Picking the Right Stem Cell With Gold Nanoparticles


Sumeet Mahajan and his laboratory at the University of Southampton has collaborated with researchers from Cambridge University to use gold nanoparticles to identify different types of stem cells in the body.

For stem cell therapies to work, clinicians must be able to identify the right stem cells population to repair damaged tissues and organs. This identification process is typically very invasive and often leaves the cells dead or damaged. For example, the use of fluorescent probes to tag and track individual cells is very powerful but quite harmful to cells.

Mahajan has been investigating the use of Surface Enhanced Raman Spectroscopy or SERS to examine cells on a molecular level and distinguish adult stem cells from other cells without damaging them.

If a metal surface is roughened, and detectable molecules are placed on it, the signal those detectable molecules provide is enhanced by almost a million fold. This enhanced signal allows for the detection of very small quantities, and even though SERS has been used in several different industries, this is the first time it has been used in therapeutics.

Mahajan has been thinking about treating bone marrow preparations with gold nanoparticles in order to distinguish the stem cells from all the other cells. Such a technique would allow for the transplantation of only the stem cells and not other material that might not help the patient.

Also, scientist studying neurodegenerative diseases such as Parkinson’s disease must transplant dopamine-using neurons. Therefore, identifying specific types of dopaminergic cells is essential for efficacious treatments for Parkinson’s disease.

Additionally, researchers are also thinking about using Mahajan’s technique to test the efficacy of particular drugs and diagnose particular diseases. If specific cell types affected by particular diseases (e.g., diabetes, cirrhosis, etc.) could be safely identified and then tested, diagnosis might become much faster.

Mahajan is collaborating with major pharmaceutical companies to further develop more effective drugs with his SERS technique.

Tumor Suppressor Gene is Required For Neural Stem Cells to Differentiate into Mature Neurons


Cancer cells form when healthy cells accumulate mutations that either inactivate tumor suppressor genes or activate proto-oncogenes. Tumor suppressor genes work inside cells to put the brakes on cell proliferation. Proto-oncogenes work to drive cell proliferation. Loss-of-function mutations in tumor suppressor genes remove controls on cell proliferation, which causes cells to divide uncontrollably. Conversely activating mutations in proto-oncogenes removes the controls on the activity of proto-oncogenes, converting them into oncogenes and driving the cell to divide uncontrollably. If a cell accumulates enough of these mutations, they can grow in such an uncontrollable fashion that they start to gain extra chromosomes or pieces of chromosomes, which contributes to the genetic abnormality of the cell. Accumulation of more mutations allows the cell to break free from the original tumorous mass and spread to other tissues.

There are over 35 identified tumor suppressor genes and one of these, CHD5, has another role besides controlling cell proliferation. Researchers at Karolinska Institutet in Stockholm, Swede, in collaboration with other laboratories at Trinity College in Dublin and BRIC in Copenhagen has established a vital role for CHD5 in normal nervous development.

Once stem cells approach the final phase of differentiation into neurons, the CHD5 protein is made at high levels. CHD5 reshapes the chromatin structure into which DNA is packaged in cells, and in doing so, it facilitates or obstructs the expression of other genes.

Ulrika Nyman, postdoc researcher in Johan Holmberg’s laboratory, said that when they switched of CHD5 expression in stem cells from mouse embryos at the time when the brain develops, the CHD5-less stem cells were unable to turn off those genes that are usually expressed in other tissues, and equally unable to turn on those genes necessary for making mature neurons. Thus these CHD5-less stem cells were trapped in a nether-state between stem cells and neurons.

CHD5 function in stem cell differentiationretinoic

The gene that encodes the CHD5 protein is found on chromosome 1 (1p36) and it is lost in several different cancers, in particular neuroblastomas, a disease found mainly in children and is thought to arise during the development of the peripheral nervous system.

Neuroblastomas that lack this part of chromosome 1 that contains the CHD5 gene are usually more aggressive and more rapidly fatal.

Treatment with retinoic acid forces immature nerve cells and some neuroblastomas to mature into specialized nerve cells. However, when workers from Holmberg’s laboratory prevented neuroblastomas from turning up their expression of CHD5, they no longer responded to retinoic acid treatment.

Holmberg explained, “In the absence of CHD5, neural tumor cells cannot mature into harmless neurons, but continue to divide, making the tumor more malignant and much harder to treat. We now hope to be able to restore the ability to upregulate CHD5 in aggressive tumor cells and make them mature into harmless nerve cells.”

Cardiac Stem Cells Offer New Hope for Treatment of Heart Failure


Scientists from the United Kingdom have, for the first time, highlighted the natural regenerative abilities of a group of stem cells that live in our hearts. This particular study shows that these cells are responsible for repairing and regenerating muscle tissue that has been damaged by a heart attack. Such damage to the heart can lead to heart failure.

There is a robust debate as to the regenerative capacity of cardiac stem cells (CSCs) in the hearts a adult human beings. While many scientists are convinced that CSCs in the hearts of newborns have good regenerative ability, many remain unconvinced that adult CSCs can do similar things (see Zaruba, M.M., et al., Circulation 121, 1992–2000 and Jesty, S.A., et al., Proc. Natl. Acad. Sci. USA 109, 13380–13385). Nevertheless, an earlier paper showed that when introduced into heart muscle after a heart attack, CSCs will regenerate the lost heart muscle and blood vessels lost in the infarct (see Beltrami, A.P., et al., Cell 114, 763–776). Resolving this disagreement requires a different type of experiment.

In this paper, Bernardo Nadal-Ginard and colleagues from the and his collaborators at the Stem Cell and Regenerative Biology Unit at the Liverpool John Moores University in Liverpool and his collaborators from Italy used a different way to affect the heart. When heart attacks are experimentally induced in the heart of rodents, the infarcts are large and they kill off large numbers of CSCs. Therefore, Nadal-Ginard and others induced severe diffuse damage of the heart muscle that also spared the CSCs. They gave the mice a large dose of a drug called isoproterenol, which acts as a “sympathomimetic.” This is confusing science talk that simply means that the drug speeds the heart rate to the point where the heart muscle exhausts itself and then starts to die off. This treatment, however, spares the CSCs (see Ellison, G.M., et al., J. Biol. Chem. 282, 11397–11409).

When the heart muscle was damaged, the CSCs differentiated into heart muscle cells and other heart-specific cells and repaired the damage in the heart. Also, the repairing cells were in the heart and were not the result of bone marrow stem cells that migrated to the bone marrow, thus putting to rest a controversy that has lasted for some years that CSCs are the result of bone marrow stem cells that migrate to the heart.

Elimination of CSCs prevents heart repair after heart damage. If, however, these heart-based stem cells are replaced after damage, the heart repairs itself and the heart recovers its function, anatomical integrity, and cellular structure.

In other experiments, removal of cardiac stem cells (CSCs) and re-injection after a heart attack shows that the CSCs can home in and repair the damaged heart.

c-kit CSCs repair heart

Since Nadal-Ginard showed that CSCs have a capacity to home to the damaged heart, less invasive treatments might be possible and that these treatments might even prevent heart failure after a heart attack in the future.

In a healthy heart, the quantity of CSCs is sufficient to repair heart muscle tissue. However, once the heart is damaged many of the CSCs are also damaged and cannot multiply or produce new muscle tissue. In these cases it could be possible to replace damaged CSCs with new ones that have been grown in the laboratory and administered intravenously.,

These new approaches involved maintaining or increasing the activity of CSCs in order to renew heart muscle and replace old, damaged cells. This new strategy will only require intravenous administration of CSCs and not require open heart procedures that require such a long time to recover.

These findings are very promising. The nest step is a clinical trial, which is due to start early 2014 and is aimed at assessing the safety and effectiveness of CSCs for preventing and treating heart failure in humans.

FDA Approves the First Stem Cell Clinical Trial for Multiple Sclerosis


The Tirsch Multiple Sclerosis (MS) Research Center of New York has received Investigational New Drug (IND) approval from the Food and Drug Administration to launch a Phase I trial that uses a patient’s own neural stem cells to treat MS.

MS is a chronic disease that results when a patient’s own immune system attacks the myelin insulation that covers many nerves. This damages the myelin sheath and causes degeneration of the nervous system. Some 2.1 million people worldwide are afflicted with MS.

“To my knowledge, this is the first FDA-approved stem cells trial in the United States to investigate direct injection of stem cells into the cerebrospinal fluid of MS patients, and represents an exciting advance in MS research and treatment,” said Saud A. Sadiq, senior research scientist at Tisch and the study’s principal investigator.

The groundbreaking study will evaluate the safety of using stem cells harvested from the patient’s own bone marrow. Once harvested, these stem cells will be injected into the cerebrospinal fluid that surrounds the spinal cord in 20 participants who meet the inclusion criteria for this trial.

Since this is a phase 1 study, it is an open safety and tolerability study. The Tisch MS Research Center and affiliated International Multiple Sclerosis Management Practice (IMSMP) will host all the activities associated with this study.

The clinical application of autologous neural precursors in MS is the culmination of a decade of stem cell research headed by Sadiq and his colleague Violaine Harris, a research scientist at Tisch.

Preclinical testing found that the injection of these cells seems to decrease inflammation in the brain and may also promote myelin repair and neuroprotection.  In a 2012 publication in the Journal of the Neurological Sciences, Harris and others showed that mesenchymal stem cell-derived neural progenitor cells could promote repair and recovery after intrathecal injection into mice with EAE (experimental autoimmune encephalitis), which is a MS-like disease in mice.  They were able to ascertain that intrathecal injection of mesenchymal stem cell-derived neural progenitor cells significantly correlated with reduced immune cell infiltration in the brain, reduced area of demyelination, and increased number of neural progenitor cells in EAE mice.  This successful preclinical study was the impetus for this clinical trial.

Sadiq said, “This study exemplifies the Tisch MS Research Center’s dedication to translational research and provides a hope that established disability may be reversed in MS.” All study participants will undergo a single bone marrow collection procedure, from which mesenchymal stem cell-derived neural progenitor cells (MSC-NPs) will be isolated. expanded, and tested prior to injection.

All patients will receive three rounds of injections at three-month intervals. Safety and efficacy parameters will be evaluated in all trial participants throughout their regular visits with their attending physicians.

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.

Urinary Stem Cells and Their Therapeutic Potential


Yuanyuan Zhang, assistant professor of regenerative medicine at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine, has extended earlier work on stem cells from urine that suggests that these cells might be more therapeutically useful than previously thought.

These urinary stem cells can be isolated from a patient’s urine sample, and they can be induced, in the laboratory, to form bladder-type cells; smooth muscle and urothelial (bladder-lining) cells. Such stem cells could certainly be used to treat urinary tract problems, even though a good deal more work is required to confirm that they can do just that.

Nevertheless, Zhang and his co-workers have discovered that these urinary tract stem cells are much more plastic than previously thought. In the laboratory, Zhang and others have managed to differentiate urinary tract stem cells into bone, cartilage, fat, skeletal muscle, nerve, and endothelial cells (the cells that line blood vessels). This suggests that urine-derived stem cells could be used in a variety of therapies.

USCs undergo multipotential differentiation in vitro. (a-c) endothelial differentiation of USCs. USCs (p3) were induced to endothelial lineage by culture in EBM-2 medium containing VEGF 50 ng/ml for 14 days. (a) In vitro vessel formation. Endothelial differentiated USCs were cultured on Matrigel for 18h to form branched networks (angiogenesis) and tubular structures. Scale bar = 100μm. (b) Expression analysis of endothelial-specific transcripts by RT-PCR. (c) Immunofluorescence staining using endothelial-specific markers revealed enhanced staining of the markers with differentiation (middle row) compared to the non-treated control (top row). Scale bar = 50μm.
USCs undergo multipotential differentiation in vitro. (a-c) endothelial differentiation of
USCs. USCs (p3) were induced to endothelial lineage by culture in EBM-2 medium containing
VEGF 50 ng/ml for 14 days. (a) In vitro vessel formation. Endothelial differentiated USCs were
cultured on Matrigel for 18h to form branched networks (angiogenesis) and tubular structures. Scale
bar = 100μm. (b) Expression analysis of endothelial-specific transcripts by RT-PCR. (c)
Immunofluorescence staining using endothelial-specific markers revealed enhanced staining of the
markers with differentiation (middle row) compared to the non-treated control (top row). Scale bar =
50μm.

Zhang said that urinary tract stem cells could be used to treat urological disorders such a kidney disease, urinary incontinence, and erectile dysfunction. However, Zhang is optimistic that they can also be used to treat a wider variety of treatment options, such as making replacement bladders, urine tubes, and other urologic organs.

Since these stem cells come from the patient’s own body, they can have a low chance of being rejected by the immune system. Also, they do not cause tumors when implanted into laboratory animals.

In their latest work, Zhang and his colleagues obtained urine samples from 17 healthy individuals whose ages ranged from five to 75 years old. Even though these stem cells are only one of a large collection of cells in urine, isolating urinary stem cells from urine only requires minimal processing.

A single USC (inset) is followed through different passages (p0-p12). The cells were expanded to a colony were cultured in KSFM-EFM medium with 5% serum and images recorded with passage. Images shown at x100
A single USC (inset)
is followed through different passages (p0-p12). The cells were expanded to a colony were cultured in
KSFM-EFM medium with 5% serum and images recorded with passage. Images shown at x100

In the laboratory, Zhang and his team differentiated the cells into derivatives of all three embryological layers (endoderm – skin and nervous tissue; mesoderm – bone, muscle, glands, and blood vessels; and endoderm – digestive system).

Differentiation of one USC clone into UCs and SMCs. (a) USCs (p3) t were used to differentiate into two distinct lineages. Culture in SMCs-lineage differentiation (2.5 ng/ml TGF-􀈕1 and 5 ng/ml PDGF-BB) and UCs-lineage differentiation (30 ng/ml EGF) medium was used for 14 days.
Differentiation of one USC clone into UCs and SMCs. (a) USCs (p3) t were used to
differentiate into two distinct lineages. Culture in SMCs-lineage differentiation (2.5 ng/ml TGF-􀈕1 and
5 ng/ml PDGF-BB) and UCs-lineage differentiation (30 ng/ml EGF) medium was used for 14 days.

After showing the multipotent nature of urinary tract stem cells in the laboratory, Zhang and others took smooth muscle cells and urothelial cells made from urinary tract stem cells and transplanted them into mice with tissue scaffolds that had been made from decellularized pig intestine. The scaffolds only had extracellular molecules and not cells. After one month, the implanted cells had formed multi-layered, tissue-like structures.

USCs were infected with BMP9 or control GFP and were injected subcutaneously into nude mice. i) Bony masses were only observed in mice implanted with BMP-transduced USCs at week 4. ii) The harvested bony masses were subjected to microCT imaging revealing the isosurface (left) and density heat maps (right).
USCs were infected with BMP9 or control GFP and were
injected subcutaneously into nude mice. i) Bony masses were only observed in mice implanted with
BMP-transduced USCs at week 4. ii) The harvested bony masses were subjected to microCT imaging
revealing the isosurface (left) and density heat maps (right).

Urinary tract stem cells or as Zhang calls them, urine-derived stem cells or USCs, have many cell surface characteristics of mesenchymal stem cells from bone marrow, but they are also like pericytes, which are cells on the outside of small blood vessels. Zhang and others suspect that USCs come from the upper urinary tract, including the kidney. Patients who have had kidney transplants from male donors have USCs with a Y chromosome in them, which suggests that the kidney is a source or one of the sources of these cells.

Determination of USC source. Several clones of USCs (p3) were cultured and analyzed for expression of kidney-lineage marker. (a) FISH (left) and amelogenin gene PCR analysis (right) analysis of USCs isolated from urine obtained from a male donor-to-female recipient kidney transplant for presence of Y-chromosome (L: DNA ladder, M: male control, F: female control, A4: USC from male donor-to-female recipient urine sample, N: negative control).
Determination of USC source. Several clones of USCs (p3) were cultured and analyzed for
expression of kidney-lineage marker. (a) FISH (left) and amelogenin gene PCR analysis (right)
analysis of USCs isolated from urine obtained from a male donor-to-female recipient kidney transplant
for presence of Y-chromosome (L: DNA ladder, M: male control, F: female control, A4: USC from
male donor-to-female recipient urine sample, N: negative control).

Even more work needs to be done before we can truly become over-the-moon excited about these cells as a source of material for regenerative medicine, Zhang’s work is certainly an encouraging start.

See Shantaram Bharadwaj, et al., Multi-Potential Differentiation of Human Urine-Derived Stem Cells: Potential for Therapeutic Applications in Urology. Stem Cells 2013 DOI: 10.1002/stem.1424.

Stem Cell Gene Therapy For An Inherited Neurological Disease


Scientists at the University of Manchester in the United Kingdom have used stem cell gene therapy to treat a fatal genetic brain disease in mice. Sanfilippo is a fatal, inherited condition that causes progressive dementia in children. This particular treatment strategy could also be used to treat other types of neurological, inherited diseases. The Manchester team hopes to bring this strategy to a clinical trial within two years.

Sanfilippo afects one in 89,000 children in the United Kingdom and is an untreatable “mucopolysaccharide disease ” or MPS disease. MPS diseases involve an abnormal storage of mucopolysaccharides. This abnormal storage results from the absence of a specific enzyme. Without the enzyme, the breakdown process of mucopolysaccharides is incomplete. Partially broken down mucopolysaccharides accumulate in the body’s cells causing progressive damage. The storage process can affect appearance, development, and the function of various organs of the body. Each MPS disease is caused by the deficiency of a specific enzyme.

Patients with Sanfilippo are unable to degrade heparan sulfate. There are four different types of Sanfilippo, which is also called MPS type III. MPS type IIIA results from a deficiency in the enzyme N-sulfoglucosamine sulfohydrolase, MPS type IIIB lacks N-Acetylglucosaminidase, MPS type IIIC has an absence in Acetyl-CoA:alpha-glucosaminide-acetyltransferase, and MPS type IIID lacks N-acetylglucosamine 6-sulphatase. In all four forms of MPS III, excessive heparan sulphate storage occurs in the brain, leading to its progressive deterioration; the amount of heparan sulphate storage in other tissues influences the extent of physical symptoms. Children eventually lose the ability to walk and swallow.

Brian Bigger from the University of Manchester’s Institute of Human Development led this research into therapies for MPS type IIIA. According to Bigger, bone marrow transplants have been used to treat similar diseases (e.g., Hurler syndrome). In this case, gene therapy was used to introduce the missing enzyme into the transplanted cells. Unfortunately, this did not work terribly well because the white blood cells from the bone marrow did not make enough of the enzyme to treat the disease.

A fraction of the white blood cells made bone marrow are called monocytes, and some of the monocytes traffic to the brain to become microglia. Since microglia are made by hematopoietic stem cells (HSCs) in the bone marrow, genetic engineering of cultured HSCs should increase expression of the missing enzyme in microglia. In previous experiments, HSCs were engineered with viruses to express the missing enzyme, but this expression was poor in microglia.

To fix this problem, Bigger and his team increased enzyme expression in the engineered HSCs in bone marrow. They used a gene control region from the “pyruvate kinase” gene, which is a very highly expressed gene. This increased expression of the missing enzyme to five times the normal levels and to 11% of normal levels in the microglia cells in the brain. The enzyme

This type of treatment corrects the inflammation in the brain and completely corrects the hyperactivity behavior in mice with Sanfilippo. Bigger adds, “We now hope to work to a clinical trial in Manchester in 2015.”

Bigger and his colleagues are manufacturing a viral vector to deliver genetic material into cells for use in humans and they hope to use this in a clinical trial with patients at Central Manchester University Hospital NHS Foundation Trust by 2015.

This stem cell gene therapy approach was recently shown by Italian scientists to improve conditions in patients with a similar disease that affects the brain called metachromatic leukodystrophy. Bigger refined the vector used bythe Italian group.

According to Bigger, this approach might have the potential to treat several neurological genetic diseases.

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.

Neural Stem Cell Proliferation Increased By Herbal Extract


When it comes to herbal medicine, count me a skeptic. Some people swear by many herbs, but when these same herbs are objectively tested under controlled conditions, they fail spectacularly or they only show modest effects.

For example, a lady in my church is absolutely certain that Echinacea will cure your cold. However, a paper by Barrett in Phytomedicine, 2003 Jan;10(1):66-86 reviews several Echinacea trials and concludes that: “Although suggestive of modest benefit, these trials are limited both in size and in methodological quality. Hence, while there is a great deal of moderately good-quality scientific data regarding E. purpurea, effectiveness in treating illness or in enhancing human health has not yet been proven beyond a reasonable doubt.” Also the prestigious Cochrane database has examined many human trials that tested Echinacea and concluded that “Echinacea preparations tested in clinical trials differ greatly. There is some evidence that preparations based on the aerial parts of Echinacea purpurea might be effective for the early treatment of colds in adults but results are not fully consistent. Beneficial effects of other Echinacea preparations, and for preventative purposes might exist but have not been shown in independently replicated, rigorous randomized trials.” For this study, see Linde K, Barrett B, Wölkart K, Bauer R, Melchart D. Echinacea for preventing and treating the common cold. Cochrane Database Syst Rev. 2006 Jan 25;(1):CD000530,

When it comes to Ginkgo biloba extracts, the use of Ginkgo for age-related dementia has a veritable history, but the Cochrane reviews concluded: “The evidence that Ginkgo biloba has predictable and clinically significant benefit for people with dementia or cognitive impairment is inconsistent and unreliable.” See Birks J, Grimley Evans J. Ginkgo biloba for cognitive impairment and dementia. Cochrane Database Syst Rev. 2009 Jan 21;(1):CD003120. doi: 10.1002/14651858.CD003120.pub3.

Therefore, it is with some skepticism that I relate the following report to you.

Neural stem cells in the subventricular zone of the hippocampal dentate gyrus on adult mammals are responsible for learning and memory. These cells stop dividing during severe depression and dementia and expand during learning.

Hippocamus anatomy

The natural growth of these stem cells is insufficient to replenish cells after a severe stroke or in the event of serious brain disease. Therefore finding a way to stimulate these is important from a clinical standpoint.

Professor Yuliang Wang from Weifang Medical University has used an extract of Ginkgo biloba called EGb761 to treat rats with dementia. In their hands, this materials seems to safely treat memory loss and cognitive impairment (see Zhang Z, Peng D, Zhu H, Wang X. Experimental evidence of Ginkgo biloba extract EGB as a neuroprotective agent in ischemia stroke rats. Brain Res Bull. 2012 Feb 10;87(2-3):193-8).

Wang and his co-workers took this work one step further and examined the effects of EGb761 on the proliferation of neural stem cells in the subventricular zone and dentate gyrus of rats with vascular dementia.

According to Wang and others, the extract promoted and prolonged the proliferation of neural stem cells in the subventricular zone and dentate gyrus of rats with vascular dementia. The cells continued to proliferate for four months and improved learning and memory in rats with vascular dementia.

If you do not believe it, see Wang JW and others, Neural Regeneration Research 2013; 8 (18): 1655-1662.

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.

Gum-Based Stem Cells For Regenerative Medicine


The gums are also known as the gingivae, and this soft tissue serves as a biological barrier that covers the oral cavity of the maxillae and mandible (upper and lower jawbones). The gingivae also harbor a stem cell population known as gingival mesenchymal stem cells or GMSCs.

“Oh that’s a big surprise,” you say, “another mesenchymal stem cell population found in the body.” Well this one is a big deal because of its tissue of origin. Most MSCs are formed during embryonic development from cells that originate from the mesoderm, the embryonic tissue that lies between the skin of the embryo and the gut. Mesoderm forms the muscles, bones, connective tissue, adrenal glands, circulatory system, kidneys, gonads, and some other vitally important tissues.

Mesoderm

However, in the head, a large number of tissues are formed from “neural crest cells.” Neural crest cells hail from the top of the neural tube, which is the beginnings of the spinal cord. The dorsal-most portion of the neural tube contains a population of cells that move out of the neural tube and colonize the embryo to form a whole host of tissues. These include: Neurons, including sensory ganglia, sympathetic and parasympathetic ganglia, and plexuses, Neuroglial cells, Schwann cells, Adrenal medulla, Calcitonin-secreting cells, Carotid body type I cells, Epidermal pigment cells, Facial cartilage and bone Facial and anterior ventral skull cartilage and bones, Corneal endothelium and stroma, Tooth papillae, Dermis, smooth muscle, and adipose tissue of skin of head and neck, Connective tissue of salivary, lachrymal, thymus, thyroid, and pituitary glands, Connective tissue and smooth muscle in arteries of aortic arch origin. Wow, that’s a lot of stuff. I think you can see that these neural crest cells are important players during embryonic development.

Neural_Crest

Songtao Shi, from the Ostrow School of Dentistry, University of Southern California and his co-workers demonstrated that approximately 90% of GMSCs are derived from cranial neural crest cells and 10% are derived from mesoderm. This is important because neural crest-based stem cells seem to have greater plasticity.

Shi and his team compared mesodermally derived MSCs with GMSCs and the neural crest derived MSCs have a greater ability to differentiate into neural cells and cartilage-making cells.

In a mouse model of colitis in which mice are fed dextran sulfate sodium, which induces colitis in the mice, the neural crest derived MSCs did a better job of relieving the inflammation associated with colitis than their mesodermally derived counterparts.

Shi admits that further research on these stem cells must be done in order to better understand them and their functional roles. Shi is especially interested in the functional interaction between the neural crest derived MSCs in the gum and the mesodermally derived MSCs. Also, their potential for suppressing inflammation in particular diseases of the immune system and wound healing needs to be examined in some detail.

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.”

New Liver Drug Gets Fast Tracked by the FDA


Liver scarring (fibrosis) and cirrhosis (deposition and build up of fatty deposits in the liver) are life-threatening events. We normally associate cirrhosis in our thinking with chronic alcoholism, but there are many other conditions that can cause liver fibrosis and cirrhosis. For example, chronic systemic lupus erythematosis, which is normally just known s lupus, can wreak havoc upon the patient’s liver. Likewise Crohn’s disease, chronic hepatitis infections, or even certain genetic can cause liver disease. Once a patient’s liver scars over to a certain point. The last stop for them is either a liver transplant, or Hospice.

Until now? A biotechnology company called Galectin Therapeutics has announced that the U.S. Food and Drug Administration (FDA) has granted their new drug, GR-MD-O2 (galactoarabino-rhamnogalacturonate – say that five times fast) so-called “Fast Track designation” as an experimental treatment for non-alcoholic steatohepatitis (NASH) with hepatic fibrosis, which is also commonly known as fatty liver disease with advanced fibrosis.

GR-MD-02 is an experimental name for a complex carbohydrate drug that targets galectin-3. Galectin-3 is a cell surface protein found on liver cells and it plays a critical protein in the pathogenesis of fatty liver disease and fibrosis. Galectin proteins are central players in those diseases that involve scaring of organs such as cancer, and inflammatory and fibrotic disorders. The drug binds to galectin proteins and disrupts their function. Preclinical data have shown that GR-MD-02 can reverse fibrosis and cirrhosis in kidney, lung, and liver.

Galectin-3
Galectin-3

What is “fast track” designation? Here how this works: Fast Track is a process designed by the FDA to speed up the development and review of drugs to treat serious conditions and fill an unmet medical need. The goal is to get important new drugs to the patient earlier. Determining whether a condition is serious is a matter of judgment, but generally is based on whether the drug will have an impact on such factors as survival, day-to-day functioning, or the likelihood that the condition, if left untreated, will progress from a less severe condition to a more serious one. The kinds of conditions that have qualified for fast tracking in the past include AIDS, Alzheimer’s, heart failure and cancer. .

To qualify for fast tracking, the drug must either treat or prevent a condition with no currently available, or if there are available therapies, a fast track drug must show some advantage over available therapy. These advantages would come in the following forms:

1. Show superior effectiveness (outcomes or improved effect on serious outcomes);
2. Avoid serious side effects of an available therapy;
3. Improve the diagnosis of a serious condition (in those cases where early diagnosis results in an improved outcome);
4. Decreases clinically significant toxicity of an available therapy
5. Ability to address emerging or anticipated public health need

If a drug is fast tracked, then is will receive more frequent meetings with FDA, more frequent written correspondence from FDA, or eligibility for Accelerated Approval and Priority Review, or some combination of these.

Galectin Therapeutics is currently conducting a phase 1 clinical trial to evaluate the safety, tolerability and efficacy for single and multiple doses of GR-MD-02 over four weekly doses of GR-MD-02 treatment in patients with fatty liver disease with advanced fibrosis. In this study, Galectin will enroll eight patients in each dose escalation cohort and there will be at least three cohorts and potentially up to five cohorts, with a maximum of 40 patients at six clinical sites in the US, which each have extensive experience in clinical trials in liver disease.

“Our preclinical data has shown that GR-MD-02 has robust treatment effects in reversing fibrosis and cirrhosis. Fast Track designation enables us to expedite the compound’s development and review process, with the ultimate goal of bringing a first-in-class treatment to the millions of Americans suffering from fatty liver disease with advanced fibrosis,” said Dr. Peter Traber, president, chief executive officer and chief medical officer of Galectin Therapeutics Inc. “We are very pleased that the FDA sees the clinical value of GR-MD-02 and seriousness of fatty liver disease, and we look forward to working closely with the FDA throughout this process.”

Bmi1 Controls Adult Stem Cell “Stemness”


Stem cell scientists from the laboratory of Ophir Klein at UC San Francisco have discovered a new role for a protein called Bmi1 that might give clues as to how to get adult stem cells to regenerate organs.

Ophir Klein, the director of the Craniofacial and Mesenchymal Biology Program and chairman of the Division of Craniofacial Anomalies at UC San Francisco, said “Scientists have known that Bmi1 is a central control switch within the adult stem cells of many tissues, including the brain, blood, lung and mammary gland. Bmi1 also is a cancer-causing gene that becomes reactivated in cancer cells.”

Crystal structure of the BMI1 protein
Crystal structure of the BMI1 protein

All stem cells are somewhat immature in comparison to their adult counterparts. Stem cells also have the capacity to divide almost indefinitely and generate specialized cells. Bmi1 acts as a molecular switch that, if pushed in one direction, drives stem cells to proliferate and grow, but if pushed in the opposite direction, keeps cell proliferation in check. Research from Klein’s lab now suggests that Bmi1 might prevent the progeny of stem cells from differentiating into the wrong cell types in the wrong location.

Downstream targets of Bmi1
Downstream targets of Bmi1

This new discovery suggests that manipulation of Bmi1 and other regulatory molecules might be some of the steps included in laboratory recipes to turn specialized cell development on and off to create new cell-based treatments for tissue lost to injury, disease, or aging.

Also, the dual role of Bmi1 in pathological settings might be intriguing. Cancers are, in many cases, driven by adult stem cells that behave abnormally. If these stem cells could be differentiated, then their growth would slow. Possibly, inactivating Bmi1 in tumor stem cells might be one strategy.

In these experiments, Klein and his colleagues examined those adult stem cells found in the large incisors of mice. Unlike humans, these teeth grow continuously and are, therefore, an attractive model for stem cell research. Klein explained, “There is a large population of stem cells, and the way the daughter cells of the stem cells are produced is easy to track – it’s if they are on a conveyor belt.” Early in life, human beings possess a stem cell population that similarly drive tooth development, but they become inactive after the adult teeth are fully formed during early childhood.

Mouse mandible showing  the large, paired incisors
Mouse mandible showing the large, paired incisors

In the current study, postdoctoral research fellows Brian Biehs and Jimmy Hu showed that at the base of the growing mouse incisor there is a stem cell population that actively expresses Bmi1. In these cells, Bmi1 suppressed a set of genes called Hox genes. When activated, the Hox genes trigger the development of specific cell types and body structures.

In the mouse incisor, Bmi1 keeps these stem cells in their stem cell state and prevent them from differentiating prematurely or inappropriately. “This new knowledge is useful in a fundamental way for understanding how cell differentiating is controlled and may help us manipulate stem cells to get them to do what we want to do,” said Klein.

As they state in the abstract of their paper: “As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.”

Thus this finding from the Klein lab may provide a vital clue for the manipulation of adult stem cells and, perhaps, cancer cells.