encountered by CKD patients.

The laboratory of James Yoo from the Institute for Regenerative Medicine at Wake Forest University has investigated the ability of cultured, human kidney cells that express EPO to improve kidney structure and function in a rodent model of CKD.

In this experiment, Yoo and his coworkers cut off the blood supply to the kidneys of hairless rats and then fed them the antibiotic gentimicin for a day (five doses). 8-10 weeks after this treatment, kidney function was reduced and the rats had all the signs of CKD.

Next, Yoo and others injected into the kidneys of these rats cultured human kidney cells. One group of rats received injections of buffer into their kidneys, some received cultured human kidney cells, and another group received cultured kidney cells that had been engineered to express EPO. These kidney cells came from a discarded organ from a 51-year-old human organ donor.

The kidneys of these rats were assayed for function and structure. One of the features of CKD is lots of protein in the urine. When the levels of protein in the urine of these rats was examined 1, 4, and 12 weeks after they had received infusions of the kidney cells, along with other markers of kidney damage, the levels of protein in the urine were high in the rats injected with buffer, lower in those injected with cultured kidney cells, and much mower in those injected with the EPO-expressing kidney cells. Also, hemoglobin levels (hemoglobin is the protein in red blood cells that ferries oxygen from the lungs to the tissues) were significantly higher in the rats injected with EPO-expressing kidney cells.

Next, Yoo and his colleagues examined the kidneys for inflammation and scarring. Scarring is relatively easy to detect because there are tissue stains that will highlight scarring (e.g., Masson’s Trichome stain). Once again, the buffer injected kidneys were loaded with scars, the kidney cell-injected kidneys had much less scarring and the rats injected with EPO-expressing kidney cells had even less scarring in most of the kidney. Also the presence of inflammatory cells in the kidney, which is indicative of cell damage, was significantly lower in kidneys injected with either type of cultured kidney cell. As an added bonus, Yoo’s group examined the markers of kidney cell damage (8-OHdG) and these were lower in the kidneys injected with cultured human kidney cells.

Did the injected cells hang around in the kidneys and contribute to the kidney? The answer seems to be, only a bit. When the rat kidneys were checked for human cells 12 weeks after injection, very few human kidney cells were found.

These experiments suggest that cultured kidney cells, particularly EPO-expressing ones, can initiate regeneration in damaged kidneys. While this experimental protocol requires adjustment and tweaking, it suggests a potential therapeutic strategy for treating CKD patients.

Bringing the Dysfunctional Bone Marrow of Diabetics Back to Life


One of the most insidious consequences of diabetes mellitus is its nocuous effects on the ability of the circulatory system to repair itself. The small vessels within our organ undergoes constant remodeling and repair in response to the wears and tears of life. Diabetes seriously decreases the ability of the circulatory system to execute this repair.

This day-to-day circulatory repair relies upon a group of bone marrow stem cells known as “bone marrow-derived early outgrowth cells or EOCs, and EOCs from patients with diabetes mellitus are impaired in their ability to repair the circulatory system (See Fadini GP, Miorin M, Facco M et al. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol 2005;45:1449–1457).

Is there are way to reverse this destructive trend? There is a protein known as SIR1, which stands for Silent Information Regulator 1. This gene product regulates aging and the formation of blood vessels, and might very well play a role in the diabetes-induced decrease in blood vessels repair and EOC impairment.

To answer this question, the laboratory of Richard E. Gilbert from the University of Toronto, Toronto, Ontario, Canada, used drugs to increase SIR1 activity in EOCs from diabetic rodents to determine if such treatments abrogated the diabetes-induced decrease in EOC function.

Gilbert’s lab isolated EOCs from normal and diabetic mice and subjected them to a variety of tests. They determined how many blood vessel-inducing molecules were made by these cells, and the EOCs from diabetic mice produced much less of such molecules and had reduced levels of SIR1.  EOCs from diabetic mice also performed poorly in blood vessel-making assays in culture dishes.

Would kicking up the levels of SIR1 in EOCs from diabetic mice improve the function of their EOCs? By using a drug to increase SIR1 activity in EOCs, GIlbert and others were able to show that increased SIR1 activity in EOCs from diabetic mice restored their production of blood-vessel-inducing molecules, and also improved their ability to make blood vessels in culture.

This extraordinary publication shows that the diminished abilities of bone marrow from diabetic or aged individuals is not irreversible. Perhaps research such as this can spur the discovery of drugs that reserve the decline of SIR1 activity in diabetics and aged patients to beef up their circulatory self-repair mechanisms.

See Darren A. Yuen, et al., “Angiogenic Dysfunction in Bone Marrow-Derived Early Outgrowth Cells from Diabetic Animals Is Attenuated by SIRT1 Activation,” Stem Cells Translational Medicine 2012;1:921–926.