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.