100% Reprogramming Rates

For the first time, stem cell scientists have reprogrammed cultured skin cells into induced pluripotent cells (iPSCs) with near-perfect efficiency.

Even several laboratories have examined protocols to increase the efficiency of cellular reprogramming, a research team at the Weizmann Institute of Science in Rehovot, Israel has managed to increase the conversion rate to almost 100%, ten times the rate normally achieved, by removing a single proteins called Mbd3. This discovery can potentially allow scientists to generate large volumes of stem cells on demand, which would accelerate the development of new treatments.

In 2006, scientists from the laboratory of Shinya Yamanaka showed that mature cells could be reprogrammed to act like embryonic stem cells (ESCs). These reprogrammed adult cells could grow in culture indefinitely and differentiate into any type of cell in the body. However the creation of iPSc lines was notoriously inefficient and labor-intensive. Low cell-conversion rates have slowed the study of the reprogramming process itself. It has also discouraged the development of protocols for producing iPSCs under GMP or “Good Manufacturing Practice” conditions for use in human patients.

However, in a series of experiments that were published in the journal Nature, Weizmann Institute stem-cell researcher Jacob Hanna and his team have reprogrammed cells with nearly 100% efficiency. Moreover, Hanna and his group showed that reprogrammed cells transition to pluripotency on a synchronized schedule.

“This is the first report showing that you can make reprogramming as efficient as anyone was hoping for,” says Konrad Hochedlinger, a stem-cell scientist at Harvard Medical School in Boston, Massachusetts. “It is really surprising that manipulating a single molecule is sufficient to make this switch, and make essentially every single cell pluripotent within a week.”

To make iPSCs from adult cells, scientists typically transfect them with a set of four genes. These genes turn on the cells’ own pluripotency program, which converts them into iPSCs. But even established techniques convert less than 1% of cultured cells. Many cells get stuck in a partially reprogrammed state, and some become pluripotent faster than others, which makes the whole reprogramming process difficult to monitor.

Hanna and his team investigated the potential roadblocks to reprogramming by working with a line of genetically-engineered mouse cells. In these cells, the reprogramming genes were already inserted into the genomes of the cells and could be activated with a small molecule. Such cells normally reprogram at rates below 10%. But when a gene responsible for producing the protein Mbd3 was repressed, reprogramming rates soared to nearly 100%.

Hanna says that the precise timing of embryonic development led him to wonder whether it is possible to “reprogram the reprogramming process.” Cells in an embryo do not remain pluripotent indefinitely, explained Hanna. Usually, Mbd3 represses the pluripotency program as an embryo develops, and mature cells maintain their expression of Mbd3. However, during cellular reprogramming, those proteins expressed from the inserted pluripotency genes induce Mbd3 to repress the cells’ own pluripotency genes.

This hamstrings reprogramming, says Hanna. “It creates a clash, and that’s why the process is random and stochastic. It’s trying to have the gas and brakes on at the same time.” Depleting the cells of Mbd3 allows reprogramming to proceed unhindered.

The team also reprogrammed cells from a human, using a method that does not require inserting extra genes. This technique usually requires daily doses of RNA over more than two weeks. With Mbd3 repressed, only two doses were required.

Stem Cells and LDL Play a Role in Atherosclerosis

Researchers at the University at Buffalo have discovered a new understanding of atherosclerosis in humans that include a key role for stem cells that promote inflammation.

Published in the journal PLOS One, this work extends to humans previous findings in lab animals by researchers at Columbia University that showed that high levels of LDL (“bad”) cholesterol promote atherosclerosis by stimulating production of hematopoietic stem/progenitor cells (HSPC’s).

“Our research opens up a potential new approach to preventing heart attack and stroke, by focusing on interactions between cholesterol and the HSPCs,” says Thomas Cimato, lead author on the PLOS One paper and assistant professor in the Department of Medicine in the UB School of Medicine and Biomedical Sciences.

Cimato noted that the role of stem cells in atherosclerosis could lead to the development of a useful therapy in combination with statins or to a novel therapy that could be used in place of statins for those individuals who cannot tolerate them.

In humans, high total cholesterol recruits stem cells from the bone marrow into the bloodstream. The cytokine IL-17, which has been implicated in many chronic inflammatory diseases, including atherosclerosis, is responsible for the recruitment of HSPCs. IL-17 boosts levels of granulocyte colony stimulating factor (GCSF), which induces the release of stem cells from the bone marrow.

According to Cimato, they observed that statins reduce the levels of HSPCs in the blood but not every subject responded similarly. “We’ve extrapolated to humans what other scientists previously found in mice about the interactions between LDL cholesterol and these HSPCs,” explains Cimato.

The fact that a finding in laboratory animals holds true for humans is noteworthy, adds Cimato. “This is especially true with cholesterol studies,” he says, “because mice used for atherosclerosis studies have very low total cholesterol levels at baseline. We feed them very high fat diets in order to study high cholesterol but it isn’t [sic] easy to interpret what the levels in mice will mean in humans and you don’t know if extrapolating to humans will be valid.”

Cimato added that the LDL concentrations in the blood of mice in their studies is much higher than what is found in patients who come to the hospital with a heart attack or stroke.

“The fact that this connection between stem cells and LDL cholesterol in the blood that was found in mice also turns out to be true in humans is quite remarkable,” he says.

Cimato explains that making the jump from rodents with very high LDL cholesterol to humans required some creative steps, such as the manipulation of the LDL cholesterol levels of subjects through the use of three different kinds of statins.

The study involved monitoring for about a year a dozen people without known coronary artery disease who were on the statins for two-week periods separated by one-month intervals when they were off the drugs.

“We modeled the mechanism of how LDL cholesterol affects stem cell mobilization in humans,” says Cimato.

Cimato and his group found that LDL cholesterol modulates the levels of stem cells that form neutrophils, monocytes and macrophages, the primary cell types involved in the formation of plaque and atherosclerosis.

The next step, he says, is to find out if HSPCs, like LDL cholesterol levels, are connected to cardiovascular events, such as heart attack and stroke.

Spiking Stem Cells to Generate Myelin

Regenerating damaged nerve tissue represents a unique challenge for regenerative medicine. Nevertheless, some experiments have shown that it is possible to regenerate the myelin sheath that surrounds particular nerves.

Myelin is a fatty, insulating sheath that surrounds particular nerves and accelerates the transmission of nerve impulses. The myelin sheath also helps neurons survive, and the myelin sheath is attacked and removed in multiple sclerosis, a genetic disease called Charcot-Marie-Tooth disease, and spinal cord injuries. Being able to regenerate the myelin sheath is an essential goal of regenerative medicine.

Fortunately, a new study from a team of UC Davis (my alma mater) scientists have brought this goal one step closer. Wenbig Deng, principal investigator of this study and associate professor of biochemistry and molecular medicine, said, “Our findings represent an important conceptual advance in stem cell research. We have bioengineered the first generation of myelin-producing cells with superior regenerative capacity.”

The brain contains two main cell types; neurons and glial cells. Neurons make and transmit nerve impulses whereas glial cells support, nourish and protect neurons. One particular subtype of glial cells, oligodendrocytes, make the myelin sheath that surrounds the axons of many neurons. Deng and his group developed a novel protocol to induce embryonic stem cells (ESCs) to differentiate into oligodendrocyte precursor cells or OPCs. Even though other researchers have made oligodenrocytes from ESCs, Deng’s method results in purer populations of OPCs than any other available method.

Making OPCs from ESCs is one thing, but can these laboratory OPCs do everything native can do? When Deng and his team tested the electrophysiological properties of their laboratory-made OPCs, they discovered that their cells lacked an important component; they did not express sodium channels. When the lab-made OPCs were genetically engineered to express sodium channels, they generated the characteristic electrical spikes that are common to native OPCs. According to Deng, this is the first time anyone has made OPCs in the laboratory with spiking properties. Is this significant?

Deng and his colleagues compared the spiking OPCs to non-spiking OPCs in the laboratory. Not only did the spiking OPCs communicate with neurons, but they also did a better job of maturing into oligodentrocytes.

Transplantation of these two OPC populations into the spinal cord and brains of mice that are genetically unable to produce myelin also showed differences. Both types of OPCs were able to mature into oligodendrocytes and produce myelin sheaths, but only the spiking OPCs had the ability to produce longer and thicker myelin sheaths.

Said Deng, “We actually developed ‘super cells’ with an even greater capacity to spike than natural cells. This appears to give them an edge for maturing into oligodendrocytes and producing better myelin.

Human neural tissue has a poor capacity to regenerate and even though OPCs are present, they do not regenerate tissue effectively when disease or injury damages the myelin sheath. Deng believes that replacing glial cells with the enhanced spiking OPCs to treat injuries and diseases has the potential to be a better strategy than replacing neurons, since neurons are so problematic to work with in the laboratory. Instead providing the proper structure and environment for neurons to live might be the best approach to regenerate healthy neural tissue. Deng also said that many diverse conditions that have not been traditionally considered to be myelin-based diseases (schizophrenia, epilepsy, and amyotrophic lateral sclerosis) are actually now recognized to involve defective myelin.

On that one, I think Deng is dreaming. ALS is caused by the death of motor neurons due to mechanisms that are intrinsic to the neurons themselves. Giving them all the myelin in the world in not going to help them. Also, OPCs made from ESCs will be rejected out of hand by the immune system if they are used to regenerate myelin in the peripheral nervous system. The only hope is to keep them in the central nervous system, but even there, any immune response in the brain will be fatal to the OPCs. This needs to be tested with iPSCs before it can be considered for clinical purposes.

Transformation of Non-Beating Human Cells into Heart Muscle Cells Lays Foundation for Regenerating Damaged Hearts

After a heart attack, the cells within the damaged part of the heart stop beating and become ensconced in scar tissue. Not only does this region not beat, it does not conduct the signal to beat either and that can not only lead to a slow, sluggish heartbeat, it can also cause irregular heart rates or arrhythmias.

Now, however, scientists have demonstrated that this damage to the heart muscle need not be permanent. Instead there is a way to transform those cells that form the human scar tissue into cells that closely resemble beating heart cells.

Last year, researchers from the laboratory of Deepak Srivastava, MD, the director of Cardiovascular and Stem Cell Research at the Gladstone Institute, transformed scar-forming heart cells (fibroblasts) into beating heart-muscle cells in live mice. Now they report doing the same to human cells in a culture dishes.

“Fibroblasts make up about 50 percent of all cells in the heart and therefore represent a vast pool of cells that could one day be harnessed and reprogrammed to create new muscle,” said Dr. Srivastava, who is also a professor at the University of California, San Francisco. “Our findings here serve as a proof of concept that human fibroblasts can be reprogrammed successfully into beating heart cells.”

In 2012, Srivastava and his team reported that fibroblasts could be reprogrammed into beating heart cells by injecting just three genes (collectively known as GMT, which is short for Gata4, Mef2c, and Tbx5), into the hearts of live mice that had been damaged by a heart attack (Qian L, et al., Nature. 2012 31;485(7400):593-8). From this work, they reasonably concluded that the same three genes could have the same effect on human cells.

“When we injected GMT into each of the three types of human fibroblasts (fetal heart cells, embryonic stem cells and neonatal skin cells) nothing happened—they never transformed—so we went back to the drawing board to look for additional genes that would help initiate the transformation,” said Gladstone staff scientist Ji-dong Fu, Ph.D., the study’s lead author. “We narrowed our search to just 16 potential genes, which we then screened alongside GMT, in the hopes that we could find the right combination.”

The research team began by injecting all candidate genes into the human fibroblasts. They then systematically removed each one to see which were necessary for reprogramming and which were dispensable. In the end, they found that injecting a cocktail of five genes—the 3-gene GMT mix plus the genes ESRRG and MESP1—were sufficient to reprogram the fibroblasts into heart-like cells. They then found that with the addition of two more genes, called MYOCD and ZFPM2, the transformation was even more complete.

To help things along, the team used a growth factor known as Transforming Growth Factor-Beta (TGF-Beta) to induce a signaling pathway during the early stages of reprogramming that further improved reprogramming success rates.

“While almost all the cells in our study exhibited at least a partial transformation, about 20 percent of them were capable of transmitting electrical signals—a key feature of beating heart cells,” said Dr. Fu. “Clearly, there are some yet-to-be-determined barriers preventing a more complete transformation for many of the cells. For example, success rates might be improved by transforming the fibroblasts within living hearts rather than in a dish—something we also observed during our initial experiments in mice.”

The immediate next steps are to test the five-gene cocktail in hearts of larger mammals. Eventually, the team hopes that a combination of small, drug-like molecules could be developed to replace the cocktail, which would offer a safer and easier method of delivery.

This latest study was published online August 22 in Stem Cell Reports.

First Patient Treated in Study that Tests Stem Cell-Gene Combo to Repair Heart Damage

The first patient has been treated in a groundbreaking medical trial in Ottawa, Canada, that uses a combination of stem cells and genes to repair tissue damaged by a heart attack. The first test subject is a woman who suffered a severe heart attack in July and was treated by the research team at the Ottawa Hospital Research Institute (OHRI). Her heart had stopped beating before she was resuscitated, which caused major damage to her cardiac muscle.

The therapy involves injecting a patient’s own stem cells into their heart to help fix damaged areas. However, the OHRI team, led by cardiologist Duncan Stewart, M.D., took the technique one step further by combining the stem cell treatment with gene therapy.

“Stem cells are stimulating the repair. That’s what they’re there to do,” Dr. Stewart said in an interview. “But what we’ve learned is that the regenerative activity of the stem cells in these patients with heart disease is very low, compared to younger, healthy patients.”

Stewart and his colleagues will supply the stem cells with extra copies of a particular gene in an attempt to restore some of that regenerative capacity. The gene in question encodes an enzyme called endothelial nitric oxide synthase (eNOS). Nitric oxide is a small, gaseous molecule that is made from the amino acid arginine by the enzyme nitric oxide synthase. Nitric oxide or NO signals to smooth muscle cells that surround blood vessels to relax, which causes blood vessels to dilate and this increases blood flow. In the damaged heart, NO also helps build up new blood vessels, which increase healing of the cardiac muscle. Steward added, “That, we think, is the key element. We really think it’s the genetically enhanced cells that will provide the advantage.”

Nitric oxide synthesis

The study will eventually involve 100 patients who have suffered severe heart attacks in Ottawa, Toronto and Montreal.

Producing blood cells from stem cells could yield a purer, safer cell therapy

The journal Stem Cells Translational Medicine has published a new protocol for reprogramming induced pluripotent stem cells (iPSCs) into mature blood cells. This protocol uses only a small amount of the patient’s own blood and a readily available cell type. This novel method skips the generally accepted process of mixing iPSCs with either mouse or human stromal cells. Therefore, is ensures that no outside viruses or exogenous DNA contaminates the reprogrammed cells. Such a protocol could lead to a purer, safer therapeutic grade of stem cells for use in regenerative medicine.

The potential for the field of regenerative medicine has been greatly advanced by the discovery of iPSCs. These cells allow for the production of patient-specific iPSCs from the individual for potential autologous treatment, or treatment that uses the patient’s own cells. Such a strategy avoids the possibility of rejection and numerous other harmful side effects.

CD34+ cells are found in bone marrow and are involved with the production of new red and white blood cells. However, collecting enough CD34+ cells from a patient to produce enough blood for therapeutic purposes usually requires a large volume of blood from the patient. However, a new study outlined But scientists found a way around this, as outlined by Yuet Wai Kan, M.D., FRS, and Lin Ye, Ph.D. from the Department of Medicine and Institute for Human Genetic, University of California-San Francisco has devised a way around this impasse.

“We used Sendai viral vectors to generate iPSCs efficiently from adult mobilized CD34+ and peripheral blood mononuclear cells (MNCs),” Dr. Kan explained. “Sendai virus is an RNA virus that carries no risk of altering the host genome, so is considered an efficient solution for generating safe iPSC.”

“Just 2 milliliters of blood yielded iPS cells from which hematopoietic stem and progenitor cells could be generated. These cells could contain up to 40 percent CD34+ cells, of which approximately 25 percent were the type of precursors that could be differentiated into mature blood cells. These interesting findings reveal a protocol for the generation iPSCs using a readily available cell type,” Dr. Ye added. “We also found that MNCs can be efficiently reprogrammed into iPSCs as readily as CD34+ cells. Furthermore, these MNCs derived iPSCs can be terminally differentiated into mature blood cells.”

“This method, which uses only a small blood sample, may represent an option for generating iPSCs that maintains their genomic integrity,” said Anthony Atala, MD, Editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “The fact that these cells were differentiated into mature blood cells suggests their use in blood diseases.”

Overexpression of a Potassium Channel in Heart Muscle Cells Made From Embryonic Stem Cells Decreases Their Arrhythmia Risk

Embryonic stem cells have the capacity to differentiate into every cell in the adult body. One cell type into which embryonic stem cells (ESCs) can be differentiated rather efficiently is cardiomyocytes, which is a fancy term for heart muscle cells. The protocol for making heart muscle cells from ESCs is well worked out, and the conversion is rather efficient and the purification schemes that have been developed are also rather effective (for example, see Cao N, et al., Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions. Cell Res. 2013 Sep;23(9):1119-32. doi: 10.1038/cr.2013.102 and Mummery CL et al., Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res. 2012 Jul 20;111(3):344-58).

Using these cells in a clinical setting has two large challenges. The first is that embryonic stem cell derivatives are rejected by the immune system of the recipient, thus setting up the patient for a graft versus host response to the implanted tissue, thus making the patient even sicker than when they started. The second problem is that heart muscle cells made from ESCs are immature and cause the heart to beat abnormally fast thus causing “tachyarrythmias” and died within the first two weeks after the transplant (see Liao SY, et al., Heart Rhythm 2010 7:1852-1859).

Both of these problems are large problems, but the laboratory of Ronald Li at the University of Hong Kong at used a genetic engineering trick to make heart muscle cells from mouse embryonic stem cells to seemingly fix this problem.

Li and his colleagues engineered mouse ESCs with a gene for a potassium rectifier channel that could be induced with drugs. Then they differentiated these genetically ESCs into heart muscle cells. This potassium rectifier channel (Kir2.1) is not present in immature heart muscle cells and putting it into these cells might cause them to beat at a slower rate.

These engineered ESC-derived heart muscle cells were tested for their electrophysiological properties first. Without the drug that induces KIR2.1, the heart muscle cells showed very abnormal electrical properties. However, once the drug was added, their electrical properties looked much more normal.

Then they induced heart attacks in laboratory animals and implanted their engineered ESC-derived heart muscle cells 1 hour after the heart attacks were induced. Animals not given the drug to induce the expression of Kir2.1 faired very poorly and had episodes of tachyarrythmia (really fast heart beat) and over half of them died by 5 weeks after the implantation. Essentially the implanted animals did worse than those animals that had had a heart attack that were not treated. However, those animals that were given the drug that induces the expression of Kir2.1 in heart muscle cells did much better. The survival rate of these animals was higher than the untreated animals after about 7 weeks after the procedure. Survival rates increased by only a little, but the increase was significant. Also, the animals that died did not die of tachyarrythmias. In fact the rate of tachyarrythmias in the animals given the inducing drug (which was doxycycline by the way) had significantly lower levels of tachyarrythmia than the other two groups.

Other heart functions were also significantly affected. The ejection fraction in the animals that ha received the Kir2.1-expression heart muscle cells was 10-20% higher than the control animals. Also the density of blood vessels was substantially higher in both sets of animals treated with ESC-derived heart muscle cells. The echocardiogram of the hearts implanted with the Kir2.1-expressing heart muscle cells was altogether more normal than that of the others.

This paper is a significant contribution to the use of ESC-derived cells to treat heart patients. The induction of heart arrhythmias by ESC-derived heart muscle cells is a documented risk of their use. Li and his colleagues have effectively eliminated that risk in this paper by forcing the expression of a potassium rectifier channel in the ESC-derived heart muscle cells. Also, because these cells were completely differentiated and did not have any interloping pluripotent cells in their culture, tumor formation was not observed.

There are a few caveats I would like to point out. First of all, the increase in survival rate above the control is not that impressive. The improvement in heart function parameters is certainly encouraging, but because the survival rates are not that higher than the control mice that received no treatment, it appears that these benefits were only conferred to those mice who survived in the first place.

Secondly, even though the heart attacks were induced in the ventricles of the heart, Li and his colleagues injected a mixture of heart muscle cells that included atrial, ventricular, nodal and heart fibroblasts. This provides an opportunity for beat mismatches and a “substrate for ventricular tachycardia” as Li puts it. In the future, the transplantation of just ventricular heart muscle cells would be cleaner experiment. Since these mice were not observed long enough to observe potential arrythmias that might have arisen from the presence of a mixed population in the ventricle.

Finally, in adapting this to humans might be difficult, since the hearts of mice beat so much faster than those of humans. It is possible that even if human cardiomyocytes were engineered with Kir2.1-type channels, that arrythmias might still be a potential problem.

Despite all that, Li’s publication is a large step forward.

Scientists Grow Small Chunks of Brain Tissue From Induced Pluripotent Stem Cells

Induced pluripotent stem cells are made from adult cells by means of genetic engineering techniques that introduce into the cells a combination for four different genes that drive the cells to de-differentiate into a cell that has many of the characteristics of embryonic stem cells without the destruction of embryos.

A new study from the laboratory of Juergen Knoblich at the Institute of Molecular Biotechnology in Vienna has mixed induced pluripotent stem cells (iPSCs) to form structures of the human brain. He largely left the cells alone to allow them to form the brain tissue, but he also placed them in a spinning bioreactor that constantly circulates the culture medium and provides nutrients and oxygen to the cells. One other growth factor he supplied to the cells was retinoic acid, which is made by the meninges that surround our brains. All of this and the cells not only divided, differentiated and assembled, but they formed brain structures that had all the connections of a normal brain. These brain-like chunks of tissue are called “mini-brains” and the recent edition of the journal Nature reports their creation.

“It’s a seminal study to making a brain in a dish,” says Clive Svendsen, a neurobiologist at the University of California, Los Angeles. Svendsen was not involved in this study, but wishes he was. Of this study, Svendsen exclaimed, “That’s phenomenal” A fully formed artificial brain is still years and years away, but the pea-sized neural clumps developed in Knoblich’s laboratory could prove useful for researching human neurological diseases.

Researchers have previously used pluripotent human stem cells to grow structures that resemble the developing eye (Eiraku, M. et al. Nature 472, 51–56 (2011), and even tissue layers similar to the cerebral cortex of the brain (Eiraku, M. et al. Cell Stem Cell 3, 519–532 (2008). However, this latest advance has seen bigger and more complex neural-tissue clumps by first growing the stem cells on a synthetic gel that resembled natural connective tissues found in the brain and elsewhere in the body. After growing them on the synthetic gel, Knoblich and his colleagues transferred the cells to a spinning bioreactor that infuses the cells with nutrients and oxygen.

“The big surprise was that it worked,” said Knoblich. The clump formed structures that resembled the brains of fetuses in the ninth week of development.

Under a microscope, the blobs contained discrete brain regions that seemed to interact with one another. However, the overall arrangement of the different proto-brain areas varied randomly across tissue samples. These structures were not recognizable physiological structures.

A cross-section of a brain-like clump of neural cells derived from human stem cells.
A cross-section of a brain-like clump of neural cells derived from human stem cells.

“The entire structure is not like one brain,” says Knoblich, who added that normal brain maturation in an intact embryo is probably guided by growth signals from other parts of the body. The tissue balls also lacked blood vessels, which could be one reason that their size was limited to 3–4 millimeters in diameter, even after growing for 10 months or more.

Despite these limitations, Knoblich and his collaborators used this system to model key aspects of microcephaly, which is a condition that causes extremely stunted brain growth and cognitive impairment. Microcephaly and other neurodevelopmental disorders are difficult to replicate in rodents because the brains of rodents develop differently than those of humans.

Knoblich and others found that tissue chunks cultured from stem cells derived from the skin of a single human with microcephaly did not grow as large as clumps grown from stem cells derived from a healthy person. When they traced this effect, they discovered that it was due to the premature differentiation of neural stem cells inside the microcephalic tissue chunks, which depleted the population of progenitor cells that fuels normal brain growth.

The findings largely confirm prevailing theories about microcephaly, says Arnold Kriegstein, a developmental neurobiologist at the University of California, San Francisco. But, he adds, the study also demonstrates the potential for using human-stem-cell-derived tissues to model other disorders, if cell growth can be controlled more reliably.

“This whole approach is really in its early stages,” says Kriegstein. “The jury may still be out in terms of how robust this is.”

Tissue Kallikrein-Modified Human EPCs Improve Cardiac Function

When cells are implanted into the heart after a heart attack, the vast majority of them succumb to the hostile environment in the heart and die. Twenty-four hours after implantation there is a significant loss of cells (see Wu et al Circulation 2003 108:1302-1305). That fact that implanted bone marrow or fat-based stem cells benefit the heart despite their evanescence is a remarkable testimony to their healing power.

To mitigate this problem, stem cell scientists have used a variety of different strategies to increase the heartiness and survival of implanted stem cells. Two main strategies have emerged: preconditioning cells and genetically engineering cells. Both strategies increase the survival of implanted stem cells (see here, and here).

When it comes to genetically engineering stem cells, Lee and Julie Chao from the Medical University of South Carolina in Charleston, South Carolina have used endothelial progenitor cells (EPCs) from human umbilical cord blood to treat mice that had suffered heart attacks, except that these cells were genetically engineered to express “Tissue Kallikrein” or TK. TK is encoded by a gene called KLKB1, which is on chromosome 4 at region q34-35 (in human genetics, the long arm of a chromosome is the “q” arm and the small arm is the “p” or petite arm). TK is initially synthesized as an inactive precursor called prekallikrein. Prekallikrein must be clipped in order to be activated and the proteases (proteases are protein enzymes that cut other proteins into smaller fragment) that do so are either clotting factor XII, which plays a role in blood clotting, and PRCP, which is also known as Lysosomal Pro-X carboxypeptidase.

TK is a protease that degrades a larger protein called kininogen in two smaller peptides called bradykinin and kallidin, both of which are active signaling molecules. Bradykinin and kallidin cause relaxation of smooth muscles, thus lowering blood pressure, TK can also degrade plasminogen to form the active enzyme plasmin.

So why engineer EPCs to express TK? As it turns out, TK activates an internal protein in cells called Akt, and activated Akt causes cells to survive and prevents them from dying (see Krankel et al., Circulation Research 2008 103:1335-1343; Yao YY, et al., Cardiovascular Research 2008 80: 354-364; Yin H et a., J Biological Chem 2005 280: 8022-8030).

The first experiments were test tube experiments in which TK EPCs were incubated with cultured heart muscle cells to determine their ability to prevent cell death. When cultured heart muscle cells were exposed to hydrogen peroxide, they died left and right, but when they were incubated with the TK-EPCs and hydrogen peroxide, far fewer of them died.

Upper panel consists of cells stained with a TUNEL stain, which designates those cells that are dead or dying.  The bottom panel are DAPI stained cells, which is a nuclear stain that marks all available cells dead or live. From left to right, normal cells, cell exposed to hydrogen peroxide, cells exposed to hydrogen peroxide plus the genes for TK, and finally, cells exposed to hydrogen peroxide and TK-EPCs.
Upper panel consists of cells stained with a TUNEL stain, which designates those cells that are dead or dying. The bottom panel are DAPI stained cells, which is a nuclear stain that marks all available cells dead or live.
From left to right, normal cells, cell exposed to hydrogen peroxide, cells exposed to hydrogen peroxide plus the genes for TK, and finally, cells exposed to hydrogen peroxide and TK-EPCs.

When these cells were exposed to low levels of oxygen, a similar result was observed, expect that the cells co-incubated with TK-EPCs showed significantly less cell death.

When TK-EPCs were injected into the infarct border zones of the heart just after they had heart attacks, the results seven days after the heart attacks were striking. The heart function of the control mice was lousy to say the least. The heart walls had thinned, their ejection fractions were in the tank (~23%) and their echocardiograms were far from normal. However, the TK-EPC-injected mice had a relatively normal echocardiogram, thick heart wall, pretty good ejection fractions (52% and oppose to the 76% of mice that had never had a heart attack), and good heart function in general. Also, the size of the infarcts was reduced in those animals whose hearts had been injected with TK-EPCs.

Representative Masson’s trichrome staining. Original magnification is 10. (f) Echocardiographic measurements for determination of LV function from M-mode measurements. (g) MDA in the ischemic mouse heart at day 7 after MI. Values are expressed as mean±s.e.m. (n¼6, *Po0.05 vs Ad.Null-hEPC- and medium-treated group; #Po0.05 vs medium-treated group).
Representative Masson’s trichrome staining. Original magnification is 10. (f) Echocardiographic measurements for determination of LV function from M-mode measurements. (g) MDA in the ischemic mouse heart at day 7 after MI. Values are expressed as mean±s.e.m. (n¼6, *Po0.05 vs Ad.Null-hEPC- and medium-treated group; #Po0.05 vs medium-treated group).

There were two other bonuses to using TK-EPCs. First, as expected, the density of new blood vessels was substantially higher in hearts that received injections of TK-EPCs. Secondly, the TK-EPCs definitely survived better than their non-genetically engineered counterparts.

Ex-vivo optical imaging study. (a, b) Representative NIR fluorescent images in explanted organs at days 2 or 7 following implantation of DiDlabeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (a: 2 days after cell delivery; b: 7 days after cell delivery). (c) Quantitative analysis of NIR fluorescent signals in explanted hearts among each group at two time points. All values are expressed as mean±s.e.m. (n¼3–4, *Po0.01 vs control group).
Ex-vivo optical imaging study. (a, b) Representative NIR fluorescent images in explanted organs at days 2 or 7 following implantation of DiDlabeled hEPCs into the ischemic myocardium of nude mice. Bars represent maximum radiance. (a: 2 days after cell delivery; b: 7 days after cell delivery). (c) Quantitative analysis of NIR fluorescent signals in explanted hearts among each group at two time points. All values are expressed as mean±s.e.m. (n¼3–4, *Po0.01 vs control group).

These results also confirm that TK works in heart muscle cells by activating the Akt protein inside the cells.  This establishes that TK works through the Akt pathway.

Once again, we see that transplantation of stem cells after a heart attack can improve the function and structure of the heart after a heart attack.  Indeed this strategy seems to work again and again.  These experiments were done in mice and therefore, they must be successful in a larger animal, like a pig before they can be deemed efficacious and safe for use in human clinical trials.  Even so, these results are hopeful.

Culture Medium from Endothelial Progenitor Cells Heals Hearts

Endothelial Progenitor Cells or EPCs have the capacity to make new blood vessels but they also produce a cocktail of healing molecules. EPCs typically come from bone marrow, but they can also be isolated from circulating blood, and a few other sources.

The laboratory of Noel Caplice at the Center for Research in Vascular Biology in Dublin, Ireland, has grown EPCs in culture and shown that they make a variety of molecules useful to organ and tissue repair. For example, in 2008 Caplice published a paper in the journal Stem Cells and Development in workers in his lab showed that injection of EPCs into the hearts of pigs after a heart attack increased the mass of the heat muscle and that this increase in heart muscle was due to a molecule secreted by the EPCs called TGF-beta1 (see Doyle B, et al., Stem Cells Dev. 2008 Oct;17(5):941-51).

In other experiments, Caplice and his colleagues showed that the culture medium of EPCs grown in the laboratory contained a growth factor called “insulin-like growth factor-1” or IGF1. IGF1 is known to play an important role in the healing of the heart after a heart attack. Therefore, Caplice and his colleagues tried to determine if IGF1 was one of the main reasons EPCs heal the heart.

To test the efficacy of IGF1 from cultured EPCs, Caplice’s team grew EPCs in the laboratory and took the culture medium and tested the ability of this culture medium to stave off death in oxygen-starved heart muscle cells in culture. Sure enough, the EPC-conditioned culture medium prevented heart muscle cells from dying as a result of a lack of oxygen.

When they checked to see if IGF1 was present in the medium, it certainly was. IGF1 is known to induce the activity of a protein called “Akt” inside cells once they bind IGF1. The heart muscle cells clearly had activated their Akt proteins, thus strongly indicating the presence of IGF1 in the culture medium. Next they used an antibody that specifically binds to IGF1 and prevents it from binding to the surface of the heart muscle cells. When they added this antibody to the conditioned medium, it completely abrogated any effects of IGF1. This definitively demonstrates that IGF1 in the culture medium is responsible for its effects on heart muscle cells.

Will this conditioned medium work in a laboratory animal? The answer is yes. After inducing a heart attack, injection of the conditioned medium into the heart decreased the amount of cell death in the heart and increased the number of heart muscle cells in the infarct zone, and increased heart function when examined eight weeks after the heart attacks were induced. The density of blood vessels in the area of the infarct also increased as a result of injecting IGF1. All of these effects were abrogated by co-injection of the antibody that specifically binds IGF1.

From this study Caplice summarized that very small amounts of IGF1 (picogram quantities in fact) administered into the heart have potent acute and chronic beneficial effects when introduced into the heart after a heart attack.

These data are good enough grounds for proposing clinical studies. Hopefully we will see some in the near future.

How Stem Cells Maintain Skin

Professor Kim Jensen from BRIC, University of Copenhagen and Cambridge University has used careful mapping studies to challenge current ideas of how the skin renews itself.

Skin is a rather complex organ system that consists of many cell types and structures. Skin includes proliferating cells in the stratum germanitivum, differentiating cells in the upper layers of the epidermis, hair cells, fat, sensory neurons, Langerhans cells, and sweat and sebaceous glands.

Jensen explained, “Until now, the belief was that the skin’s stem cells were organized in a strict hierarchy with a primitive stem cells type at the top of the hierarchy, and that this cell gave rise to all other cell types of the skin. However, our results show that there are differentiated levels of stem cells and that it is their close micro-environment that determines whether they make hair follicles, fat- or sweat glands.”

Jensen’s work completes what was a “stem cell puzzle.” As Jensen put it, “our data complete what is already known about the skin and its maintenance. Researchers have until now tried to fit their results into the old model for skin maintenance. However, the results give much more meaning when we relate them to the new model that our research purposes.”

To give an example of what Jensen is talking about, over-proliferation of skin cells can initiate skin cancer, but the stem cells of the skin that help maintain the integrity of the skin will lack any detectable genetic changes. According to Jensen, the reason these stem cells lack detectable genetic changes in that they do not take part in over-proliferation.

To demonstrate this, Jensen used a unique technique to label skin cells. They made a mouse strain that expresses a glowing protein from the control region of the Lrig1 gene. The Lrig1 gene is expressed in all proliferating skin stem cell populations. Therefore, making a mouse strain in which all cells expressing Lrig1 also express a glowing protein is a sure-fire way to label the skin stem cell populations.

Jensen and his cohorts used several experimental strategies. First, they simply mapped out the glowing cells in the skin. Jensen and his colleagues discovered that the skin contains several stem cell populations that reside in distinct compartments.  These different compartmentalized skin stem cells contributed to specific tissues and their domains did not over lap.

Basic RGB


When the mice were wounded, the proliferating stem cells freely crossed over into each other’s domains and helped heal and remake structures that they normally would not make.  This shows that upon wounding, the stem cells compartment boundaries break down as the stem cells proliferate to recreate the compartments that might have been lost as a result of wounding.  Therefore, Jensen’s work shows that Lrig1 marks stem cells in the epidermis, and that these stem cells have a unique lineage potential.  Secondly, the epidermis is maintained in discrete compartments by these multiple stem cell populations.  These stem cell populations largely keep to themselves and do not invade other compartments.  Therefore, stem cell compartmentalization underlies maintenance of the tissue complexity of the skin and not “hierarchy.”  This simply means that where the stem cells live is far more important to skin stem cell function than who their parents were.  Finally, wounding alters stem cell fate and break down the boundaries.

Wounding does more than that.  When Jensen and his colleagues made a mouse with an activated form of the ras gene that was expressed in skin, the skin showed no signs of tumor formation.  This is odd, since activating mutations in ras are extremely common in human and mouse tumors and cultured cells with activated ras mutations grow like cancer cells.  However, if the skin of these mice with the activated ras gene in their skin is wounded, then tumors form.  Therefore, wounding not only breaks down the compartments in which stem cells reside, it also potentiates cancer formation.

Jensen said of his results, “Our research will now take two directions.  We will establish mathematical models for organ maintenance in order to measure what stem cells are doing in the skin.  Also, we will expand our investigations in cancer initiation, hoping for results that can contribute to cancer diagnostics and improved treatment.”

Using Sleeping Stem Cells to Treat Aggressive Leukemias

British scientists have discovered that aggressive forms of leukemia (blood cancers) do not displace normal stem cells from the bone marrow, but instead, put them to sleep. If the normal stem cells are asleep, it implies that they can be awakened. This offers a new treatment strategy for acute myeloid leukemia or AML.

This work comes from researchers at Queen Mary, University of London with the support of Cancer Research UK’s London Research Institute.

In the United Kingdom, approximately 2,500 people are diagnosed with AML each year. The disease strikes young and old patients and the majority of patients die from AML.

In healthy patients, the bone marrow contains hematopoietic stem cells (HSCs) that divide to form either a common myeloid precursor (CMP) or a common lymphoid precursor (CLP) that differentiate into various kinds of white blood cells or red blood cells or lymphocytes. Individuals afflicted with AML, however, have bone marrow invaded by leukemic myeloid blood cells. Since red blood cells are derived from the myeloid lineage, AML causes red blood cell deficiencies (anemia), and the patient becomes tired, and is at risk for excessive bleeding. AML patients are also more vulnerable to infection those white blood cells that fight infections are not properly formed.

HSC differentiation2

David Taussig from the Barts Center Institute at Queen Mary, University of London said that the widely accepted explanation for these symptoms is that the cancerous stem cells displace or destroy the normal HSCs.

However, Taussig and his colleagues have found in bone marrow samples from mice and humans with AML contain plenty of normal HSCs. Thus, AML is not destroying or displacing the HSCs. Instead, the cancerous stem cells appear to be turning them off so that they cannot form HSCs. If Taussig and his coworkers and collaborators had determine how these leukemic myeloid blood cells are shutting off the normal HSCs, they might be able to design treatments to turn them back on.

Such a treatment strategy would increase the survival of AML patients. Only 40% of younger patients are cured of AML, and the cure rate for older patients in much lower. Current treatments that include chemotherapy and bone marrow transplants are not terribly successful with older patients.

Taussig’s group examined the levels of HSCs in the bone marrow of mice that had been transplanted with human leukemic myeloid cells from AML patients. They discovered that the numbers of HSCs stayed the same, but these same HSCs failed to transition through the developmental stages that result in the formation of new blood cells. When Taussig and his group examined bone marrow from 16 human AML patients, they discovered a very similar result.

Even though AML treatment has come a long way in the last ten years, there is still an urgent need for more effective treatments to improve long-term survival. This present study greatly advances our understanding of what’s going on in the bone marrow of AML patients. The future challenge is to turn this knowledge into treatments.

Under normal circumstances, stress on the body will boost HSC activity. For example, when the patient hemorrhages, the HSCs kick into action to produce more red blood cells that were lost during the bleed. However, the cancer cells in the bone marrow are somehow over-riding this compensatory mechanism and the next phase of this research will determine exactly how they do it.

Nanometer Scaffolds Regulate Neural Stem Cells

In the laboratory, stem cells can grow in liquid culture quite well in many cases, but this type of culture system, though convenient and rather inexpensive, does not recapitulate the milieu in which stem cells normally grow inside our bodies. Inside our bodies, stem cells stick to all kinds of surfaces and interact with and move over a host of complex molecules. Many of the molecules that stem cells contact have profound influences over their behaviors. Therefore, reconstituting or approximating these environments in the laboratory is important even though it is very difficult.

Fortunately nanotechnology is providing ways to build surfaces that approximate the kinds of surfaces stem cells encounter in our bodies. While this field is still in its infancy, stem cell-based nanotechnology may provide strategies to synthesize biologically relevant surfaces for stem cell growth, differentiation, and culture.

One recent contribution to this approach comes from Jihui Zhou and his team from the Fifth Hospital Affiliated to Qiqihar Medical University. Zhou and his co-workers prepared randomly oriented collagen nanofiber scaffolds by spinning them with an electronic device. Collagen is a long, fibrous protein that is found in tendons, ligaments, skin, basement membranes (the substratum upon which sheets of cells sit), bones, and is also abundant in cornea, blood vessels, cartilage, intervertebral disc, muscles, and the digestive tract. Collagen is extremely abundant in the human body; some 30% of all the proteins in our bodies are collagen. It is the main component in connective tissues.

There are many different types of collagen. Some types of collagen form fibers, while others for sheets. There are twenty-eight different types of collagen. Mutations in the genes that encode collagens cause several well-known genetic diseases. For example, mutations in collagen I cause osteogenesis imperfecta, the disease made famous by the Bruce Willis/Samuel T. Jackson movie, “Unbreakable.” Mutations in Collagen IV cause Alport syndrome, and mutations in either collagen III or V cause Ehlers-Danlos Syndrome.

Wen cells make fibrous collagen, they weave three collagen polypeptides together to form a triple helix protein that is also heavily crosslinked. This gives collagen its tremendous tensile strength.

Collagen fibers
Collagen fibers

In this experiment, electronic spinning technology made the collagen fibers and these fibers had a high swelling ratio when placed in water, high pore size, and very good mechanical properties.

Zhou grew neural stem cells from spinal cord on these nanofiber scaffolds and the proliferation of the neural stem cells was enhanced as was cell survival. Those genes that increase cell proliferation (cyclin D1 and cyclin-dependent kinase 2) were increased, as was those genes that prevent cells from dying (Bcl-2). Likewise, the expression of genes that cause cells to die (caspase-3 and Bax) decreased.

Thus novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit programmed cell death without inducing differentiation of the stem cells. These scaffolds do this by inducing the expression of proliferation- and survival-promoting genes.

Artificial Bones From Umbilical Cord Stem Cells

I am back from vacation. We visited some colleges in Indiana for my daughter who will be a senior this year. She really liked Taylor University and Anderson University. We’ll see if the tuition exchange works out.

Now to blogging.

Scientists from Granada, Spain have patented a hew biomaterial that consists of activated carbon cloth that just happens to be able to support the growth of cells that have the ability to regenerate bone. These results came from experiments that were conducted outside any living animals, but they hope to confirm these results in a living animal in the near future.

This new biomaterial facilitates the growth of bone-making cells derived from umbilical cord stem cells. This activated carbon cloth acts as a scaffold for cells that differentiate into “osteoblasts,” which are bone-building cells. This activated carbon cloth gives the osteoblasts a proper surface upon which to promote the growth of new bone.

Bone loss as a result of cancer, trauma, or degenerative bone diseases requires replacement bone to heal to damaged bone. Making new bone in the laboratory that can be transplanted is an optimal strategy for treating these patients.

Even though this laboratory-made bone was not used in living laboratory animals to date, the laboratory results look quite impressive. In the future, such techniques could help manufacture medicines or other sources of material to repair bone or lost cartilage. Once such artificial bone has been made in the laboratory, the Spanish team hopes to transplant it into rats or rabbits to determine if it can regenerate bone in such creatures.

Presently, no materials exist to replace lost bone. The method used to make bone by the research team from Granada uses a three-dimensional support that facilitates the production of those cell types that regenerate bone without the need for additional growth factors.

The growth of these umbilical cord stem cells on activated carbon cloth produced a product that could produce organic bone, but also mineralize the organic bone matrix. This patent could have numerous clinical applications in regenerative medicine and the Granada group hopes to obtain funding to continue this work and achieve their ultimate objective: to regenerate bones by implanting biomaterial in patients with bone diseases.

Using Human Induced Pluripotent Stem Cells to Study Diamond Blackfan Anemia

Diamond-Blackfan Anemia or DBA results from mutations in a gene on chromosome 19 (in most cases). Mutations in the ribosomal protein S19 affects the ability of blood cells to make protein and causes low numbers of red blood cells. DBA patients are dependent on blood transfusions, but some are cured, to some extent at least, by bone marrow transplants. Unfortunately, some DBA patients have severe side effects from bone marrow transplants, which means that bone marrow transplants are not a panacea for all DBA patients.

Fortunately, Michell J. Weiss and his colleagues at the Children’s Hospital of the Philadelphia (CHOP) have used human induced pluripotent stem cells (iPSCs) to study DBA at the molecular level and even develop the beginnings of a cure for DBA patients. Weiss collaborated with Monica Bessler, Philip Mason, and Deborah French, all of whom work at CHOP.

Remember that red blood cells are made inside the bone marrow of the patient by hematopoietic stem cells (HSCs). HSCs divide to renew themselves, and to produce a daughter cell that will differentiate into one of several different types of blood cells. As a kind of gee-wiz number, a healthy adult person will produce approximately 10[11]–10[12] (100 billion to 1 trillion) new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation.

In DBA patients, the bone marrow is empty of red blood cells. In order to get a better idea why, Weiss and his team isolated fibroblasts from the skin of DBA patients, and used genetic engineering techniques to convert them into iPSCs. When Weiss and his group tried to differentiate these iPSCs derived from DBA patients into red blood cells, they were not able to make normal red blood cells. However, Weiss and his colleagues used different genetic engineering techniques to fix the mutation in these iPSCs. After fixing the mutation, these cells could be differentiated into red blood cells. This experiment showed that it is possible to repair a patient’s defective cells.

This is a proof-of-principle experiment and there are many hurdles to overcome before this type of experiment can be done in the clinic to DBA patients. However, these iPSCs can play a vital role in deciphering some of the mysteries surrounding this disease. For example, two family members may have exactly the same mutation, but only one of them shows the disease whereas the other does not. Since iPSCs are specific to the patient from whom they were made, Weiss and his group hope to compare the molecular differences between them and understand the difference in expression of this disease.

Also, these cells offer a long-lasting model system for testing new drugs or gene modifications that may offer new treatments that are personalized to individual patients.

Weiss and his research group used this same technology to test drugs for the often aggressive childhood leukemia, JMML or Juvenile Myelomonocytic Leukemia. Once again, iPSCs were made from JMML patients and differentiated into myeloid cells, which divided uncontrollably just as the original myeloid cells from JMML patients.

Weiss and his colleagues used these cells to test two drugs, both of which are active against JMML. One of them is an inhibitor of the MEK kinase that was quite active against these cells. This illustrates how iPSCs can be used to test personalized treatment regimes for patients.

The stem cell core facility at CHOP is also in the process of making iPCS lines for several inherited diseases: dyskeratosis congenita, congenital dyserythropoietic anemia, thrombocytopenia absent radii, Glanzmann’s thrombasthenia, and Hermansku-Pudlak syndrome.

The even longer term goal is the use these lines to specifically study the behavior of such cells in culture and under certain conditions, test various drugs on them, and to develop treatment strategies on them as well.

A More Efficient Way to Make Human Induced Pluripotent Stem Cells

Stem cell researchers at the University of California, San Diego have designed a simple, reproducible, RNA-based method of generating human induced pluripotent stem cells (iPSCs). This new technique broad applications for the successful production of iPSCs for use in therapies and human stem cell studies.

Human iPSCs are made from adult cells by genetically engineering adult cells to overexpress four different genes (Oct4, Klf4, Sox2, and c-Myc). This overexpression drives the cells to de-differentiate into pluripotent stem cells that have many of the same characteristics as embryonic stem cells, which are made from embryos. However, because iPSCs are made from the patient’s own cells, the chances that the immune system of the patient will reject the implanted cells is low.

The problem comes with the overexpression of these four genes. Initially, retroviruses have been used to reprogram the adult cells. Unfortunately, retroviruses plop their DNA right into the genome of the host cell, and this change is permanent. If these genes get stuck in the middle of another gene, then that cell has suffered a mutation. Secondly, if these genes are stuck near another highly-expressed gene, then they too might be highly expressed, thus driving the cells to divide uncontrollably.

Several studies have shown that in order to reprogram these cells, these four genes only need to be overexpressed transiently. Therefore, laboratories have developed ways of reprogramming adult cells that do not use retroviruses. Plasmid-based systems have been used, adenovirus and Sendai virus-based systems, which do not integrate into the genome of the host cell, have also been used, and even RNA has been used (see Federico González, Stéphanie Boué & Juan Carlos Izpisúa Belmonte, Nature Reviews Genetics 12, 231-242).

The UC San Diego team led by Steven Dowdy has used Venezuelan equine virus (VEE) that they engineered to express the reprogramming genes required to make iPSCs from adult cells. Because this virus does not integrate into the host genome, and expresses RNA in the host cell only transiently, it seems to be a safe and effective way to make buckets of messenger RNA over a short period of time.

The results were impressive. The use of this souped-up VEE produced good-quality iPSCs very efficiently. Furthermore, it worked on old and young human cells, which is important, since those patients who will need regenerative medicine are more likely to be young patients than old patients. Also, changing the reprogramming factors is rather easy to do as well.

Japanese first Ever Induced Pluripotent Stem Cell Clinical Trial Given the Green Light

The first clinical trial that utilizes induced pluripotent stem cells has been given a green light. For this clinical trial six patients who suffer from age-related macular degeneration will donate skin biopsies and the cells from these skin biopsies will be used to generate induced pluripotent stem (iPS) cells in the laboratory. After those iPS cell lines are screened for safety (normal numbers of chromosomes, no mutations in critical genes, etc.), they will be differentiated into retinal cells. The retinal cells will be transplanted into the retinas of these six patients.

This clinical trial was approved by Japan Health Minister Norihisa Tamura and it will be next summer by Masayo Takahashi. Dr. Takahashi is a retinal regeneration expert and a colleague of the man who first developed iPS cells, Shinya Yamanaka. Yamanaka won the Nobel Prize for his discovery of iPSCs last year. In fact, this clinical trial epitomizes, in the eyes of many, the determination of Japanese scientists and politicians to dominate the iPS cell field. This national ambition kicked into high gear after Yamanaka shared the Nobel Prize for Physiology or Medicine last October for his iPS cell work.

Norhisa Tamura, Japanese Minister of Health
Norihisa Tamura, Japanese Minister of Health
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.

“If things continue this way, this will be the first in-clinic study in iPS cell technology,” says Doug Sipp of the Riken Center for Developmental Biology (CDB). The CDB, Takahashi’s institute, will co-run the trial with Kobe’s Institute for Biomedical Research and Innovation. “It’s exciting.”

Sipp, however, also noted that this move has not surprised anyone in Japan, since the Japanese stem cell community has heavily invested in iPS cells. Nevertheless, since Takahashi yet to formally publish the details of her trial, some have questioned whether she is actually ready to move forward. IPS cells are viewed as the perfect compromise for regenerative medicine. They are adult, and therefore do not require the destruction of human embryos for their establishment, and they are also pluripotent like an embryonic cell, which makes them relatively powerful sources for regenerative medicine.

Critics, however, warn that iPS cells were only discovered in 2007. To date, they remain difficult to create and culture and they can become tumorous in many hands. However, many labs have a great deal of expertise and skill when it comes to handling and deriving iPS cells. These labs derive and culture iPS cells routinely. In fact, Sipp notes that Riken’s CDB alone has produced world-class work with all kinds of stem cells, including embryonic stem (ES) cells, which are the models for iPS cells.

Additionally, Sipp and others point out that a scientist who has collaborated with Takahashi in the past, Riken’s Yoshiki Sasai, is doing groundbreaking work with ES cells and the eye. The British journal Nature has called Sasai “The Brainmaker,” and has said that his research is “wowing” the world.

The Japanese government has also soundly funded Takahashi’s trail. The health ministry’s recent stimulus plan set aside more money for stem cells (in particular iPS cells) than anything else. According to the journal Nature, the Japanese government sequestered 21.4 billion yen ($215 million) for stem cell research. Of this pot of money, the health ministry provided 700 million yen ($7 million) for a cell-processing center to support Takahashi before her trial was even approved. Two centers devoted to iPS cells are slated to be built with 2.2 billion yen ($22 million). The AFP reports the prime minister has set aside a breathtaking $1.18 billion, for iPS-cell work. Yamanaka has told Nature that the Japanese government seems to be “telling us to rush iPS cell-related technologies to patients as quickly as possible.”

Robert Lanza, CSO of Advanced Cell Technology, might once have been the logical bet to be first to the clinic with iPS cells. Unlike Takahashi, he has three ES cell trials under his belt, and has started talks with the FDA about transplanting iPS cell-derived platelets, but his iPS proposal is taking longer. Lanza bitterly noted, not without justification, “We don’t have the prime minister and emperor to speed things along for us.”

Since 2007, the year that Yamanaka reported the derivation of iPS cells from adult cells, Japan has focused on iPS cells. Yamanaka showed that increasing the expression of four genes could change limited adult human cells into potent, embryonic-like cells. “At Yamanaka’s institute alone, there are at least 20 teams focusing on iPS cells now,” Sipp says. There are teams at Riken, the Universities of Tokyo and Keio, and others. “A lot is happening here.” In fact, the Center for IPS Cell Research and Application was created expressly for Yamanaka.

Takahashi has reported part of the design of her clinical trial at scientific meetings. She told the International Society for Stem Cell Research in June 2012 she had created iPS-cell derived retinal pigment epithelial (RPE) cells for transplantation. RPE cells lie behind the photoreceptors in the retina, and the photoreceptors have their ends embedded into the RPE. The RPE cells replenish and nourish the photoreceptors, and without the RPE cells, the photoreceptors die from the damage incurred by exposure to light.

Retinal Pigmented Epithelium

Death of the RPE cells cause eventual death of photoreceptors and that results in blindness. At the International Society for Stem Cell Research conference, Takahashi reported her that her iPS cell-derived RPEs possess proper structure and gene expression. They also do not produce tumors when transplanted into mice, and survive at least six months when transplanted into the retinas of monkeys. The vision of these animals, however, was not tested. She did note that some AMD patients’ sight improves when RPE cells are moved from the eye’s periphery to its center.

Retinal pigment epithelial cells derived from iPS cells.
Retinal pigment epithelial cells derived from iPS cells.

Takahashi has published many iPS and ES cell papers. These papers include two papers with Yamanaka: one on creating retinal cells from iPS cells, and one on creating safe iPS cells. However she has not published trial details, which is not required, but such a landmark trial should be transparent, as argued by many stem cell experts.

Still, according to Sipp, Takahashi has submitted a relevant paper to a top journal for review, which shows that this clinical trial is purely a determination of the safety of the procedure. Lanza has reported his trials in the journal The Lancet, and similar, but small, trials are doing well. His three ES cell trials treated Stargardt’s macular dystrophy and Age-related Macular Degeneration. Lanza’s trial, however, treated “dry” macular degeneration, while Takahashi’s trial will treat “wet” Age-related Macular Degeneration, which is good news for Takahashi.

Paul Knoepfler, a UC Davis stem cell scientist who runs a widely read blog site, has written that the ministry overseeing Takahashi’s trial will reportedly monitor some key factors: gene sequencing and tumorigenicity. But Knoepfler, like others, would like to see more details.

The Japanese Health Ministry and the US FDA recently agreed to devise a joint regulatory framework for retinal iPS cell clinical trials, which will come on line 2015. Takahashi’s trial is set for 2014.

Making Induced Pluripotent Stem Cells With Small Molecules

A Journal article in the August 9th edition of Science Magazine features work from the laboratories of Yang Zhao and Hongkui Deng, both of whom are from the College of Life Sciences and Peking-Tsinghua Center for Life Sciences at Peking University in Beijing, China. Zhao and Deng and colleagues used small molecules to transform adult cells into induced pluripotent stem cells.

To review, induced pluripotent stem cells are derived from adult cells by genetically engineering the adult cells to express a cocktail of four genes (OCT4, Klf4, Sox2, and c-Myc). To introduce these genes into cells, viruses are normally used, but other techniques are also available. The resultant cells look and act like embryonic stem cells, but they do not require the death of embryos.

In this paper, Deng and colleagues took mouse embryonic fibroblasts (skin cells cultured from mouse embryos) and used them to screen over 10,000 small molecules for their ability to substitute for the OCT4 gene in the production of iPSCs. If this sounds labor intensive, that’s because it is. To conduct the screen, they used mouse embryonic fibroblasts that were infected with viruses that expressed Sox2, Klf4, and c-Myc. These genes are not enough to convert adult cells into iPSCs. However, with these chemicals, these three genes could produce iPSCs from mouse embryonic fibroblasts (MEFs). They identified at least three molecules; Forskolin, 2-methyl-5-hydroxytryptamine and a synthetic molecule called D4476, that could substitute for OCT4.

Thus, by using chemicals, they could get away from using one of the genes required to de-differentiate adult cells into iPSCs. Could they whittle down the number of genes even further? Previously, Deng and Zhao published a paper in which a chemical cocktail was used to substitute for the other three genes so that conversion into iPSCs was achieved by introducing only the OCT4 gene into cells (Li, YQ et al., CELL RESEARCH 21(1): 196-204. They called this cocktail “VC6T.” Therefore, they used VC6T and Forskolin, on their MEFs and the beginnings of de-differentiation occurred, but not much else.

Could chemicals be identified that would take the cells the rest of the way to iPSCs? Another chemical screen examined this possibility. In this test, the MEFs were rigged so that they expressed OCT4 when the cells were treated with the antibiotic doxycycline. By giving the cells doxycycline for 4-8 days, and then testing chemicals to take the cells the rest of the way, they identified a slew of compounds that, when given to the OCT4-expressing MEFs, they became iPSCs.

Then came the real test – make iPSCs with just chemicals and no introduced genes. Could it be done? When they gave the MEFs some of the chemicals identified in the last screen (they called it DZNep), plus VC6T, the expression of OCT4 went up, but the cells simply did not look like iPSCs. So, they changed the culture medium to a “2i” culture system that inhibits some key regulatory proteins in the cells. When they used this same chemical cocktail in a 2i culture system, it worked and iPSCs were produced. Deng and Zhao called these stem cells “chemically induced pluripotent stem cells” or CiPSCs.

(A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars, mean ± SD (n = 3 biological repeat wells). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars, mean ± SD (n = 2 biological repeat wells). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. Scale bars, 100 μm. For (D), cells for reprogramming were replated on day 12.
(A and B) Numbers of iPSC colonies induced from MEFs infected by SKM (A) or SK (B) plus chemicals or Oct4. Error bars, mean ± SD (n = 3 biological repeat wells). (C) Morphology of MEFs for chemical reprogramming on day 0 (D0) and a GFP-positive cluster generated using VC6TF on day 20 (D20) after chemical treatment. (D) Numbers of GFP-positive colonies induced after DZNep treatment on day 36. Error bars, mean ± SD (n = 2 biological repeat wells). (E to G) Morphology of a compact, epithelioid, GFP-positive colony on day 32 (D32) after treatment (E), a primary CiPSC colony on day 40 (D40) after treatment (F), and passaged CiPSC colonies (G). (H) Schematic diagram illustrating the process of CiPSC generation. Scale bars, 100 μm. For (D), cells for reprogramming were replated on day 12.

Next, they optimized the dosages of these chemicals in order to increase the efficiency of iPSC production. They were able to increase the efficiency of iPSC production to 5% (1 of every 20 colonies of cells), which is respectable. They also identified yet another small molecule that beefed up iPSC production by another 40-fold. Also, this chemical cocktail was able to make iPSCs from mouse adult fibroblasts, fat-derived stem cells, and fibroblasts from newly born mice.

When the CiPSC lines were characterized, they made all the right genes to be designated as pluripotent stem cells, and they had normal numbers of normal-looking chromosomes all the way through 13 passages.

When injected into mice with dysfunctional immune systems, the CiPSCs made tumors that were mixtures of tissues of all over the body. When they were transferred into early mouse embryos, they could contribute to the bodies of developing mice, and they could even contribute to the production of eggs and sperm, When baby mice were completely made from CiPSCs, those mice were fertile and had babies of their own. This is the ultimate test of pluripotency and the CiPSCs passed it with flying colors.

A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied.
A) Hematoxylin and eosin staining of CiPSC-derived teratoma (clone CiPS-30). (B to D) Chimeric mice (B, clone CiPS-34), germline contribution of CiPSCs in testis, (C, clone CiPS-45) and F2 offspring (D, clone CiPS-34). Scale bars, 100 μm. (E) Genomic PCR analyzing pOct4-GFP cassettes in the tissues of chimeras. (F) Survival curves of chimeras. n, total numbers of chimeras studied.

Other experiments in this paper examined why these chemicals induced pluripotency in adult cells, but these experiments, though interesting, are lost in the fact that this research group has generated iPSCs without using any viruses, or genetic engineering technology. These CiPSCs are true pluripotent stem cells and they were generated without killing any embryos or introducing genes that might drive cells to become abnormal.

If this can be replicated with human cells, it would be earth-shattering for regenerative medicine.

Researchers Create Inner Ear Structures From Stem Cells

Indiana University scientists have used mouse embryonic stem cells to make key structures of the inner ear. This accomplishment provides new insights into the sensory organ’s developmental process and sets the stage for laboratory models of disease, drug discovery and potential treatments for hearing loss, and balance disorders.

Eri Hashino, professor of otolaryngology at the University of Indiana School of Medicine, and his co-workers, were able to use a three-dimensional cell culture method that directed the stem cells to form inner-ear sensory epithelia that contained hair cells and supporting cells and neurons that detect sound, head movements and gravity.

In the past, other attempts to grow inner-ear hair cells in standard culture systems have not succeeded. Apparently the cues required to form inner-ear hair bundles, which are essential for detecting auditory or vestibular signals, are absent in cell-culture dishes.

Inner ear hair cells
Inner ear hair cells

To conquer this barrier, Hashino and his team changed their culture system. The suspended the cells as aggregates in a specialized culture medium and this mimicked conditions normally found in the body as the inner ear develops.

Another strategy that paid off was to precisely time the application of several small molecules that coaxed the stem cells to differentiate from one stage to the next into precursors for the inner ear.

a, Schematic of vestibular end organs and type I/II vestibular hair cells. vgn, vestibular ganglion neurons. b, c, Pax2 (b) and Calb2 (c) are expressed in all Myo7a+ stem-cell-derived hair cells on day 20. CyclinD1 (cD1) is expressed in supporting cells. d–g, The structural organization of vesicles with Calb2+ Myo7a+ hair cells mimics the E18 mouse saccule (sagittal view) in vivo. nse, nonsensory epithelium. h, Tuj1+ neurons extending processes to hair cells. i, The synaptic protein Snap25 is localized to the basal end of hair cells. j, The postsynaptic marker Syp colocalizes with Ctbp2 (arrowheads and inset). hcn, hair cell nucleus. k, Quantification of synapses on day 16, 20 and 24 hair cells (n > 100 cells, *P < 0.05, ***P < 0.001; mean ± s.d.). l, Overview of in vitro differentiation. Scale bars, 50 μm (d, f, h), 25 μm (b, c, e, g), 10 µm (i), 5 µm (j).  Also, BMP = Bone morphogen protein, FGF = fibroblast growth factor, LGN = Small molecule that inhibits BMP signaling, Wnt = small secreted glycoprotein involved in cell signaling.
a, Schematic of vestibular end organs and type I/II vestibular hair cells. vgn, vestibular ganglion neurons. b, c, Pax2 (b) and Calb2 (c) are expressed in all Myo7a+ stem-cell-derived hair cells on day 20. CyclinD1 (cD1) is expressed in supporting cells. d–g, The structural organization of vesicles with Calb2+ Myo7a+ hair cells mimics the E18 mouse saccule (sagittal view) in vivo. nse, nonsensory epithelium. h, Tuj1+ neurons extending processes to hair cells. i, The synaptic protein Snap25 is localized to the basal end of hair cells. j, The postsynaptic marker Syp colocalizes with Ctbp2 (arrowheads and inset). hcn, hair cell nucleus. k, Quantification of synapses on day 16, 20 and 24 hair cells (n > 100 cells, *P < 0.05, ***P < 0.001; mean ± s.d.). l, Overview of in vitro differentiation. Scale bars, 50 μm (d, f, h), 25 μm (b, c, e, g), 10 µm (i), 5 µm (j). Also, BMP = Bone morphogen protein, FGF = fibroblast growth factor, LGN = Small molecule that inhibits BMP signaling, Wnt = small secreted glycoprotein involved in cell signaling.

Even though the added growth factors made a big difference to the success of this experiment, it was the three-dimensional suspension culture system that provided many important mechanical cues. The tension caused by the pull of the cells on each other played a very important role in directing the differentiation of the cells to become inner-ear precursors.

Karl A Koehler, first author of this paper and a graduate student in the medical neuroscience program at IU School of Medicine said: “The three-dimensional culture allows the cells to self-organize into complex tissues using mechanical cues that are found during embryonic development.”

Hashino added that they were “surprised to see that once stem cells are placed in 3-D culture, these cells behave as if they knew not only how to self-organize into a pattern remarkably similar to the native inner ear.” Hashino continued: “Our initial goal was to make inner-ear precursors in culture, but when we did testing we found thousands of hair cells in a culture dish.”

Electrophysiological testing of these stem cell-derived hair cells showed that they were, in fact, functional, and were similar to those that sense gravity and motion. Moreover, neurons like those that normally link the inner-ear cells to the brain had also developed in their cell culture system, and were connected to the hair cells.

Hashino thinks that additional research is needed to determine how to derived inner-ear cells involved in auditory sensation might be made from stem cells, and how such techniques might be adapted to make human inner ear cells.

Genomic Imprinting Maintains A Reserve Pool of Blood-Forming Stem Cells

Hematopoietic stem cells or HSCs reside in the bone marrow and give rise to the wide variety of specialized blood cells that inhabit our bloodstreams. Within the bone marrow, HSCs come in two varieties: an active arm of HSCs that proliferate continually to replace our blood cells and a reserve arm that sits and quietly waits for their time to come.

New research from the Stowers Institute at Kansas City, Mo, in particular a research team led by Linheng Li, discovered a mechanism that helps maintain the balance between those HSCs kept in reserve and those on active duty.

According to Dr. Li, genomic imprinting, a process that specifically shuts off one of the two gene copies found in each mammalian cell , prevents the HSCs held in reserve from being switched to active duty prematurely.

Li explained: “Active HSCs form the daily supply line that continually replenishes worn-out blood and immune cells while the reserve pool serves as a backup system that replaces damaged active HSCs and steps in during times of increased need. In order to maintain a long-term strategic reserve of hematopoietic stem cells that lasts a lifetime it is very important to ensure that the back-up crew isn’t mobilized all at once. Genomic imprinting provides an additional layer of regulation that does just that.”

Sexual reproduction produces progeny that have once set of chromosomes from the mother and one set of chromosomes from the father. The vast majority of genes are expressed from both sets of chromosomes. However, in placental mammals and marsupial mammals a small subset of genes are imprinted, which means that they receive a mark during the development of eggs and sperm and these marks shut down expression of those genes in either the sperm pronucleus or the egg pronucleus. Therefore, after the fusion of the sperm and the egg and the eventual fusion of the egg and sperm pronuclei, these imprinted genes are only expressed from one copy of genes. Some are only expressed from the paternal chromosomes and others are only expressed from the maternal chromosome. Imprinting is essential for normal development in mammals.

The importance of genetic imprinting is shown if an egg loses its pronucleus and is then fertilized by two sperms. The resulting zygote has two copies of paternal chromosomes and no copies of the maternal chromosomes. Such an embryo is called an andogenote, and the embryo fails to form but the placenta overgrows. If this occurs during human development, it can lead to a so-called “molar pregnancy” or “hydatiform mole.” This fast growing placental tissue can become cancerous and lead to uterine cancer. For that reason, molar pregnancies are usually dealt with expeditiously.

However, if the sperm that fertilizes the egg is devoid of a pronucleus, and the egg pronucleus duplicates, then the resulting zygotes can two copies of the maternal chromosomes, and this entity is known as a gynogenote, and it develops with a poorly formed placenta that dies early in development.

In previous experiments in mice, Li and his colleagues indicated that the expression of several imprinted genes changes as HSCs transition from quiescent reserve cells to multi-lineage progenitor cells.

In their current study, Li and other Stowers Institute researchers examined a differentially imprinted control region, which drives the reciprocal expression of a gene called H19 from the maternal chromosome and IGF2 (insulin-like growth factor-2) from the paternal chromosome.

The first author of this study, Aparna Venkatraman developed a mouse model that allowed her to specifically delete the imprinted copy from the maternal chromosome. Thus, in these mice, H19, which restricts growth, was no longer active and Igf2,, which promotes cell division, was now active from the paternal and the maternal chromosome. To access the effect of this loss of imprinting on the maintenance of HSCs, Venkatraman examined the numbers of quiescent HSCs and active HSCs. in mouse bone marrow.

Venkatraman explained: “A large number of quiescent HSCs was activated simultaneously when the epigenetic control provided by genomic imprinting was removed. It created a wave of activated stem cells that moved through different maturation stages.”

She followed this experiment with a closer look at the Igf2 gene. Misregulation of Igf2 leads to overgrowth syndromes such as Beckwith-Wiedmann Syndrome. It exerts its growth promoting effects through the Igf1 receptor, which induces an intracellular signaling cascade that stimulates cell proliferation.

IGF signaling pathway
IGF signaling pathway

The expression of the Igf1 receptor itself is regulated by H19, which encodes a regulatory microRNA (miR-675) that represses translation of the Igf1 receptor gene and therefore prevents production of Igf1 receptor protein. Venkatraman explained that once the “imprinting block is lifted, the Igf2-Igf1r signaling pathway is activated.” Venkatraman continued: “The resulting growth signal triggers the inappropriate activation and proliferation of quiescent HSCs, which eventually leads to the premature exhaustion of the reserve [HSC] pool.”

Interestingly, the roundworm, Caenorhabditis elegans, provided the first clues that diminished insulin/IGF signaling can increase lifespan and delay aging. Li again: “Here the IGF pathway is conserved by subject to imprinting, which inhibits its activation in quiescent reserve stem cells. This ensures the long-term maintenance of the blood system, which in turn supports the longevity of the host.”