Adjustable Gels Used to Determine Those Molecules That Drive Stem Cell Differentiation


Scientists have been very interested in the details of stem cell differentiation. To that end, several laboratories have designed hydrogels that mimic the stiffness of biological tissue in order to grow stem cells and study their differentiation.

In one enterprising laboratory, led by Rein Ulijn of the City University of New York and the University of Strathclyde, scientists have used a novel culture-based gel system to study mesenchymal stem cell differentiation and identify those metabolites used by stem cells when they select bone and cartilage cell fates. When these molecules are provided to standard stem cell cultures, these molecules can guide stem cells to generate desired cell types. This new study illustrates how new biomaterials can provide an exacting model system that can help scientists precisely determine those identifying factors that drive stem cell differentiation.

Stem-cell scientists have known that the rigidity of a hydrogel surface can instruct stem cells to differentiate. A rigid surface, as it turns out, can result in bone cell formation, whereas soft surfaces induce the differentiation of cells into neuron-like cells. With this information, Ulijn and others developed a protocol that generates gels by combining small building-block molecules that spontaneously form a network of nanosized fibers. Furthermore, by varying the concentration of these building blocks, the stiffness of these gels can be adjusted. By mimicking the stiffness of bone (40 kilopascal) or cartilage (15 kilopascal), the gel stimulates stem cells applied to its surface to differentiate accordingly.

“This paper is a great example of how chemistry can help make step changes in biology,” said Matthew Dalby of the University of Glasgow and co-senior author. “As a biologist, I needed simple yet tunable cell-culture gels that would give me a defined system to study metabolites in the laboratory. Rein had developed the chemistry to allow this to happen.”

The available gels for growing stem cells are typically derived from animal products. Unfortunately, this can affect the reproducibility of results, since different preparations of particular animal products can have rather different properties. Synthetic components usually require coatings or coupling of cell-adhesive ligands. However, the gel developed by Ulijn’s group is composed of two simple synthetic peptide derivatives. One component binds to copies of itself with high directional preference, which results in the spontaneous formation of nanoscale fibers when the molecules are dissolved in water. The second components consists of a surfactant-like molecule that binds to the fiber surface and presents simple, cell-compatible chemical groups to any cells.

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The components are held together by relatively weak and reversible interactions, e.g., hydrogen bonding and aromatic stacking. Interestingly, variants of these gels are commercially available through a spinoff company called Biogelx, Ltd., where Ulijn serves as chief scientific officer.

“We wanted a platform that provides nanofiber morphology and as-simple-as-possible chemistry and tunable stiffness to serve as a blank-slate background so that we could focus on changes in stem cell metabolism,” said Ulijn. “Matt and his team performed metabolomics analysis to find out how the key metabolites within a stem cell are used up during the differentiation process.”

Particular transcription factors are often the ingredients scientists use to induce stem cell fate in the case of induced pluripotent stem cells. However, Dalby and Ulijn think that certain metabolites might drive those pathways that cause the different intracellular concentrations of transcription factors that drive the various differentiation pathways.

One metabolite featured in the study is cholesterol sulfate. Cholesterol sulfate is used up during osteogenesis on a rigid matrix and can also be used to convert stem cells into bone-like cells in cell culture.

In their paper, Ulijn and his coworkers showed how small molecules, like cholesterol sulfate, can put into motion those cell-signaling pathways that culminate in the activation of the transcription factors that drive the transcription of major bone-related genes. The expression of these bone-specific genes drives bone formation, and this demonstrates a connection between the metabolites and the activation of transcription factors.

It must be noted that this gel does not precisely recapitulate the microenvironment inside the body. Therefore, it is unclear if the stem cells grown on it behave differently on the designed gel surfaces than they would in the body.

Although the full list of metabolites derived from the analysis is preliminary, “it could certainly point researchers in the right direction,” Ulijn said. “Our ambition is to simplify drug discovery by using the cell’s own metabolites as drug candidates,” Dalby said.

This paper was published here: Alakpa et al., “Tunable Supramolecular Hydrogels for Selection of Lineage Guiding Metabolites in Stem Cell Cultures,” Chem, 2016 DOI:10.1016/j.chempr.2016.07.001.

Hematopoietic Stem Cells Use a Simple Heirarchy


New papers in Science magazine and the journal Cell have addressed a long-standing question of how the descendants of hematopoietic stem cells in bone marrow make the various types of blood cells that course through our blood vessels and occupy our lymph nodes and lymphatic vessels.

Hematopoietic stem cells (HSCs) are partly dormant cells that self-renew and produce so-called “multipotent progenitors” or MPPs that have reduced ability to self-renew, but can differentiate into different blood cell lineages.

The classical model of how they do this goes like this: the MPPs lose their multipotency in a step-wise fashion, producing first, common myeloid progenitors (CMPs) that can form all the red and white blood cells except lymphocytes, or common lymphoid progenitors (CLPs) that can form lymphocytes (see the figure below as a reference). Once these MPPs form CMPs, for example, the CMP then forms either an MEP that can form either platelets or red blood cells, or a GMP. which can form either granulocytes or macrophages. The possibilities of the types of cells the CMP can form in whittled down in a step-by-step manner, until there is only one choice left. With each differentiation step, the cell loses its capacity to divide, until it becomes terminally differentiated and becomes platelet-forming megakarocyte, red blood cell, neutrophil, macrophage, dendritic cells, and so on.

hematopoiesis-from-multipotent-stem-cell

These papers challenge this model by arguing that the CMP does not exist. Let me say that again – the CMP, a cell that has been identified several times in mouse and human bone marrow isolates, does not exist. When CMPs were identified from mouse and human none marrow extracts, they were isolated by means of flow cytometry, which is a very powerful technique, but relies on the assumption that the cell type you want to isolate is represented by the cell surface protein you have chosen to use for its isolation. Once the presumptive CMP was isolated, it could recapitulate the myeloid lineage when implanted into the bone marrow of laboratory animals and it could also produce all the myeloid cells in cell culture. Sounds convincing doesn’t it?

In a paper in Science magazine, Faiyaz Notta and colleagues from the University of Toronto beg to differ. By using a battery of antibodies to particular cell surface molecules, Notta and others identified 11 different cell types from umbilical cord blood, bone marrow, and human fetal liver that isolates that would have traditionally been called the CMP. It turns out that the original CMP isolate was a highly heterogeneous mixture of different cell types that were all descended from the HSC, but had different developmental potencies.

Notta and others used single-cell culture assays to determine what kinds of cells these different cell types would make. Almost 3000 single-cell cultures later, it was clear that the majority of the cultured cells were unipotent (could differentiate into only one cell type) rather than multipotent. In fact, the cell that makes platelets, the megakarocyte, seems to derive directly from the MPP, which jives with the identification of megakarocyte progenitors within the HSC compartment of bone marrow that make platelets “speedy quick” in response to stress (see R. Yamamoto et al., Cell 154, 1112 (2013); S. Haas, Cell Stem Cell 17, 422 (2015)).

Another paper in the journal Cell by Paul and others from the Weizmann Institute of Science, Rehovot, Israel examined over 2700 mouse CMPs and subjected these cells to gene expression analyses (so-called single-cell transriptome analysis). If the CMP is truly multipotent, then you would expect it to express genes associated with lots of different lineages, but that is not what Paul and others found. Instead, their examination of 3461 genes revealed 19 different progenitor subpopulations, and each of these was primed toward one of the seven myeloid cell fates. Once again, the presumptive CMPs looked very unipotent at the level of gene expression.

One particular subpopulation of cells had all the trappings of becoming a red blood cell and there was no indication that these cells expressed any of the megakarocyte-specific genes you would expect to find if MEPS truly existed. Once again, it looks as though unipotency is the main rule once the MPP commits to a particular cell lineage.

Thus, it looks as though either the CMP is a very short-lived state or that it does not exist in mouse and human bone marrow. Paul and others did show that cells that could differentiate into more than one cell type can appear when regulation is perturbed, which suggests that under pathological conditions, this system has a degree of plasticity that allows the body to compensate for losses of particular cell lineages.

A model of the changes in human My-Er-Mk differentiation that occur across developmental time points. Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.
A model of the changes in human My-Er-Mk differentiation that occur across developmental time points.
Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

Fetal HSCs, however, are a bird of a different feather, since they divide quickly and reside in fetal liver.  Also, these HSCs seem to produce CMPs, which is more in line with the classical model.  Does the environmental difference or fetal liver and bone marrow make the difference?  In adult bone marrow, some HSCs nestle next to blood vessels where they encounter cells that hang around blood vessels known as “pericytes.”  These pericytes sport a host of cell surface molecules that affect the proliferative status of HSCs (e.g., nestin, NG2).  What about fetal liver?  That’s not so clear – until now.

In the same issue of Science magazine, Khan and others from the Albert Einstein College of Medicine in the Bronx, New York, report that fetal liver also has pericytes that express the same cell surface molecules as the ones in bone marrow, and the removal of these cells reduces the numbers of and proliferative status of fetal liver HSCs.

Now we have a conundrum, because the same cells in bone marrow do not drive HSC proliferation, but instead drive HSC quiescence.  What gives? Khan and others showed that the fetal liver pericytes are part of an expanding and constantly remodeling blood system in the liver and this growing, dynamic environment fosters a proliferative behavior in the fetal HSCs.

When umbilical inlet is closed at birth, the liver pericytes stop expressing Nestin and NG2, which drives the HSCs from the fetal liver to the other place were such molecules are found in abundance – the bone marrow.

These models give us a better view of the inner workings of HSC differentiation.  Since HSC transplantation is one of the mainstays of leukemia and lymphoma treatment, understanding HSC biology more perfectly will certainly yield clinical pay dirt in the future.

 

Fat-Derived Stem Cells Form Muscle in Muscular Dystrophy Mice


Stem cell therapy for Duchenne muscular dystrophy (DMD) has been plagued by poor cell engraftment into diseased muscles. Additionally, there are no reports to date describing the efficient generation of muscle progenitors from fat-derived stem cells (ADSCs) that can contribute to muscle regeneration.

A study by Cheng Zhang and others from Sun Yat-sen University in Guangzhou, China, Guangdong Province has examined the ability of progenitor cells differentiated from ADSCs using forskolin, basic fibroblast growth factor, the glycogen synthase kinase 3β inhibitor 6-bromoindirubin-3′-oxime as well as the supernatant of ADSC cultures to form workable muscle cells.

When these fat-derived stem cells were treated as described above, they formed a proliferative population of muscle progenitors from ADSCs that had characteristics similar to muscle satellite cells. Furthermore, in culture, these cells were capable of terminal differentiation into multinucleated myotubes.

When these fat-derived stem cells were transplanted into mice that had an inherited type of DMD, the progenitor cells successfully engrafted in skeletal muscle for up to 12 weeks, and generated new muscle fibers, restored dystrophin expression, and contributed to the satellite cell compartment.

These findings highlight the potential application of ADSCs for the treatment of muscular dystrophy. They also illustrate the ability of ADSCs to differentiate into functional skeletal muscle cells when treated properly in culture. These same cells might serve as a treatment for DMD patients.

This article was published in Hum. Mol. Genet. (2015) doi: 10.1093/hmg/ddv316.

Cartilage-Making Stem Cells from Joints


Chiharo Akazawa from the Tokyo Medical and Dental University and his colleagues have tested two types of mesenchymal stem cells from human patients for their ability to make bone, cartilage, or fat. Their tests illustrated what has been shown several time before; mesenchymal stem cells tend to differentiate into the tissues that most closely resemble their tissue of origin.

Akazawa and his colleagues previously discovered a way to effectively isolated mesenchymal stem cells from bone marrow, which is no small feat because mesenchymal stem cells (MSCs) are a minority of the cells in bone marrow (Mabuchi and others (2013), Stem Cell Reports 1: 152-165). In a recent paper in the journal PLoS ONE, Akazawa and others used this technique to isolate MSCs from bone marrow and from synovial membrane – the fluid-filled sac that encases joints. In large joints, this synovium is large and called a “bursa.”.

In culture, the bone marrow-derived MSCs from several different human donors showed a marked tendency to form bone, but they did not make good cartilage or fat. The synovial MSCs, on the other hand, did not do so well at making bone, but made very good fat and cartilage. These differentiation trends were observed in MSCs culture for several different human donors. All cells were collected during arthroscopic surgery.

Since the synovial membrane of patients suffering from osteoarthritis undergoes, increased cell division, it is possible that the number of stem cells also increases. Alternatively, using MSCs from healthy donors who do not have arthritis may be even more preferable. Nevertheless, MSCs from synovial membrane show excellent cartilage-making potential and they may be a suitable source of cell for cartilage regeneration.

Toxic Gas Prompts Mesenchymal Stem Cells to Become Bone Cells


Hydrogen sulfide smells like rotten eggs and is toxic to human life at moderate concentrations. Therefore, imagine the surprise of researchers when they discovered that low concentrations of this poisonous gas actually stimulate mesenchymal stem cells from bone marrow to differentiate into bone-making cells.

In a paper published in the journal Cell Stem Cell, Yi Liu from the Ostrow School of Dentistry at the University of Southern California and colleagues have discovered that hydrogen sulfide (H2S), acts as a “gaseous signaling molecule” that mesenchymal stem cells actually produce at sub-lethal concentrations.

H2S acts as a “gasotransmitter” that regulates multiple signaling pathways. To determine the extent of these pathways, Liu and his colleagues made mice that were unable to synthesize any H2S. The H2S-deficient mice showed distinct abnormalities in bone marrow mesenchymal stem cells. Namely, mesenchymal stem cells (MSCs) from H2S-deficient mice were unable to properly self=-renew or differentiate into bone-making cells (osteoblasts).

When Liu and others dug a little deeper, they found that H2S deficiency results in aberrant influx of intracellular Ca2+. Problems with calcium handling arose because calcium channels have amino acids that actually react with the H2S. This reaction between the calcium channels and H2S opens the channels and allows entry of calcium into the cell. Now cells contain a host of enzymes that need calcium to operate properly.  Without the reaction of the calcium channels with H2S, calcium does not influx into the cell and the differentiation of mesenchymal stem cells into bone-making cells stops.

schematic diagram R1.ppt

Why is this important? Consider some of the diseases of bone, such as osteoporosis, in which the bones thin and become fragile. Restoring mesenchymal stem cell function in osteoporotic patients with treatments of H2S levels at nontoxic levels may provide treatments for diseases such as osteoporosis that might arise from H2S deficiencies.

Thus by understanding stem cell biology better, we can potentially treat a disease like osteoporosis with small amounts of a stinky gas. Incredible, isn’t it?

Skin Tissue Grown From Human Stem Cells


A research team from King’s College, London, in collaboration with the San Francisco Veteran Affairs Medical Center has succeeded in growing the epidermal layer of skin in culture, this cultured skin has many of the mechanical and biological properties of actual human skin.

The outermost layer of the skin, known as the epidermis forms a protective barrier between the external environment and the body. It protects against water loss and prevents the entry of microorganisms.

Tissue engineers have been able to grow skin cells (keratinocytes) in culture, but getting them to organize into an organ that resembles biological skin has proven rather difficult. However, the ability to test drugs on cultured skin that greatly mimics human skin has been the goal of such research for several years.

For this present project, keratinocytes were made from induced pluripotent stem cells that were derived from skin cells obtained from biopsies. These keratinocytes made from induced pluripotent stem cells (iPSCs) were very similar to keratinocytes made from embryonic stem cells and primary keratinocytes isolated from skin biopsies.

To form a three-dimensional structure like skin, the keratinocytes were cultured in a high-to-low humidity environment and they assembled into a layer structure that looked like human skin. When this cultured skin was compared with skin made from embryonic stem cell-derived keratinocytes or from keratinocytes isolated from skin biopsies, there were no significant structural differences.

Scientists hope to use this cultured skin to study congenital skin diseases like ichthyosis (characterized by dry, flaky skin) or atopic dermatitis. Growing large quantities of skin in culture will also allow drugs and cosmetics to be effectively tested for safety without the use of expensive and sometimes highly variable animal models.

This technology would also allow different laboratories to grow skin from different ethnic groups that have distinct types of skin with variable biological properties.

Long-Term Survival of Transplanted Human Neural Stem Cells in Primate Brains


A Korean research consortium has transplanted human neural stem cells (hNSCs) into the brains of nonhuman primates and ascertained the fate of these cells after being inside the brains of these animals for 22 and 24 months. They discovered that the implanted hNSCs had not only survived, but differentiated into neurons and never caused any tumors.

This important study is slated to be published in the journal Cell Transplantation.

To properly label the hNSCs so that they were detectable inside the brains of the animals, Lee and others loaded them with magnetic nanoparticles to enable them to be followed by magnetic resonance imaging (MRI). Also, they did not use immunosuppressants when they transplanted their hNSCs into the animals. This study is the first to examine the long-term survival and differentiation of hNSCs without the need for immunosuppression.

“Stroke is the fourth major cause of death in the US behind heart failure, cancer, and lower respiratory disease,” said study co-author Dr. Seung U. Kim of University of British Columbia Hospital’s department of neurology in Canada. “While tissue plasminogen activator (tPA) treatment within three hours after a stroke has shown good outcomes, stem cell therapy has the potential to address the treatment needs of those stroke patients for whom tPA treatment was unavailable or did not help.”

Based on the ability of hNSCs to differentiate into a variety of types of nerves cells, Lee and his colleagues thought that these cells have remarkable potential to treat damaged brain tissue and replace what was lost after a stroke, head injury or other type of brain trauma. Cell regeneration therapy can potentially treat brain-specific diseases like Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), spinal cord injury and stroke.

Dr. Kim and colleagues in Korea grafted magnetic particle-labeled hNSCs into the brains of laboratory primates and evaluated their performance to assess their survival and differentiation over 24 months. Of particular interest was determining their ability to differentiate into neurons and to determine whether the cells caused tumors.

“We injected hNSCs into the frontal lobe and the putamen of the monkey brain because they are included in the middle cerebral artery (MCA) territory, which is the main target in the development of the ischemic lesion in animal stroke models,” commented Dr. Kim. “Thus, research on survival and differentiation of hNSCs in the MCA territory should provide more meaningful information to cell transplantation in the MCA occlusion stroke model.”

Lee’s team said that they chose NSCs for transplantation because the existence of multipotent NSCs “has been known in developing rodents and in the human brain with the properties of indefinite growth and multipotent potential to differentiate” into the three major CNS cell types – neurons, astrocytes and oligodendrocytes.

“The results of this study serve as a proof-of-principle and provide evidence that hNSCs transplanted into the non-human primate brain in the absence of immunosuppressants can survive and differentiate into neurons,” wrote the researchers. “The study also serves as a preliminary study in our planned preclinical studies of hNSC transplantation in non-human primate stroke models.”

“The absence of tumors and differentiation of the transplanted cells into neurons in the absence of immunosuppression after transplantation into non-human primates provides hope that such a therapy could be applicable for use in humans.” said Dr. Cesar V. Borlongan, Prof. of Neurosurgery and Director of the Center of Excellence for Aging & Brain Repair at the University of South Florida. “This is an encouraging study towards the use of NSCs to treat neurodegenerative disorders”.

An Improved Way to Make Motor Neurons in the Laboratory from Stem Cells


A research team from the University of Illinois at Urbana-Champaign has reported that they can produce human motor neurons from stem cells much more quickly and efficiently than previous methods allowed. This finding was published in the journal Nature Communications and it will almost certainly provide new ways to model human motor neuron development, diseases of the nervous system, and ways to treat spinal cord injuries.

The new protocol described in the Nature Communications paper includes adding critical signaling molecules to precursor cells a few days earlier than specified by previous methods. This innovation increases the proportion of healthy motor neurons derived from stem cells from 30 to 70 percent. It also cuts in half the time required to make motor neurons.

“We would argue that whatever happens in the human body is going to be quite efficient, quite rapid,” said University of Illinois cell and developmental biology professor Fei Wang, who led the study with visiting scholar Qiuhao Qu and materials science and engineering professor Jianjun Cheng. “Previous approaches took 40 to 50 days, and then the efficiency was very low – 20 to 30 percent. So it’s unlikely that those methods recreate human motor neuron development.”

The new method designed by Qu generated a larger population of mature, functional motor neurons in 20 days. According to Wang, this new approach will allow scientists to induce mature human motor neuron development in cell culture, and to identify the factors that drive this process

Because stem cells can differentiate into a wide variety of cell types, they are unique compared to mature, adult cells. Making neurons from either embryonic stem cells or induced pluripotent stem cells requires the addition of signaling molecules to the cells at critical moments in culture.

Previously, Wang and his colleagues discovered a molecule called compound C that converts stem cells into “neural progenitor cells,” or NPCs. NPCs represent an early stage in neuronal development, and further manipulation of NPCs can drive them to become neurons, but differentiating NPCs into motor neurons presents another set of problems.

Other published studies have established that the addition of two important signaling molecules, six days after exposure to compound C, to NPCs in culture can generate motor neurons, but at rather poor efficiencies. In this newly published study, Qu showed that adding the signaling molecules at Day 3 worked better: The NPCs differentiated into motor neurons quickly and efficiently. Thus, Day 3 represents a previously unrecognized NPC cell stage.

This new approach has immediate applications in the laboratory. Amyotrophic lateral sclerosis or ALS is a neurological disease that causes motor neurons to die off. By using Wang and Qu’s cell culture system to make neurons from the skin cells of ALS, and watching them develop into motor neurons, scientists and physicians will divine other new insights into disease processes. Therefore, any method that improves the speed and efficiency of generating the motor neurons will be a boon to neuroscientists. These cells can also be used to screen for drugs to treat motor neuron diseases, and might even be used to therapeutically restore lost function in patients someday.

“To have a rapid, efficient way to generate motor neurons will undoubtedly be crucial to studying – and potentially also treating – spinal cord injuries and diseases like ALS,” Wang said.

UC Davis Stem Cell Scientists Make Bladder Cells from Pluripotent Stem Cells


Patients who suffer from malformation of the spinal cord or have suffered a severe spinal cord injury sometimes have bladder malfunction as well. Replacing a poorly functioning bladder is a goal of regenerative medicine, but it is not an easy goal. The bladder is lined with a special cell population called “urothelium.” Urothelium is found throughout the urinary tract and it is highly elastic. Persuading stem cells to form a proper urothelium has proved difficult.

Urothelium
From http://ocw.tufts.edu/data/4/221158/221174_xlarge.jpg

Now scientists from the University of California, Davis (my alma mater), have succeeded in devising a protocol for differentiating human pluripotent stem cells into urothelium. The laboratory of Eric Kurzock, chief of the division of pediatric urologic surgery at UC Davis Children’s Hospital, published this work in the journal Stem Cells Translational Medicine. This work is quite exciting, since it provides a way to potentially replace bladder tissue for patients whose bladders are too small or do not function properly.

Kurzock explained: “Our goal is to use human stem cells to regenerate tissue in the lab that can be transplanted into patients to augment or replace their malfunctioning bladders,”

In order to make bladder cells in the laboratory, Kurzrock and his coworkers used two different types of human pluripotent stem cells. First, they used two types of induced pluripotent stem cells (iPS cells). The first came from laboratory cultures of human skin cells that were genetically engineered and cultured to form iPS cultures. The second iPS line was derived from umbilical cord blood cells that had been genetically reprogrammed into an embryonic stem cell-like state.

Even though further work is needed to establish that bladder tissues made from such stem cells are safe or effective for human patients, Kurzrock thinks that iPS cell–derived bladder grafts made from a from a patient’s own skin or umbilical cord blood cells represent the ideal tissue source for regenerative bladder treatments. This type of tissue would be optimal, he said, because it lowers the risk of immunological rejection that typifies most transplants.

One of the truly milestone developments in this research is the protocol Kurzrock and his colleagues developed to direct pluripotent stem cells to differentiate into bladder cells. This protocol was efficient and, most importantly, allowed the stem cells to proliferate in culture over a long period of time. This is crucial in order to have enough material for therapeutic purposes.

“What’s exciting about this discovery is that it also opens up an array of opportunities using pluripotent cells,” said Jan Nolta, professor and director of the UC Davis Stem Cell program and a co-author on the new study. “When we can reliably direct and differentiate pluripotent stem cells, we have more options to develop new and effective regenerative medicine therapies. The protocols we used to create bladder tissue also provide insight into other types of tissue regeneration.”

To hone their urothelium-differentiation protocol, Kurzrock and his colleagues used human embryonic stem cells obtained from the National Institutes of Health’s human stem cell repository. These cells were successfully differentiated into bladder cells. Afterwards, the Kurzrock group used the same protocol to coax iPS cells made from skin and umbilical cord blood into urothelium. Not only did these cells look like urothelium, but they also expressed the protein “uroplakin,” which is unique to the bladder and helps make it impermeable to toxins in urine.

In order to bring this protocol to the clinic, the cells must proliferate, differentiate and express bladder-specific proteins without depending on any animal or human products. They must do all these things independent of signals from other human cells, said Kurzrock. Therefore, for future research, Kurzrock and his colleagues plan to modify their laboratory cultures so that they will not require any animal and human products, which will allow use of the cells in patients.

Kurzrock’s primary goal as a physician is with children who suffer from spina bifida and other pediatric congenital disorders. Currently, when he surgically reconstructs a child’s defective bladder, he must use a segment of their own intestine. Because the function of intestine, which absorbs food, is almost the opposite of bladder, bladder reconstruction with intestinal tissue may lead to serious complications, including urinary stone formation, electrolyte abnormalities and cancer. According to Kurzrock, developing a stem cell alternative not only will be less invasive, but should prove to be more effective, too, he said.

Another patient group who might benefit from this research is bladder cancer patients. More than 70,000 Americans each year are diagnosed with bladder cancer, according to the National Cancer Institute. “Our study may provide important data for basic research in determining the deviations from normal biological processes that trigger malignancies in developing bladder cells,” said Nolta. More than 90 percent of patients who need replacement bladder tissue are adults with bladder cancer. Kurzrock said “cells from these patients’ bladders cannot be used to generate tissue grafts because the implanted tissue could carry a high risk of becoming cancerous. On the other hand, using bladder cells derived from patients’ skin may alleviate that risk. Our next experiments will seek to prove that these cells are safer.”

Stem Cells from Muscle Can Repair Nerve Damage After Injury


Researchers from the University of Pittsburgh School of Medicine have discovered that stem cells derived from human muscle tissue can repair nerve damage and restore function in an animal model of sciatic nerve injury. These data have been recently published online in the Journal of Clinical Investigation, but more importantly, this work demonstrates the feasibility of cell therapy for certain nerve diseases, such as multiple sclerosis.

Presently there are few treatments for peripheral nerve damage. Peripheral nerve damage can leave patients with chronic pain, impaired muscle control and decreased sensation.

The senior author of this work, Henry J. Mankin, serves as the Chair in Orthopedic Surgery Research, Pitt School of Medicine, and deputy director for cellular therapy, McGowan Institute for Regenerative Medicine, and said, “This study indicates that placing adult, human muscle-derived stem cells at the site of peripheral nerve injury can help heal the lesion. The stem cells were able to make non-neuronal support cells to promote regeneration of the damaged nerve fiber.”

Muscle-derived stem cells

Workers in Mankin’s laboratory, in collaboration with Dr. Mitra Lavasani, assistant professor of orthopedic surgery, Pitt School of Medicine, grew human muscle-derived stem/progenitor cells in culture by using a culture medium suitable for nerve cells. In culture, Lavasani, Mankin and their colleagues found that when these muscle-derived stem cells were grown in the presence of specific nerve-growth factors, they differentiated into neurons and glial cells. Glial cells act as support cells from neurons. One type of glial cell that these muscle-derived stem cells could differentiate into was Schwann cells, which are the cells that form the myelin sheath around the axons of neurons to accelerate the speed at which nerve impulses are conducted.

Schwann Cell

Mankin and his colleagues then injected these human muscle-derived stem/progenitor cells into mice that had a quarter-inch injury in their right sciatic nerve. The sciatic nerve controls right leg movement. Six weeks later, the nerve had fully regenerated in stem-cell treated mice, but the untreated group showed only limited nerve regrowth and functionality. In other tests, 12 weeks after treatments, the stem cell-treated mice were able to keep their treated and untreated legs balanced at the same level while being held vertically by their tails. When the treated mice ran through a special maze, analyses of their paw prints showed that their gait, which had been abnormal, was now completely normal. Finally, treated and untreated mice experienced loss of muscle mass after nerve damage, but only the stem cell-treated mice regained normal muscle mass by 72 weeks after nerve damage.

sciatic-nerve

“Even 12 weeks after the injury, the regenerated sciatic nerve looked and behaved like a normal nerve,” Dr. Lavasani said. “This approach has great potential for not only acute nerve injury, but also conditions of chronic damage, such as diabetic neuropathy and multiple sclerosis.”

Drs. Huard and Lavasani and the team are now trying to understand how the human muscle-derived stem/progenitor cells triggered injury repair. They are also developing delivery systems, such as gels, that could hold the cells in place at larger injury sites.

The co-authors of this paper included Seth D. Thompson, Jonathan B. Pollett, Arvydas Usas, Aiping Lu, Donna B. Stolz, Katherine A. Clark, Bin Sun, and Bruno Péault, all of whom are from the University of Pittsburgh.

Personalized Stem Cells for Curing Parkinson’s Disease


Stem cell treatments for curing Parkinson’s disease have been one of the dreams of stem cell scientists ever since the first embryonic stem cells were derived from mouse embryos in 1981. Unfortunately, this proved to be one of the harder therapeutic nuts to crack. Several experiments have shown that while feasible, getting the recipe right has required a fair amount of tweaking.

brain-labels

Parkinson’s disease (PD) results from the progressive death of neurons in the midbrain that release a neurotransmitter called dopamine, To review briefly, the brain consists of the forebrain, midbrain and hindbrain. The forebrain consists of the two large cerebral hemispheres that occupy the vast majority of the space within your skull. In addition to the left and right cerebral hemispheres is the diencephalon that consists of the thalamus, subthalamus, hypothalamus, and epithalamus. The thalamus serves as a relay station for a whole variety of nerve fiber tracts, the hypothalamus regulates visceral activities by way of other brain regions and the autonomic nervous system. and the epithalamus connects the limbic system to the rest of the brain. The midbrain, which lies below the diencephalon, is part of the brain stem and dopamine produced in two regions of the midbrain, the substantia nigra and ventral tegmental area play roles in motivation and habituation, and refinement of the control of voluntary movement, The hindbrain consists of the metencephalon and the myelencephalon, both of which contain mutiple fiber tracts and nuclei for vital functions.

Midbrain 2

The death of dopamine-producing neurons in the pars compacta region of the substantia nigra region of the midbrain causes PD. The par compacta sends nerve fibers to the cerebral hemispheres, in particular to cluster of neurons called the basal ganglia. The basal ganglia do not initiate movement, but they refine movement and stabilize the limbs and other body parts while moving. Thus the basal ganglia normally exert a constant inhibitory influence on a wide range of movements. preventing movement at inappropriate times. When someone decides to move, this inhibition is reduced for the required motor system, thereby releasing it for activation. Dopamine releases this inhibition, and therefore high levels of dopamine tend to promote movement and low levels of dopamine demand greater exertion to generate any given movement. Thus the net effect of dopamine depletion is to produce hypokinesia, or less overall movement.

Basal ganglia

Now that we have some knowledge of PD and what causes it, we can examine how to cure it. Since the death of dopamine-secreting neurons causes PD, replacing death or moribund neurons should be possible. Several preclinical studies in laboratory animals and clinical studies with human patients has shown that this is possible.

Rodents can contract a synthetic form of PD if they are treated with a drug called 6-hydroxydopamine. This drug kills off their dopamine-secreting neurons and creates a PD-like disease. Embryonic stem cells can be differentiated in the laboratory into dopamine-secreting neurons, which can then be transplanted into the midbrain. In PD rats, this strategy has reversed the symptoms of PD, but tumor growth has been a nagging problem. The biggest problem is that isolating fully differentiated dopamine-secreting cells has proven difficult because of a lack of good, solid indicators that say to the scientists, “This one is a dopamine-secreting neuron and this one is not.” Thus, isolating nice, clean cultures of only dopamine-secreting cells has been kind of tough to do.

Fortunately, Doi and others in the Takahashi lab at the University of Kyoto showed that prolonged maturation culture system (42 days long) can eliminate most of the tumor-making cells. However, this culture system is laboriously long. Now, Takahashi and Doi and others have struck again in a paper published in Stem Cell Reports in which they used induced pluripotent stem cells to derive dopamine-secreting neurons to treat PD rats.  Because induced pluripotent stem cells are made from a patient’s own adult cells and are converted into embryonic-like stem cells by means of a combination of genetic engineering and cell culture techniques, they are patient-specific and do not require the dismembering of human embryos.

The novelty of this paper is that Doi and others used a protein that acts as an earmark for dopamine-secreting midbrain neurons and this protein is called CORIN. CORIN is a protease, which simply means that it clips other proteins into small pieces. Nevertheless, by using the CORIN protein, Takahashi, Doi and others successfully and efficiently isolated dopamine-secreting midbrain neurons from other cells in their cultures.  Additionally, Doi and the gang were able to differentiate the induced pluripotent stem cells into dopamine-secreting progenitor cells.  This means that the cells were poised to differentiate into dopamine-secreting neurons, but were not quite there yet.  This way, the cells would grow in culture, but upon transplantation, they would differentiate into dopamine-secreting neurons rather than form tumors.  High numbers of cells are required for clinical purposes and this technique allows the for production of large number of cells.

The technique used in this paper also produced the cells under conditions that were safe, scalable and potentially usable for clinical use. These high-quality cells never produced any tumors and produced definitive behavioral improvements in the implanted laboratory animals. The problems that remain are one of scale. The grafts of dopamine-secreting cells that survived in the midbrains of these mice were relatively small (about 1 square millimeter in size or the thickness of a dime).  This is probably due to the fact that the cells differentiate when transplanted rather than growing.  Therefore, this technique will need to be adapted to somehow increase the size of the graphs of dopamine-secreting neurons.  In some PD patients such small graphs will probably work just fine, but in others, probably not.  The other issue is that these implanted cells might be subjected to the same bad intracerebral environment as the original cells and die off quickly, thus abrogating any positive clinical effect they might have.  This is another issue that will need to be examined.

The work goes on, without the need to destroy any embryos.

See Daisuke Doi at al., Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation. Stem Cell Reports 2014, 2: 337-350.

Orthopedic Regeneration With a Combination of Stem Cells, Gene Therapy, and Tissue Engineering


A Duke University research team has combined synthetic scaffolding materials with gene delivery techniques to generate replacement cartilage precisely where it’s needed in the body.

The ingenious strategy utilized by this research project circumvents the need for large quantities of growth factors, which are expensive and difficult to apply after implantation. The Duke team led by Farshid Guilak, director of orthopedic research at Duke University Medical Center, used gene therapy to make stem cells that synthesize their own growth factors.

In brief, Guilak and his collaborators used genetically engineered viruses to transfer genes to stem cells embedded in a synthetic matrix. Upon infection, the stem cells grew and differentiated as needed, but the scaffolding provided the necessary structural cues for the stem cells to move to the proper configuration and form cartilage with the proper shape and biomechanical properties.

Guilak has devoted several years to developing biodegradable synthetic scaffolds that mimic the mechanical properties of cartilage. After testing many different scaffolds, he settled on a 3D woven poly(ε-caprolactone) scaffold, which is completely biodegradable and provides an excellent structural matrix for the synthesis of cartilage.  However, an additional challenge for engineering good cartilage is to coax stem cells embed themselves in the scaffold while differentiating into cartilage-making cells, known as chondrocytes, after the scaffold has been implanted into a living organism.

One widely used strategy is to treat the stem cells with growth factors to induce chrondrocyte formation and cartilage production. Such cartilage can be implanted after it has been grown in the laboratory. However, this approach has some inherent limitations.

Guilak explained that “a major limitation in engineering tissue replacements has been the difficulty in delivering growth factors to the stem cells once they are implanted in the body.” Guilak continued: “There’s a limited amount of growth factor that you can put into the scaffolding, and once it’s released, it’s all gone. We need a method for long-term delivery of growth factors, and that’s where the gene therapy comes in.”

To tackle this perennial problem, Guilak tapped a talented colleague of his, Charles Gersbach, an assistant professor of biomedical engineering, who happens to also be a gene therapy expert.

Gersbach looked at the tissue engineering problem in an entirely new way and suggested that if the mountain will not come to Mohammed (that is to say if the growth factors cannot be given to stem cells after implantation), then Mohammed should grow his own mountain (the stem cells should be genetically engineered to make their own growth factors). Unfortunately, the conventional gene therapy methods are too complex to be commercially feasible. Typically, stem cells are collected, infected with genetically modified viruses that introduces new genes into them, grown to large numbers, and applied to synthetic cartilage scaffolds and implanted into the patient. Sounds like a headache? That’s because it is.

Fortunately, Gersbach had a slick gene therapy trick up his lab coat sleeve: “There are a few challenges with that process, one of them being that there are way too many extra steps,” said Gersbach. “So we turned to a technique I had previously developed that affixes the viruses that deliver the new genes onto a material’s surface.”

A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold.  Photo source:  http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.
A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold. Photo source: http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.

This new study combines Gersbach’s gene therapy technique—dubbed biomaterial-mediated gene delivery—to induce those human mesenchymal stem cells embedded in Guilak’s synthetic cartilage scaffolding to produce growth factor proteins (in particular a molecule called transforming growth factor β3  or TGF-β3). Based on the results of their experiments, the technique works and that the resulting synthetic, composite cartilage-like material is at least as good biochemically and biomechanically as if the growth factors were introduced in the laboratory.

“We want the new cartilage to form in and around the synthetic scaffold at a rate that can match or exceed the scaffold’s degradation,” said Jonathan Brunger, a graduate student who has spent time in both Guilak’s and Gersbach’s laboratories developing and testing the new technique. “So while the stem cells are making new tissue (in the body), the scaffold can withstand the load of the joint. In the ideal case, one would eventually end up with a viable cartilage tissue substitute replacing the synthetic material.”

This particular study examines cartilage regeneration, but Guilak and Gersbach hope that their technique could be applied to the regeneration of many different kinds of tissues, especially orthopaedic tissues such as tendons, ligaments and bones. Also, because the platform comes ready to use with any stem cell, it presents an important step toward commercialization.

“One of the advantages of our method is getting rid of the growth factor delivery, which is expensive and unstable, and replacing it with scaffolding functionalized with the viral gene carrier,” said Gersbach. “The virus-laden scaffolding could be mass-produced and just sitting in a clinic ready to go. We hope this gets us one step closer to a translatable product.”

Citation: “Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.” Brunger, J.M., Huynh, N.P.T., Guenther, C.M., Perez-Pinera, P., Moutos, F.T., Sanchez-Adams, J., Gersbach C.A., and Guilak F. PNAS Plus, 2014. DOI: 10.1073/pnas.1321744111/-/DCSupplemental

Making Heart Muscle from Skeletal Muscle Stem Cells


Several experiments in animals and a few clinical trials in human patients have shown that implanting skeletal muscle cells isolated from muscle biopsies into the heart after a heart attack can help the heart to some degree, but the implanted skeletal muscle cells do not integrate into the existing heart muscle mass and the skeletal muscle cells do not differentiate into heart muscle cells.

Experiments like those mentioned above utilized muscle satellite cells. Muscle satellite cells are a resident stem cell population that respond to muscle damage and divide to form skeletal muscle cells form new muscle. Satellite cells are a perfect example of a unipotent stem cell, which is to say a cell that makes one type of terminally differentiated cell type.

Skeletal muscles, however, have another cell population called muscle-derived stem cells or MDSCs. MDSCs express an entirely different set of cell surface proteins than satellite cells, and have the capacity to differentiate into skeletal muscle, smooth muscle, bone, tendon, nerve, endothelial and hematopoietic cells. MDSCs grow well in culture, tolerate low oxygen conditions quite well, and show excellent regenerative potential.

Other laboratories have managed to culture MDSCs in collagen and produce beating heart muscle cells. Others have observed MDSCs forming a proper myocardium under certain conditions. Several studies have established the ability to MDSCs to treat laboratory animals that have suffered a heart attack. The most recent work from Sekiya and others has established that cell sheets made from MDSCs can reduce dilation of the left ventricle, increased capillary density, and promoted recovery without causing erratic heat beat patterns.

Despite their obvious efficacy. MDSCs remain difficult to isolate in high enough numbers to therapeutic purposes. None of the cell surface molecules sported by MDSCs are unique to those cells. Therefore, getting clean cultures of MDSCs remains a challenge. Still, these cells represent some of the best hopes for regenerative medicine in the heart. These cells do form heart muscle cells and heal ailing hearts. They can be grown in bioreactors to high numbers and can also be combined with engineered materials to shore up a damaged heart and mediate its regeneration. While the use of MDSCs is still in its infancy, the promise certainly is there.

Repopulation of Damaged Livers With Skin-Derived Stem Cells


Patients with severe liver disease must receive a liver transplant. This major procedure requires that the patient survives major surgery and then takes anti-rejection drugs for the rest of their lives. In general, liver transplant patients tend to fair pretty well. The one-year survival rate of liver transplant patients approaches 90% (see O’Mahony and Goss, Texas Heart Institute Journal 2012 39(6): 874-875).

A potentially better way to treat liver failure patients would be to take their own liver cells, convert them into induced pluripotent stem cells (iPSCs), differentiate them into liver cells, and use these liver cells to regenerate the patient’s liver. Such a treatment would contain a patient’s own liver cells and would not require anti-rejection drugs.

Induced pluripotent stem cells or iPSCs are made from genetically-engineered adult cells that have had four specific genes (Oct4, Klf4, Sox2, and c-Myc) introduced into them. As a result of the heightened expression of these genes, some of the adult cells dedifferentiate and are reprogrammed into cells that resemble embryonic stem cells. Normally, this procedure is relatively inefficient, slow, and induces new mutations into the engineered cells. Also, when iPSCs are differentiated into liver cells (hepatocytes), they do not adequately proliferate after differentiation, and they also fail to properly function the way adult hepatocytes do.

New work from laboratories at the University of California, San Francisco (UCSF), has differentiated human hepatocytes by means of a modified technique that bypasses the pluripotency stage. These cells were then used to repopulate mouse livers.

“I really like this paper. It’s a step forward in the field,” said Alejandro Soto-Gutiérrez, assistant professor of pathology at the University of Pittsburgh, who was not involved in the work. “The concept is reprogramming, but with a shortcut, which is really cool.”

Research teams led by Holger Willenbring and Sheng Ding isolated human skin cells called fibroblasts and infected them with engineered viruses that forced the expression of three genes: OCT4, SOX2, and KLF4. These transduced cells were grown in culture in the presence of proteins called growth factors and small molecules in order to induce reprogramming of the cells into the primary embryonic germ layer known as endoderm. In the embryo, the endoderm is the inner-most layer of cells that forms the gastrointestinal tract and its associated structures (liver, pancreas, and so on). Therefore, the differentiation of adult cells into endodermal progenitor cells provides a handy way to form a cell type that readily divides and can differentiate into liver cells.

“We divert the cells on their path to pluripotency,” explained coauthor Holger Willenbring, associate professor of surgery at UCSF. “We still take advantage of what is intrinsic to reprogramming, that the cells are becoming very plastic; they’ve become flexible in what kind of cell type they can be directed towards.”

The authors called these cells induced multipotent progenitor cells (iMPCs). The iMPCs were easily differentiated into endodermal progenitor cells (iMPC-EPCs). These iMPC-EPCs were grown in culture with a cocktail of small molecules and growth factors to increase iMPC-EPC colony size while concomitantly maintain them in an endodermal state. Afterwards, Willenbring and others cultured these cells with factors and small molecules known to promote liver cell differentiation. When these iMPC-Hepatocytes (Heps) were transplanted into mice with damaged livers, the iMPC-Hep cells continued to divide at least nine months after transplantation. Furthermore, the transplanted cells matured and displayed gene expression profiles very similar to that of typical adult hepatocytes. Transplantation of iMPC-Heps also improved the survival of a mouse model of chronic liver failure about as well as did transplantation of adult hepatocytes.

“It is a breakthrough for us because it’s the first time that we’ve seen a cell that can actually repopulate a mouse’s liver,” said Willenbring. Willenbring strongly suspects that iMPCs are better able to repopulate the liver because the derivation of iMPC—rather than an iPSC—eliminates some steps along the path to generating hepatocytes. These iMPCs also possess the ability to proliferate in culture to generate sufficient quantities of cells for therapeutic purposes and, additionally, can functionally mature while retaining that proliferative ability to proliferate. Both of these features are important prerequisites for therapeutic applications, according to Willenbring.

Before this technique can enter clinical trials, more work must be done. For example: “The key to all of this is trying to generate cells that are identical to adult liver cells,” said Stephen Duncan, a professor of cell biology at Medical College of Wisconsin, who was not involved in the study. “You really need these cells to take on all of the functions of a normal liver cell.” Duncan explained that liver cells taken directly from a human adult might be able to repopulate the liver in this same mouse model at levels close to 90 percent.

Willenbring and his colleagues observed repopulation levels of 2 percent by iMPC-Heps, which is substantially better than the 0.05 percent repopulation typically accomplished by hepatocytes derived from iPSCs or embryonic stem cells. However: “As good as this is, the field will need greater levels of expansion,” said Ken Zaret of the Institute for Regenerative Medicine at the University of Pennsylvania, who did not participate in the work. “But the question is: What is limiting the proliferative capacity of the cells?”

Zaret explained that it is not yet clear whether some aspect of how the cells were programmed that differed from how they normally develop could have an impact on how well the population expands after transplantation. “There still is a ways to go [sic],” he said, “but [the authors] were able to show much better long-term repopulation with human cells in the mouse model than other groups have.”

See S. Zhu et al., “Mouse liver repopulation with hepatocytes generated from human fibroblasts,” Nature, doi:10.1038/nature13020, 2014.

Sweat Glands Are A Source of Stem Cells for Wound Healing


Stem Cells from human sweat glands serve as a remarkable source for wound healing treatments according to a laboratory in Lübeck, Germany.

Professor Charli Kruse, who serves as the head of the Fraunhofer Research Institute for Marine Biotechnology EMB, Lübeck, Germany, and his colleagues isolated cultured pancreatic cells in the course of their research to look into the function of a protein called Vigilin. When the pancreatic cells were grown in culture, they produced, in addition to other pancreatic cells, nerve and muscle cells. Thus the pancreas contains a stem cell population that can differentiate into different cell types.

Kruse and his group decided to investigate other glands contained a similar stem cell population that could differentiate into other cell types.

Kruse explained: “We worked our way outward from the internal organs until we got to the skin and the sweat glands. Again, this yielded the same result: a Petri dish full of stem cells.”

Up to this point, sweat glands have not received much attention from researchers. Mice and rats only have sweat glands on their paws, which makes them rather inaccessible. Human beings, on the other hand, have up to three million sweat glands, predominantly on the soles of out feet, palms of the hand, armpits, and forehead.

Ideally, a patient could have stem cells taken from her own body to heal an injury, wound, or burn, Getting to these endogenous stem cell populations, however, represents a challenge, since it requires bone marrow biopsies or aspirations, liposuction, or some other invasive procedure.

Sweat glands, however, are significantly easier to find, and a short inpatient visit to your dermatologist that extracts three millimeters of underarm skin could provide enough stem cells to grow in culture for treatments.

Stem cells from sweat glands have the capacity to aid wound healing. Kruse and his group used sweat gland-based stem cells in laboratory animals. The Kruse group used skin biopsies from human volunteers and separated out the sweat gland tissues under a dissecting scope. Then the sweat gland stem cells were grown in culture and induced to differentiate into a whole host of distinct cell types.

Then Kruse’s team grew these sweat gland stem cells in a skin-like substrate that were applied to wounds on the backs of laboratory animals. Those animals that had received stem cell applications healed faster than those that received no stem cells.

If the stem cells were applied to the mice with the artificial substrate, the cells moved into the bloodstream and migrated away from the site of the injury. In order to help heal the wound the cells had to integrate into the skin and participate in the healing process.

“Not only are stem cells from sweat glands easy to cultivate, they are extremely versatile, too,” said Kruse.

Kruse and his team are already in the process of testing a treatment for macular degeneration using sweat gland-based stem cells. “In the long-term, we could possibly set up a cell bank for young people to store stem cells from their own sweat glands/ They would then be available for use should the person need new cells, following an illness,l perhaps, or in the event of an accident,” Kruse said.

Teaching Old Neural Stem Cells New Tricks


In our brains, cells called neurons produce nerve impulses and are responsible for thinking, learning memory, reasoning, and so on. Neurons do not exist in isolation, but in combination with cells called glial cells that support the neurons, nourish them, and protects them from stress damage. Neurons and glial cells are replenished by brain-specific neural stem cell populations in the brain.

Unfortunately, the neural stem cell population in our brains tends to produce far fewer neurons as they age. This deficit of new neurons can play a role in the onset of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Also, our own “senior moments” when we forget where we placed our iPod or car keys comes from a loss of neurons as we age.

Fortunately, some recent research might change this trend. A team from Japan’s Keio University, and the Riken national research institute, has reported the discovery of a small RNA molecule (micro-RNA) that controls neuron production in young mice. When this micro-RNA was manipulated in older mice, their neural stem cells started to make neurons again. The Japanese team also has reasons to believe that the same mechanism is at work in human brains as well. This research was reported in the journal Proceedings of the National Academies of Science. The mechanism is believed to exist in humans as well.

Senior Author Hideyuki Okano said, “We observed the neurogenic-to-gliogenic switching in developing NSCs.” Translation: Okano and his team examined embryonic mouse brains and their neural stem cell (NSC) populations. They found what many other groups have previously observed: that the developing embryonic brain NSCs create neurons first, then switch over to making glial cells later. Okano’s team also discovered the microRNA-17/106-p38 axis that is responsible for this initial neuron-to-glial cell switch during embryonic development.

When they manipulated this embryonic microRNA-17/106-p38 pathway in older, post-natal NSCs in culture, these older post-natal NSCs switched from making glial cells to producing neurons.

In culture, NSCs are difficult to control, since getting large supplies of neurons from cell cultures that various research groups call NSCs is very difficult.

Nevertheless, “there is general agreement that neurogenesis (make neurons) largely precedes gliogenesis (making glial cells) during CNS development in vertebrates,” Okano explained. And adult NSCs, according to Okano, clearly can produce neurons in the body, “whereas they exhibit strong gliogenic characteristics under culture conditions in vitro (that is, in the laboratory).”

Adult NSCs in two regions of the brain—the subventricular zone and hippocampus—also “make neurons, even though transplant studies have shown us that the adult CNS is a gliogenic environment.”

Subventricular Zone

So it seems clear that old NSCs can make neurons, at least under certain conditions. However, it is very difficult to determine the age at which NSCs begin making substantially more glial cells than neurons. According to Okano, “It is difficult to clearly explain the association between total glial cell number and changes in NSC abilities. Moreover, there is less evidence about gliogenic ability of aged NSCs because most of studies about NSCs have mainly focused on the neurogenic ability. “

Still, Okano says: “There are some reports about decline of neurogenesis ability of NSCs with age. These reports indicate that reduction in paracrine Wnt3 factors, and increase of (chemokine) CCL11 concentration in blood, impaired adult neurogenesis in the hippocampus, for example.”

Could the group’s microRNA approach improve memory in humans? Okano believes so, but says more work needs to be done.

“We observed the neurogenic effect by overexpression of miR-17 in primary cultured neurospheres” – spheres of a variety of cells, including NSCs—“derived from the SVZ at postnatal day 30. Similar phenomenon by overexpression of miR-106b-25 cluster has been reported by another group.”

Okano also warns that his approach has only been attempted in cultured cells. He cautioned, “There is no evidence using knock-out mice. Therefore, the functions of them in adult neurogenesis and learning/memory functions are still unclear.”

Next, Okano’s group will develop “a useful method for precise manipulation of cytogenesis from NSCs. “

However, he says, “we think that further understanding of basic molecular mechanisms underlying the neural development is also an important issue.” He will study the ways in which his microRNA system interacts with other glia-producing genes. He wants to fully understand the mechanisms underlying “the end of neurogenic competence and acquisition of gliogenic competence.”

Finally, the group will “examine the significance of miR-17/p38 pathway in various somatic stem cells other than NSCs,” he says.

Stimulus-Triggered Acquisition of Pluripotency Cells: Embryonic-Like Stem Cells Without Killing Embryos or Genetic Engineering


Embryonic stem cells have been the gold standard for pluripotent stem cells. Pluripotent means capable of differentiating into one of many cell types in the adult body. Ever since James Thomson isolated the first human embryonic stem cell lines in 1998, scientists have dreamed of using embryonic stem cells to treat diseases in human patients.

However, deriving human embryonic stem cell lines requires the destruction or molestation of a human embryo, the smallest, youngest, and most vulnerable member of our community. In 2006, Shinya Yamanaka and his colleges used genetic engineering techniques to make induced pluripotent stem (iPS) cells, which are very similar to embryonic stem cells in many ways. Unfortunately, the derivation of iPSCs introduces mutations into the cells.

Now, researchers from Brigham and Women’s Hospital (BWH), in Boston, in collaboration with the RIKEN Center for Developmental Biology in Japan, have demonstrated that any mature adult cell has the potential to be converted into the equivalent of an embryonic stem cell. Published in the January 30, 2014 issue of the journal Nature, this research team demonstrated in a preclinical model, a novel and unique way to reprogram cells. They called this phenomenon stimulus-triggered acquisition of pluripotency (STAP). Importantly, this process does not require the introduction of new outside DNA, which is required for the reprogramming process that produces iPSCs.

“It may not be necessary to create an embryo to acquire embryonic stem cells. Our research findings demonstrate that creation of an autologous pluripotent stem cell – a stem cell from an individual that has the potential to be used for a therapeutic purpose – without an embryo, is possible. The fate of adult cells can be drastically converted by exposing mature cells to an external stress or injury. This finding has the potential to reduce the need to utilize both embryonic stem cells and DNA-manipulated iPS cells,” said senior author Charles Vacanti, MD, chairman of the Department of Anesthesiology, Perioperative and Pain Medicine and Director of the Laboratory for Tissue Engineering and Regenerative Medicine at BWH and senior author of the study. “This study would not have been possible without the significant international collaboration between BWH and the RIKEN Center,” he added.

The inspiration for this research was an observation in plant cells – the ability of a plant callus, which is made by an injured plant, to grow into a new plant. These relatively dated observations led Vacanti and his collaborators to suggest that any mature adult cell, once differentiated into a specific cell type, could be reprogrammed and de-differentiated through a natural process that does not require inserting genetic material into the cells.

“Could simple injury cause mature, adult cells to turn into stem cells that could in turn develop into any cell type?” hypothesized the Vacanti brothers.

Vacanti and others used cultured, mature adult cells. After stressing the cells almost to the point of death by exposing them to various stressful environments including trauma, a low oxygen and acidic environments, researchers discovered that within a period of only a few days, the cells survived and recovered from the stressful stimulus by naturally reverting into a state that is equivalent to an embryonic stem cell. With the proper culture conditions, those embryonic-like stem cells were propagated and when exposed to external stimuli, they were then able to redifferentiate and mature into any type of cell and grow into any type of tissue.

To examine the growth potential of these STAP cells, Vacanti and his team used mature blood cells from mice that had been genetically engineered to glow green under a specific wavelength of light. They stressed these cells from the blood by exposing them to acid, and found that in the days following the stress, these cells reverted back to an embryonic stem cell-like state. These stem cells then began growing in spherical clusters (like plant callus tissue). The cell clusters were introduced into developing mouse embryos that came from mice that did not glow green. These embryos now contained a mixture of cells (a “chimera”). The implanted clusters were able to differentiate into green-glowing tissues that were distributed in all organs tested, confirming that the implanted cells are pluripotent.

Thus, external stress might activate unknown cellular functions that set mature adult cells free from their current commitment to a particular cell fate and permit them to revert to their naïve cell state.

“Our findings suggest that somehow, through part of a natural repair process, mature cells turn off some of the epigenetic controls that inhibit expression of certain nuclear genes that result in differentiation,” said Vacanti.

Of course, the next step is to explore this process in more sophisticated mammals, and, ultimately in humans.

“If we can work out the mechanisms by which differentiation states are maintained and lost, it could open up a wide range of possibilities for new research and applications using living cells. But for me the most interesting questions will be the ones that let us gain a deeper understanding of the basic principles at work in these phenomena,” said first author Haruko Obokata, PhD.

If human cells can be made into embryonic stem cells by a similar process, then someday, a simple skin biopsy or blood sample might provide the material to generate embryonic stem cells that are specific to each individual, without the need for genetic engineering or killing the smallest among us. This truly creates endless possibilities for therapeutic options.

Vascular Progenitors Made from Induced Pluripotent Stem Cells Repair Blood Vessels in the Eye Regardless of the Site of Injection


Johns Hopkins University medical researchers have reported the derivation of human induced-pluripotent stem cells (iPSCs) that can repair damaged retinal vascular tissue in mice. These stem cells, which were derived from human umbilical cord-blood cells and reprogrammed into an embryonic-like state, were derived without the conventional use of viruses, which can damage genes and initiate cancers. This safer method of growing the cells has drawn increased support among scientists, they say, and paves the way for a stem cell bank of cord-blood derived iPSCs to advance regenerative medical research.

In a report published Jan. 20 in the journal Circulation, Johns Hopkins University stem cell biologist Elias Zambidis and his colleagues described laboratory experiments with these non-viral, human retinal iPSCs, that were created generated using the virus-free method Zambidis first reported in 2011.

“We began with stem cells taken from cord-blood, which have fewer acquired mutations and little, if any, epigenetic memory, which cells accumulate as time goes on,” says Zambidis, associate professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. The scientists converted these cells to a status last experienced when they were part of six-day-old embryos.

Instead of using viruses to deliver a gene package to the cells to turn on processes that convert the cells back to stem cell states, Zambidis and his team used plasmids, which are rings of DNA that replicate briefly inside cells and then are degraded and disappear.

Next, the scientists identified and isolated high-quality, multipotent, vascular stem cells that resulted from the differentiation of these iPSC that can differentiate into the types of blood vessel-rich tissues that can repair retinas and other human tissues as well. They identified these cells by looking for cell surface proteins called CD31 and CD146. Zambidis says that they were able to create twice as many well-functioning vascular stem cells as compared with iPSCs made with other methods, and, “more importantly these cells engrafted and integrated into functioning blood vessels in damaged mouse retina.”

Working with Gerard Lutty, Ph.D., and his team at Johns Hopkins’ Wilmer Eye Institute, Zambidis’ team injected these newly iPSC-derived vascular progenitors into mice with damaged retinas (the light-sensitive part of the eyeball). The cells were injected into the eye, the sinus cavity near the eye or into a tail vein. When Zamdibis and his colleagues took images of the mouse retinas, they found that the iPSC-derived vascular progenitors, regardless of injection location, engrafted and repaired blood vessel structures in the retina.

“The blood vessels enlarged like a balloon in each of the locations where the iPSCs engrafted,” says Zambidis. Their vascular progenitors made from cord blood-derived iPSCs compared very well with the ability of vascular progenitors derived from fibroblast-derived iPSCs to repair retinal damage.

Zambidis says that he has plans to conduct additional experiments in diabetic rats, whose conditions more closely resemble human vascular damage to the retina than the mouse model used for the current study, he says.

With mounting requests from other laboratories, Zambidis says he frequently shares his cord blood-derived iPSC with other scientists. “The popular belief that iPSCs therapies need to be specific to individual patients may not be the case,” says Zambidis. He points to recent success of partially matched bone marrow transplants in humans, shown to be as effective as fully matched transplants.

“Support is growing for building a large bank of iPSCs that scientists around the world can access,” says Zambidis, although large resources and intense quality-control would be needed for such a feat. However, Japanese scientists led by stem-cell pioneer Shinya Yamanaka are doing exactly that, he says, creating a bank of stem cells derived from cord-blood samples from Japanese blood banks.