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 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.


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.

Mesenchymal Stem Cell Transplantation Improves Heart Remodeling After a Heart Attack

Stem cell scientists from the University of Maryland, Baltimore have used bone marrow mesenchymal stem cells (MSCs) to treat sheep that had suffered a heart attack. They found that the injected stem cells prevented the heart from deteriorating.

This work was a collaboration between the laboratories of Mark Pittenger, ZhonGjun Wu and Bartley Griffith from the Department of Surgery and the Artificial Organ Laboratory.

After a heart attack, the region of the heart that was deprived of oxygen undergoes cell death and is replaced by a heart scar. However, the region next to the dead cells also undergo problematic changes. The cells in these regions adjacent to dead region must contract more forcibly in order to compensate for the noncontracting dead region. These cells enlarge, but some undergo cell death due to inadequate blood supply. There are other changes that can occur, such as abnormalities in Calcium ion handling and poor contractability.

Thus, the problems that result from a heart attack can spread throughout the heart and cause heart failure. In this experiment, the U of Maryland scientists injected MSCs into the sheep hearts four hours after a heart attack to determine if the stem cells could prevent the region adjacent to the dead heart cells from deteriorating.

In this experiment, bone marrow MSCs were isolated from sheep bone marrow and put through a battery of tests to ensure that they could differentiate into bone, cartilage, and fat. Once the researchers were satisfied that the MSCs were proper MSCs, they induced heart attacks in the sheep, and then injected ~200 million MSCs into the area right next to the region of the heart that died.

After 12 weeks, tissue biopsies from these sheep hearts were taken and examined. Also, the sheep hearts were measured for their heart function and structure.

The sheep that did not receive any MSC injections continued to deteriorate and showed signs of stress. The cells adjacent to the dead region expressed a cadre of genes associated with increased cell stress. Furthermore, there was increased cell death and evidence of scarring in the region adjacent to the death region. There was also evidence of Calcium ion-handling problems in the adjacent tissue and increased cell death.

On the other hand, the hearts of the sheep that had received injections of MSCs into the area adjacent to the dead region showed a reduced expression of those genes associated with increased cell stress. Also, these hearts contracted better than those that had not received stem cell injections. There was also less cell death, less scarring, and no evidence of Calcium ion-handling problems.

Changes that occur in the heart after a heart attack are collectively referred to as “remodeling.” Remodeling begins regionally, in those areas near the dead heart cells, but these deleterious changes spread to the rest of the heart, resulting in heart failure. The injections of MSCs into the area next to the dead region clearly prevented remodeling from occurring.

This pre-clinical study is a remarkable study for another reason: the MSCs used in this study were allogeneic. Allogeneic is a fancy way of saying that they did not come from the same animal that suffered the heart attack, but from some other healthy animal. Therefore, the delivery of a donor’s MSCs into the heart of a heart attack patient could potentially prevent heart remodeling.

The main problem with this experiment is that the MSCs were injected directly into the heart muscle. In humans, such a procedure requires special equipment and carries potential risks that include perforation of the heart wall, rupture of the heart wall, or further damaging the heart muscle. Therefore, if such a technology could be adapted to a more practical delivery system in humans, then certainly human clinical trials should be forthcoming.

See Yunshan Zhao, et al., “Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction.” Stem Cells Translational Medicine 2012;1:685-95.

Merry Christmas to All My Readers!!

Luke 2:1-20:

In those days Caesar Augustus issued a decree that a census should be taken of the entire Roman world. (This was the first census that took place while[a] Quirinius was governor of Syria.) And everyone went to their own town to register.

So Joseph also went up from the town of Nazareth in Galilee to Judea, to Bethlehem the town of David, because he belonged to the house and line of David. He went there to register with Mary, who was pledged to be married to him and was expecting a child. While they were there, the time came for the baby to be born, and she gave birth to her firstborn, a son. She wrapped him in cloths and placed him in a manger, because there was no guest room available for them.

And there were shepherds living out in the fields nearby, keeping watch over their flocks at night. An angel of the Lord appeared to them, and the glory of the Lord shone around them, and they were terrified. But the angel said to them, “Do not be afraid. I bring you good news that will cause great joy for all the people. Today in the town of David a Savior has been born to you; he is the Messiah, the Lord. This will be a sign to you: You will find a baby wrapped in cloths and lying in a manger.”

Suddenly a great company of the heavenly host appeared with the angel, praising God and saying,

“Glory to God in the highest heaven,
and on earth peace to those on whom his favor rests.”
When the angels had left them and gone into heaven, the shepherds said to one another, “Let’s go to Bethlehem and see this thing that has happened, which the Lord has told us about.”

So they hurried off and found Mary and Joseph, and the baby, who was lying in the manger. When they had seen him, they spread the word concerning what had been told them about this child, and all who heard it were amazed at what the shepherds said to them. But Mary treasured up all these things and pondered them in her heart. The shepherds returned, glorifying and praising God for all the things they had heard and seen, which were just as they had been told

A very Merry Christmas to all.  God Bless You – All of You!!!

Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate The Healing of Large Skin Wounds

Bioprinting is a contrived term that describes the deposition of cells on surfaces by means of inkjet printer technology. Because the inkjet squirts small quantities of ink in a precisely specified shape and pattern, inkjets can be adapted to the application of cells on living surfaces or on scaffolds fashioned in the form of living organs or tissues.

Shay Soker at the Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina, has published a remarkable study that uses inkjet technology to deposit stem cells over large skin wounds. His study shows that bioprinting is a potentially very efficient way to deliver stem cells to wounds.

There are on estimate a half a million burns treated in the US each year. Extensive burns and so-called full thickness skin wounds are usually very traumatic for patients. The mortality rates of burns are about 5% and cost ~2 billion per year. Present strategies for treating burns tend to produce extensive scarring and relatively poor cosmetic outcomes.

Tissue engineering approached have the potential to provide more effective treatments for such injuries. Graft products such as Dermagraft and TransCyte from Advanced BioHealing and Apligraft from Organogenesis are cellularized graft products composed or a polymer scaffold that is seeded with cells. Unfortunately, these are expensive to make. Cell spraying and bioprinting, which deposits cells encased in hydrogel spheres all around the wound are a cheaper and potentially more attractive approach to wound therapy.

Soker’s team used stem cells from amniotic fluid and mesenchymal stem cells for this experiments. These stem cells were grown in culture, mixed in fibrin-collagen hydrogels, and bioprinted to surgically-produced wounds on the backs of hairless (nude) mice. The wounds all closed at approximately the same rate over a two-week period for those wounds treated with amniotic-fluid stem cells or mesenchymal stem cells. Wound closing was slow for those treated with only hydrogels.

Amniotic Fluid Stem Cells
Amniotic Fluid Stem Cells

After the wounds closed, biopsies of the wounds showed that the wounds that had been treated with amniotic fluid stem cells were filled with small blood vessels. Wounds bioprinted with mesenchymal stem cells did not have quite as many blood vessels as those seen in mice treated with amniotic stem cells, and those treated only with hydrogels had hardly any. However, when the biopsies were examined in detail to find the stem cells, they were not to be found. Therefore, the stem cells were not incorporated into the wounds, but induced healing through molecules that they secreted.

Not satisfied with this, Soker and his colleagues examined the gene expression patterns of the amniotic fluid stem cells and compared them to the gene expression patterns of mesenchymal stem cells. As expected, the amniotic fluid stem cells had oodles and oodles of growth factors. Fibroblast growth factors, Insulin-like growth factors, Vascular endothelial growth factor, Hepatic growth factor, and several others were made by amniotic fluid stem cells. Mesenchymal stem cells made their fair share of growth factors, but not nearly as many ans their amniotic fluid counterparts.

From these experiments, Soker concluded that even though bioprinting is a new technology, is can deliver cells effectively to surface wounds. Also, the stem cells do not directly contribute to the healing of the wound, but induce other cells to migrate into the wound and heal it. The delivery of bioprinted cells in hydrogels has the potential to rebuild a tissue from the ground up.

See Aleksander Skardai, et al., “Bioprinted Amniotic-Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds,” Stem Cells Translational Medicine 2012;1:792-802.

John Gurdon Embraces Human Cloning

Wesley Smith has reported that Nobel Laureate John Gurdon, who shared the Nobel Prize in Medicine this year with Japanese induced pluripotent stem cell discoverer Shinya Yamanaka, has come out in favor of human cloning.

From the story in the Daily Mail:
‘I take the view that anything you can do to relieve suffering or improve human health will usually be widely accepted by the public – that is to say if cloning actually turned out to be solving some problems and was useful to people, I think it would be accepted,’ he said. During his public lectures – which include speeches at Oxford and Cambridge Universities – he often asks his audience if they would be in favour of allowing parents of deceased children, who are no longer fertile, to create another using the mother’s eggs and skin cells from the first child, assuming the technique was safe and effective.

‘The average vote on that is 60 per cent in favour,’ he said. ‘The reasons for “no” are usually that the new child would feel they were some sort of a replacement for something and not valid in their own right. ‘But if the mother and father, if relevant, want to follow that route, why should you or I stop them?’


Smith then quotes from his magnificent book “Consumers Guide to a Brave New World,” which all my readers to RUN out to buy and read over and over again:

Scientists would have to clone thousands of embryos and grow them to the blastocyst stage [one week] to ensure that part of the process leading up to transfer into a uterus could be “safe,” monitoring and analyzing each embryo, destroying each one in the process. Next, cloned embryos would have to be transferred into the uteruses of women volunteers [or implanted in an artificial womb]. The initial purpose would be analysis of development, not bringing the pregnancy to a live birth. Each of these clonal pregnancies would be terminated at various points of development, each fetus destroyed for scientific analysis. The surrogate mothers would also have to be closely monitored and tested, not only during the pregnancies but also for a substantial length of time after the abortions.

Finally, if these experiments demonstrated that it was probably safe to proceed, a few clonal pregnancies would be allowed to go to full term. Yet even then, the born cloned babies would have to be constantly monitored to determine whether any health problems develop. Each would have to be followed (and undergo a battery of tests both physical and psychological) for their entire lives, since there is no way to predict if problems [associated with gene expression] might arise later in childhood, adolescence, adulthood, or even into the senior years.


Smith, in my view, is spot on. Therapeutic cloning will not stop at using cloned blastocysts to make patient-specific embryonic stem cell lines. The reason for this is that even though cells made from differentiated embryonic stem cells can have therapeutic value, such cells can also be rejected by the immune system of the host animal. A much more fail-safe way to do this experiment is to gestate the embryos to the fetal stage and use the fetal tissues.

Once we go down the road of cloning and destroying embryos just to make embryonic stem cell lines from them, what’s to keep us from aborting fetuses just to get their cells? This slippery slope is real and speaks volumes, none of it good, about a society that sacrifices its youngest and more vulnerable members to serve the needs of others. It cheapens human life to the nth degree and at its lowest point, it simple murder.

Gurdon, however, speaks of reproductive cloning to replace children lost through tragedy. While I can appreciate the sentiment, sentiment is an extremely poor reason basis for ethics. Folks, biology is not destiny. Cloning experiments in animals have shown us that even cloned embryos made from material taken from the same mother, that are genetically identical are neither identical to their mothers nor are they identical to each other. Random events that occur during development and the way each individual responds to their environment shapes them in a unique manner. The cloned sheep Dolly was completely unlike her cloned siblings in personality, behavior, or overall appearance. The same can be said for CC (for “Carbon Copy”), the first cloned cat, which looked unlike her mother and had a very different personality.

Yet these cloned children are asked from the second they are born to replace another child who is unlike them. The cloned child is a human person and while the right for each person to be authentically who there are in an inherent right of all human beings, this very right is denied these cloned kids – they are born for the very reason that they can be someone else. This is a violation of everything it means to be human, and it is the very reason no good thing can come from human cloning.

Gurdon is a brilliant scientist, but as we have seen before, great scientists sometimes make terrible ethicists.

A Patient-Friendly Way to Make Stem Cells

Scientists at Cambridge University in the laboratory Amer Ahmed Rana have used blood samples to isolate cells from which patient-specific stem cells were made. Because blood samples are far more routine than tissue or organ biopsies, they can provide a much more patient-friendly way to secure material for the production of patient-specific stem cells.

Induced pluripotent stem cells (iPSCs) are made from adult cells by genetic engineering techniques that introduce four specific genes into them. The adult cells then de-differentiate to a more developmentally primitive state and if these cells survive and are successfully cultured, they will form an iPSC line.

Rana and his co-workers cultured blood drawn from several heart patients to isolate a blood cells known as a “late outgrowth endothelial progenitor cell” or L-EPC. Endothelial cells are those cells that compose blood vessels, and endothelial progenitor cells or EPCs are the stem cell population that make endothelial cells. EPCs are found in bone marrow, but some are also found in the peripheral circulation.

There are two main types of EPCs: early-outgrowth and late-outgrowth EPCs. Early-outgrowth EPCs are among the first cells to form spindle-shaped clusters of cells only a few days after being placed in culture. Early-outgrowth EPCs secrete high levels of blood vessel-inducing molecules, but they have only a limited ability to proliferate. They also are able to ingest bacteria, like other white blood cells. Late outgrowth EPCs are much rarer and they grow very well in culture, but are unable to ingest bacteria. They also can form capillaries and repair damaged blood vessels when injected into laboratory animals. There is a debate as to whether or not these cells come from the bone marrow or are dislodged from blood vessels.

Rana and his colleagues have designed a protocol for converting L-EPCs into iPSCs that can then be differentiated into heart, or blood vessel cells rather easily. This practical and rather efficient method does not require tissue biopsies, which are painful and impractical in very young or very old patients, and only requires the cells available from a single, routine blood sample.

Also, because blood samples can be efficiently and safely frozen, the cells from the blood sample can be locked in time for later use, when the patient needs regenerative treatments. The ease of this procedure should, Rana hopes, push it further toward human clinical trials in the near future.

Growth Factors to Heal the Heart

When the heart suffers a heart attack, local areas of the heart experience cell death as a result of blockage in a coronary vessel. The cell death is followed by local inflammation which causes further cell death and produces a heart scar. This produces a situation in which a portion of the heart does not contract and also does not conduct impulses to beat. Can this dead heart tissue live again?

Several experiments have used stem cells to refurbish the dead heart tissue, and a variety of different stem cells can clearly produce new heart cells that help the heart beat better. Can growth factors that stimulate cell growth and division do a similar job?

Just injecting growth factors into the bloodstream will not do because the growth factors will not spend any appreciable time in or around the heart cells. Is there another way to do it? Yes. The answer is hydrogels.

Hydrogels are semi-solid materials that can be made and in which the growth factors can be embedded. The hydrogels are gradually degraded while they release growth factors into the heart tissue. The slow but stead release of various growth factors can induce the heart to heal itself.

Works from the laboratory of Michael E. Davis at Georgia Institute of Technology and Emory University School of Medicine in Atlanta, Georgia have published a paper in PLoS ONE describing this very strategy. Using rats that had suffered heart attacks, Davis and his group applied a polyethylene glycol-based hydrogel laced with two growth factors, hepatic growth factor (HGF) and vascular endothelial growth factor (VEGF) to the hearts of these animals.

There were no immediate effects to the application of these hydrogels as determined by electrocardiograms. However, with the passage of time, some remarkable changes to the hearts of these rats were observed. Three weeks after the application of hydrogels to rat hearts, animals treated hydrogel material only, injected with growth factors only showed no significant improvement over those rats that were not injected with anything. But those rats whose hearts had been injected with hydrogels laced with VEGF showed a 50% increase in blood vessel density and those injected with hydrogel imbued with HGF and VEGF showed a 100% increase in blood vessel density. These same rats also showed a huge reduction in the size of the heart scar (41.5 % vs 13.9% fibrosis), and also showed significant increased in heart function after three weeks.

Why did these growth factors work so well? Several experiments conducted by Davis’ group showed that the stem cell population in the heart, the cardiac progenitor cells or CPCs, were pitched into overdrive by the growth factors, In short, in the presence of these two growth factors, the cells went nuts. They went to area where the hydrogel had been applied and made new heart muscle cells and blood vessels.

Therefore, these two growth factors can be applied to the heart to elicit healing within the heart after a heart attack. The hydrogels keep the growth factors there and release them slowly so tat they can perform their healing magic.

Hopefully this experiment will lead to preclinical studies in larger animals (pigs and sheep), and then, hopefully, clinical trials in human patients.  See Salimath AS, et al., PLoS ONE 2012 7(11) e50980.

Mesenchymal Stem Cells Found Around Blood Vessels in the Liver

Mesenchymal stem cells (MSCs) are found throughout the body and it is possible that every organ in our body has a MSC population. MSCs have the ability to differentiate into three main tissues: bone, fat and cartilage. However, the efficiency of this differentiation differs from one MSC population to another. Also, some MSCs can form smooth muscle for blood vessels and there is even evidence that MSCs can form blood vessels under certain conditions (for example, see Wingate K, Bonani W, Tan Y, Bryant SJ, Tan W. Acta Biomater. 2012 8(4):1440-9. doi: 10.1016/j.actbio.2011.12.032).

One of the places MSCs are usually found is around blood vessels. MSCs like to hang out on the outside of blood vessels in some tissues, and for this reason, MSCs are sometimes called “perivascular” stem cells.

One organ that has a stem cell population is the liver, but there is disagreement as to where they reside. Now a new publication has established that cells that hang out near blood vessels in liver are the MSC population in liver.

Eva Schmelzer from the McGowan Institute for Regenerative Medicine at the University of Pittsburgh has published a fine paper in the journal Stem Cells and Development detailing, with the help of her trusty laboratory colleagues, the characterization of liver MSCs.

Briefly, Schmelzer and her colleagues obtained fetal and adult lover tissue from tissue suppliers and minced them up, digested them with the appropriate enzymes, pushed them through cell strainers and then destroyed all the contaminating red blood cells. The remaining cells were grown in a cell culture medium. The stem cells would outgrow all the other cells, which would make their isolation and purification easy.

To purify the cells, Schmelzer’s co-workers used a technique called “flow cytometry.” When they had purified the liver MSCs, they set about characterizing them.

The liver MSCs grew quite well in culture and also grew quickly. They also expressed lots of surface proteins normally found on MSCs, confirming that they are MSCs. When gene expression experiments examined what genes these MSCs expressed, they expressed some smooth muscle genes and a several other genes enriched in cells near blood vessels. When Schmelzer examined cross sections of liver to determine where these cells are located, she found them curled up next to blood vessels.

In culture, the liver MSCs did not make very good cartilage or fat. However, they did make very good smooth muscle and bone. The efficiency of MSC differentiation tends to depend on where they were isolated. The rule of thumb is that MSCs most easily differentiate into those tissues that are closest to their own tissue of origin. Therefore, we would expect bone marrow MSCs to make better bone and cartilage than fat-based MSCs, and we would expect fat-based MSCs to make better fat than bone or liver-based MSCs. The ability of liver MSCs to be so good and making bone might be a little surprising, but when we consider that bone marrow stem cells begin their lives in the liver before they migrate to the bone marrow, perhaps this finding makes more sense.

In short, the adult and fetal liver contain a MSC population that is found on the outside of the blood vessels and these cells have an excellent capacity to make bone and smooth muscle for blood vessels. Thus liver biopsies might provide do more than provide material for diagnostic purposes – they might secure cells for regenerative purposes.

The Stem Cell blog has this fine piece on the California Stem Cell Agency and its governing board. It is no secret that there are massive conflicts of interest on the board and until these are eliminated, the agency will lack public confidence and credibility. The National Institutes of Health warned them about this a while a ago, but the agency seems blithely unaware of any problem.

The Stem Cell Blog

ImageStem cell agency needs to shed conflicts
California‘s stem cell agency understands the importance of attacking chronic problems. So if it wants to survive beyond 2014, it should heed the Institutes of Medicine‘s advice to eliminate conflicts of interest on its board — and do it before awarding the remaining $1.2 billion of the $3 billion voters approved for stem cell research.

The California Institute for Regenerative Medicine asked the prestigious Institutes of Medicine, the health arm of the National Academy of Sciences, to evaluate its operations. One of the findings was that the vast majority of the agency’s 29 board members stand to benefit in various ways from their decisions on awarding research grants.

This has been suggested before, but the new report leaves no question of the ethical issue. To justify its continuation in some form, perhaps as a nonprofit or a foundation…

View original post 126 more words

A Faster, Safer Method for Producing Stem Cells

Researchers from the extremely prolific Salk Institute laboratory of Juan Carlos Izpisua Belmonte have designed a new method for generating stem cells from mature, adult cells that has the potential to boost laboratory production of stem cells. This technique could overcome an important barrier to regenerative medical therapies that would replace damaged or unhealthy tissues.

This new stem cell production technique allows for the unlimited production of stem cells and stem cell derivatives and also considerably reduces the time it takes to produce these cells; instead of taking two months, Juan Carlos’ lab can make them in two weeks.

Ignacio Sancho-Martinez, one of the first authors of this paper, said, “One of the barriers that needs to be overcome before stem cell therapies can be widely adopted is the difficulty of producing enough cells quickly enough for acute clinical application.”

Sancho-Martinez and his colleagues in the Belmonte laboratory published this new method in the journal Nature Methods.

Stem cells are important for regenerative medicine because of their pluripotency. Pluripotency refers to the ability of a stem cell to differentiate into any cell in the adult human body. Pluripotent stem cells for research and clinical uses are derived from one of two sources; embryos or from adult cells that have been reprogrammed to be pluripotent.

Pluripotent stem cells from embryos – embryonic stem cells (ESCs) – have the disadvantage of being rejected by the immune system when they are placed in the body of a patient. Therefore, scientists have attempted to develop clinical therapies with pluripotent stem cells made by reprogramming adult cells – induced pluripotent stem cells or iPSCs. Because these cells are made from the patient’s own cells, they should possess the same set of surface proteins as the patient. Therefore the patient’s immune should not recognize them as foreign.

Unfortunately, there are drawbacks to iPSCs. The method by which iPSCs are made from adult cells is rather inefficient and is also time-consuming and labor-intensive. Furthermore, once the iPSCs are made, they must be differentiated into the desired cell type. Differentiation is rarely 100% efficient and if the differentiated cells cannot be effectively isolated from the incompletely differentiated cells, they can cause tumors upon implantation.

To circumvent these problems, scientists have tried to reprogram cells to something other than a pluripotent state. Reprogramming adult cells into a “multipotent” state rather than a pluripotent state is potentially easier, faster, and does not carry the risk of tumor formation. Unlike pluripotent cells, which can become any adult cell type, multipotent cells can only differentiate into a small subset of the possible adult cells. The reprogramming of adult cells into multipotent progenitor cells is called “direct lineage conversion.”

While direct lineage conversion works rather well, it is a one-for-one conversion; one skin cell is converted into one muscle cell, and so on. This makes the technique inherently unproductive, since regenerative medical strategies will require large quantities of cells. Thus, Izpisua Belmonte’s laboratory examined ways to increase the output from direct lineage conversion.

Leo Kurian, a Salk Institute post-doctoral researcher, and one of the first co-authors on this paper, explained it this way: “Beyond the obvious issue of safety, the biggest consideration when thinking about stem cells for clinical use is productivity.”

To this end, this Salk Institute team invented a new technique that they called “indirect lineage conversion,” or ILC. During ILC, somatic cells are pushed back to an earlier stage of development that is suitable for the specification of multipotent progenitor cells. Because these multipotent progenitor cells have the capacity to divide, they can be expanded to greater numbers.

ILC has the potential to produce multiple lineages once adult cells are transferred to the a special environment designed by Belmonte’s lab. Most importantly, ILC saves time and also reduces the risk of tumor formation, since the adult cells are reprogrammed to become particular lineage progenitors rather than iPSCs. In the words of Sancho-Martinez, “We don’t push then to zero, we just push them back a bit.”

See Leo Kurian, et al., “Conversion of human fibroblasts to angioblast-like progenitor cells.” Nature Methods 2012; DOI:10.1038/nmeth.2255.

ACT to Start Clinical Trial of iPSC-Derived Platelets

Platelets are blood cells that help clot our blood when blood vessels are damaged. They are small cells, and are only about 20% of the diameter of red blood cells. There are typically about 150,000-350,000 platelets per microliter of blood. Despite these large numbers, platelets only compose a tiny fraction of the blood volume. As mentioned, the main function of platelets is to prevent bleeding. Platelets Platelets are produced in the bone marrow by the typical process of blood cell production. Hematopoietic stem cells in the bone marrow divide to renew and form a progenitor cell that differentiates into either a pro-erythrocytes, which becomes a red blood cell, or a promegakaryocyte. Promegakaryoyctes differentiate into megakaryocytes, which are the cells that form platelets. Platelets bud off the cytoplasm of the megakaryocytes. Consequently, platelets do not possess a nucleus. Each megakaryocyte produces between 5,000 and 10,000 platelets. Platelet differentiation Megakaryocyte and platelet production is regulated by a hormone called thrombopoietin, which is produced by the liver and kidneys. The average lifespan of circulating platelets is 5-9 days, and older platelets are destroyed by cells in the spleen and by Kupffer cells in the liver that gobble up the old platelets and recycle their components. Many platelets are held in reserve by the spleen, which are released when needed by contraction of the spleen, which is induced by the sympathetic nervous system (fight or flight response).

The cells that compose the inner surface of blood vessels normally inhibit platelet activation by producing various molecules, such as nitric oxide, and prostaglandin I2. Blood vessel cells also make a cell surface molecule called von Willebrand factor, which helps them adhere to the cable-like protein collagen that lies outside the blood vessels. Injury to blood vessels reduces the production of these inhibitory molecules and exposes the platelets and blood to collagen and von Willebrand factor (vWF). When the platelets contact collagen or vWF, they become activated. This activation manifests itself is several ways. First of all, the platelets dump the granules that they store. These granules contain several important molecules, but they also place new surfaced proteins on the outside of the platelets that help them clump together. Activated platelets also change their shape to become more spherical, and extensions of the surface form. This gives them a kind of star-shape.


Other molecules released from their granules include ADP, which is a platelet-activating molecule, the neurotransmitter serotonin, which induces blood vessels to constrict (this staunches blood loss), blood clotting factors (factors V and XIII for those who are interested), and some growth factors. Platelet activation also induces platelets to synthesize a molecule called thromboxane A2, which, like ADP, activates other platelets. Thus platelet activation is a self-amplifying event and gets more and more platelets involved in the act. I hope I have convinced you of the importance of platelets.

Some people have problems with insufficient numbers of platelets, and they have trouble properly clotting their blood.  Therefore, giving them more platelets is an excellent way to treat them, but a source of platelets must be found in order to give them to the patient. Enter the Massachusetts-based biotechnology company, ACT.  Advanced Cell Technologies from Marlborough, Mass., wants to test platelets made from reprogrammed cells; that is to say induced pluripotent stem cells.   Patients with some types of leukemia, anemia and other conditions need repeated infusions of platelets to avoid bleeding to death.  Additionally, the immune system of such patients can become sensitized to donated platelets, which compromises their effectiveness.

Platelets made from induced pluripotent stem cells (iPSCs) could overcome that problem because they were derived from a patient’s own cells. Platelets also seem to be the ideal cell for this technology because the platelets live for such s short time, they do not have a nucleus, and therefore, cannot cause tumors. Alan Michelson, a platelet researcher at Harvard Medical School and Boston Children’s Hospital who would lead a clinical trial in the U.S. studying ACT’s stem-cell derived platelets, said, “This would really be a dramatic advance in medicine, but it remains to be seen if this would be successful.”

Robert Lanza, ACT’s chief scientific officer, said that the company has the “capacity to make enough” platelets for the initial clinical trials but would “take time to scale up for widespread use.”  He added, “It doesn’t require any embryos. It doesn’t require eggs. It doesn’t require any destruction of embryos.” ACT has proposals for clinical trials and the company has said that testing could begin as early as the end of next year if regulators sign off.  The U.S. Food and Drug Administration has declined to comment, since federal rules prevent it from discussing any therapies that may be under development.

According to Lanza, the proposed U.S. trial would potentially infuse  normal platelets and stem-cell-derived platelets into eight patients and compare how well the cells functioned.  Because the platelets can be labeled, blood draws from the patients would determine if the iPSC-derived platelets behave like bona fide platelets.

According to Cynthia Dunbar, a stem-cell researcher at the National Heart, Lung and Blood Institute and editor of the journal Blood, if the iPSC-derived platelets worked like normal platelets, “the potential impact would be great.”

POSEIDON Clinical Trial Shows the Feasibility of Heart Patients Being Treated With Someone Else’s Mesenchymal Stem Cells

Joshua Hare from the Interdisciplinary Stem Cell Institute at the University of Miami Miller School of Medicine has headed up the POSEIDON clinical trial. Poseidon stands for the Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis Pilot Study (NCT01087996). The goal of this study was to examine the effects of mesenchymal stem cells on the hearts of patients who had suffered a heart attack some time ago and compare the effects of treating a patient with their own mesenchymal stem cells, or with those of a donor. Mesenchymal stem cells (MSCs) are known to not rile the immune system, but if they are implanted for a long period of time, there is a chance that they will elicit an immune response. However, there is evidence that the bone marrow quality of a patient decreases after a heart attack. Therefore, administering MSCs from a healthy donor might work even better than using a heart attack patient’s own MSCs.

In this trial, Hare and his colleagues took 31 patients who suffered from “ischemic cardiomyopathy,” and randomly gave them injections into their heart muscle of their own MSCs, or MSCs from a healthy donor. They monitored the patients for adverse effects and also monitored their heart function 1 year after MSC administration.

The results showed a low rate of adverse effects for both groups 30 days after treatment. One year after treatment, there were even fewer adverse effects. The patients who were given their own MSCs showed improvement over the distance walked in 6 minutes. These same patients also said that they had an increased quality of like. Both groups also showed some improvements in the remodeling of their hearts, which is the say that the enlargement of the heart was decreased. Unfortunately, there were no measurable improvements in heart function. Importantly, none of the patients who had received someone else’s MSCs showed a significant immune response against the implanted MSCs. One patient did show some antibodies against the implanted MSCs, but this is only 1/15 patients in the absence of any immunosuppression.

These results are modest, but they are good enough for a larger clinical trial. to that end, Hare will now engage in the ALLSTAR (Allogeneic Heart Stem Cells to Achieve Myocardial Regeneration) study (NCT01458405) of 270 patients with ventricular dysfunction after a heart attack. These results are reasons for cautious optimism.

Stem Cell Fixes for the Heart

Two recent papers have provided very good evidence that pluripotent stem cells can help heal a heart that has experienced a heart attack. One of these papers used induced pluripotent stem cells from rats, and the other used embryonic stem cells.

The first paper comes from the laboratory of Yoshiki Sawa, who is a professor in the Department of Surgery at the Osaka University Graduate School of Medicine in Osaka, Japan. In this paper, Sawa’s group made induced pluripotent stem cells (iPSCs) from mice and cultured them under conditions known to induce differentiation into heart muscle cells. Beating cells were detected and grown on gelatin-coated plates with Delbecco’s medium. When these cells were tested for gene expression, they made all the same genes as those found in a mouse heart.

To get the cells to form sheets of heart muscle cells, Sawa and his team plated his iPSCs on UpCell plates that are coated with a chemical that causes the cells to adhere to it at normal temperatures, but when the temperature is dropped, the cells detach from the plate. Sawa used another innovation with this culture system; he grew cell without any sugar. This caused all the non-heart cells to die off. The result was a sheet of heart muscle cells that contracted in unison.

Next, the Sawa team took induced heart attacks in a Japanese rat strain. 2 weeks after suffering the heart attack, the sheet of heart muscle cells were placed on the heart scar in half of the rats and the other half received no implants.

Four weeks after implantation of the heart muscle sheet, the differences in heart function were stark. The ejection fraction in the hearts of the animals that had received the iPSC-derived heart muscle sheets increased almost 10%. The fractional shortening, which is the degree to which the heart muscle shortens when it contracts, also increased more than 5%. Also, the amount of stretching during pumping decreased, which indicates that the heart is pumping more efficiently.

When the heart muscle from the implants were examined, they were also filled with molecules associated with the production of new blood vessels. Thus the implanted heart muscle sheets also helped heal the heart by inducing the formation of new blood vessels.

A danger of using iPSC-derived heart muscle cells is the tendency to miss undifferentiated cells and have undifferentiated cells that cause tumors. In this experiment, they noticed tumors if they only grew the cells in the sugar-free medium for a little while. However, if they grew the iPSC-heart muscle cells in sugar-free media for at least three days, all the tumor-causing cells died and implants from these sheets never formed any tumors.

This paper demonstrated the efficacy and plausibility of using patient-specific iPSCs to treat a heart that has had a heart attack some time ago.

The second paper comes from the laboratory of Marisa Jaconi in Geneva, Switzerland. In this paper, Jaconi and her gang of stem cell scientists at the Geneva University Hospitals and the Ecole Polytechnique Fédérale de Lausanne used a “cardiopatch” seeded with cardiac-committed embryonic stem cells to treat a heart attack in rats.

Because the injection of stem cells can induce arrhythmias (irregular heart beats), narrowing of blood vessels, blood vessel obstruction, and other types of damage, these two papers tried to use sheets of cells or cells embedded in biodegradable patches to treat the heart. In this paper, Jacobi and others used a hydrogel made from fibrin, which is the same material found in blood clots. Into that fibrin hydrogel, they placed mouse embryonic stem cells that had been treated with a protein called BMP-2, which drives pluripotent stem cells toward a heart cell fate.

To use these cardiopatches, Jacobi and her group induced heart attacks in a French rat strain and then applied the patch to the heart. They had two groups of rats; those that had been given heart attacks and those that had not. The sham group received either a patch with cells, a patch with iron particles (for detection with MRI) or not patch. The heart attack group received the same.

The results are a little hard to interpret, but the patch + cells definitely improved heart function. First, the hearts that had received patches with cells showed in increase in small blood vessels and blood vessel-making (CD31+) cells. Therefore the patches + cells improved heart circulation. Second, the hearts with the patch + cells showed the presence of new heart muscle cells and much mess thinning of the walls of the heart. Third, the heart functional parameters were better preserved in the patch + cells hearts. The ejection fraction decreased substantially in the hearts that did not receive cells, but in the hearts that received patch + cells, the amount of blood left in the heart after pumping and at rest did not increase nearly as much as in the other groups. These parameters are in indication of the efficiency with which the heart is pumping. The fact that the heart + cells hearts did not decrease in efficiency nearly as precipitously as the others shows that the stem cells are healing the heart.

While these results may not seem terribly robust, we must remember that the cardiopatch was only placed over a small portion of the heart. Therefore, we would not expect to see large increased in function. The fact that we do see new heart muscle cells, new blood vessels, and an arrest in the functional free fall of the heart is significant, given the small area of the heart that was cover with the cells.

The cardiopatch is a new technology and this experiment showed that the patch biodegrades quickly and without incident. It also showed that embedding cells in the patch is feasible, and that the patch is a plausible vehicle to deliver cells to the heart. This procedure also induced the formation of new heart muscle cells in the heart scar and new blood vessels too. Perhaps even more encouraging is the absence of tumors reported in this paper. Even though the ESCs were not differentiated completely into heart muscle cells, the cardiac-directed cells were differentiated enough to form either blood vessels, smooth muscle, or heart muscle. This seems to be enough to prevent the cells from forming tumors. Also, the fibrin scaffold was not deleterious to the heart, even though some studies have used other scaffolds that are damaging to the heart.

Thus cardiopatches and cardiac muscle sheets are perfectly good strategies for treating heart with stem cells. More work needs to be done, but the results are encouraging.

A New Technique to Fix Damaged Eyes With Stem Cells

Engineers at the University of Sheffield have invented a new delivery technique for delivering stem cells to eyes. They have high hopes that this technique will help repair the eyes of those patients who have suffered damage to their eyes.

The front of the eye is bordered by the transparent cornea, which transmits light to the lens. The cornea is exposed to the outside world and if there is an accident that affects the eye, the cornea is usually the part that takes a beating. The cornea undergoes constant turnover as dead cells are constantly sloughed from the cornea during blinking. At the junction between the cornea and the sclera is an area called the limbus. Located at the limbus is a population of limbal epithelial stem cells or LESCs. LESCs have many features commonly observed in other stem cells, such as small size, high nuclear to cytoplasmic ratio, and they lack expression of molecules commonly found in mature corneal cells, such as cytokeratins 3 and 12.

Human Limbus

LESCs are slow-growing, but in the event of injury they can become highly proliferative (See Lavker R.M, Sun T.T. Epithelial stem cells: the eye provides a vision. Eye. 2003;17:937–942. DOI: 10.1038/sj.eye.6700575).

LESC deficiency can result from chemical or thermal burns to the eye or as a result of certain inherited diseases. Partial or full LESC deficiency causes abnormal corneal wound healing and surface integrity. Also LESC deficiency causes the conjunctiva to grow over the cornea, and this is disastrous for the eye because the cornea is devoid of blood vessels, which is the reason why it is transparent. However the conjunctiva (the white of the eye) is filled with blood vessels and is not transparent. Thus chronic inflammation, recurrent erosion, ulceration and stromal scarring can occur and cause painful vision loss

Long term restoration of visual function requires renewal of the corneal epithelium, and this requires the placement of a new stem cell population by means of a limbus graft. From where do you get a new limbus for transplantation? Autografts use limbal cells from the good eye, but this runs the risk of scarring the cornea of the other eye.procedure is the use limbal cells from cadavers (limbal allografts). Also, making sure that the graft adheres to the requires the use of sutures, but these sutures can cause substantial amounts of irritation. Therefore, the Sheffield research group designed a new technique.

With this new technique, a disk made of biodegradable material is loaded with limbal stem cells and then placed over the eye. This disc has an outer ring pockmarked with small niches for stem cells can hide. The material in the center of the disc is thinner than that on the edges, and therefore, the center of the disc biodegrades faster. This releases the stem cells in center of the disc into the cornea where they can grow and help repair it.

Because these small niches in the disc resemble the stem cells niches found in the limbus, these discs do an excellent job of nurturing the limbal stem cells and distributing them to the cornea. Limbal grafts are either done with amniotic membrane as a carrier, but this procedure leads to increased inflammation in the eye and there is a chance that the grafts will not integrate into the limbus. The biodegradable disc groups the limbal stem cells into clusters that are more likely to ingrate into the limbus.

According to Professor Sheila MacNeil, “Laboratory tests have shown that the membranes will support cell growth, so the next stage is to trial this in patients in India, working with our colleagues in the LV Prasad Eye Institute in Hyderabad. One advantage of our design is that we have made the disc from materials already in use as biodegradable sutures in the eye so we know they won’t cause a problem in the body. This means that, subject to the necessary safety studies and approval from Indian Regulatory Authorities, we should be able to move to early stage clinical trials fairly quickly.”

In the developing world, corneal blindness is rather common in some professions and treating it is a rather pressing problem. High instances of chemical burns to the eye or accidental damage to the eye are common, but complex treatment strategies such as amniotic membrane grafts are not available to the general public.

This technique also possibilities in more developed countries, since current techniques use donor tissue to deliver the cultured cells, and this requires a tissue bank to which some people do not have access. Also, the use of the cell-impregnated disk will reduce the risk of disease transmission with grafts.