Prenatal Stem Cell Treatment Improves Mobility in Lambs With Spina Bifida


UC Davis fetal surgeon Dr. Diana Farmer has been at the forefront of treating spina bifida in infants while they are still in their mother’s womb. Now, Dr. Farmer and her colleagues have used a large animal model system to study the use of stem cells to improve the clinical outcomes of children who undergo these types of in utero procedures.

Spina bifida is a congenital birth defect that results from abnormal development of the spinal cord. During development, the spinal cord, which beings as a tube (the neural tube), is open at both ends, and these ends eventually close. However, if the posterior opening to the neural tube does not close properly, then the developing spinal cord will have severe structural defects. These structural defects adversely affect the nerves that issue from the spinal cord and spinal bifida can cause lifelong cognitive, urological, musculoskeletal and motor disabilities.

Dr. Farmer’s chief collaborator was another UC Davis science named Aijun Wang, who serves as the co-director of the UC Davis Surgical Bioengineering Laboratory.

“Prenatal surgery revolutionized spina bifida treatment by improving brain development, but it didn’t benefit motor function as much as we hoped,” said Farmer, who serves as chair of the UC Davis Department of Surgery and is the senior author of this study, which was published online in the journal Stem Cells Translational Medicine.

“We now think that when it’s augmented with stem cells, fetal surgery could actually be a cure,” said Wang.

Years ago, Farmer and her colleagues showed in an extensive clinical trial called the Management of Myelomeningocele Study (MOMS) that babies who were diagnosed with spina bifida and were eligible for in utero surgery had better outcomes that babies who underwent surgery after they were born. Babies with spina bifida who were operated on in utero had a better chance of walking, and not needing a shunt to deal with the pressure problems in the brain that some children with spina bifida experience (see N. Scott Adzick, et al., New England Journal of Medicine 2011;364(11):993-1004). Even with this study, the majority of the babies who were treated with in utero surgery were still unable to walk. To improve a baby’s chances of walking, Farmer and her collaborators turned to stem cell treatments.

Farmer and Wang combined fetal surgery with a the transplantation of stem cells from human placentas to improve neurological capabilities of babies born with spina bifida. In children, spina bifida can range from barely noticeable to rather severe. Myelomeningocele is the most common and, unfortunately, the most disabling form of spina bifida. In babies with myelomeningocele, the spinal emerges through the back and usually pulls brain tissue into the spinal column, which causes cerebrospinal fluid to fill the interior of the brain. Therefore, such patients require permanent shunts in their brains in order to drain the extra cerebrospinal fluid.

Myelomeningocele
Myelomeningocele

In this study, lambs with myelomeningocele were operated on in utero in order to return exposed spinal cord tissue into the vertebral column. Then human placenta-derived mesenchymal stromal cells (PMSCs), which have demonstrated neuroprotective qualities (see Yun HM, et al., Cell Death Dis. 2013;4:e958), were embedded in hydrogel and applied to the site of the lesion. A scaffold was placed on top to hold the hydrogel in place, and the surgical opening was closed.

Six of the animals that received the stem cell treatment were able to walk without noticeable disability within a few hours following birth. However, the six control animals that received only the hydrogel and scaffold were unable to stand.

“We have taken a very important step in expanding what MOMS started,” said Wang. “Next we need to confirm the safety of the approach and determine optimal dosing.”

Farmer and Wang will continue their efforts with funding from the California Institute for Regenerative Medicine. With additional evaluation and FDA approval, the new therapy could be tested in human clinical trials.

“Fetal surgery provided hope that most children with spina bifida would be able to live without shunts,” Farmer said. “Now, we need to complete that process and find out if they can also live without wheelchairs.”

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 Cell Treatments for Bladder Dysfunction


Spina bifida is a birth defect in which the backbone and spinal canal do not close before birth. The damage to the central nervous system affects various organs that receive nervous inputs from the spinal cord and one such organ is the urinary bladder. A condition called “neurogenic bladder” results from an inability of the nervous system to properly control the urinary bladder and the muscle tissue that lines the wall of the bladder. Neurogenic bladder can lead to spasms and a pressure build-up in the bladder. This results in urinary incontinence, and children with spina bifida and neurogenic bladder often have an urge to urinate after drinking comparatively small amounts of liquid. They can also involuntarily leak urine, and this creates a great deal of social embarrassment and emotional stress. If untreated, the long-standing and frequent pressure build-up in the bladder can lead to infections and even kidney damage.

Spina bifida

Surgical treatments for neurogenic bladder involve reconstruction of the bladder that increases its size by grafting patches from the patient’s bowel. Because the graft comes from the patient’s own body, it is unlikely that the immune system will reject these grafts. Also, the intestinal tissue patches are, on the average, strong enough to withstand the pressures in the bladder. However, there is a certain incompatibility between intestinal and bladder tissue, and this can cause long-term complications that include urinary tract infections, urinary tract stones and, rarely, cancers. Thus, researchers have been searching for newer safer patches which resemble the actual bladder wall.

Northwestern University researchers have published a study that used stem cells of children with spina bifida to generate tissue patches for bladder surgery. This paper, “Cotransplantation with specific populations of spina bifida bone marrow stem/progenitor cells enhances urinary bladder regeneration,” was published in the Proceedings of the National Academy of Sciences. Arun Sharma and colleagues from Earl Cheng’s laboratory isolated two types of cells from the bone marrow of child spina bifida patients. They isolated mesenchymal stem cells (MSCs) and CD34+ cells, which are stem and progenitor cells that usually give rise to blood cells. Sharma and her colleagues used these cells to coated molds that were made with a special polymer scaffold called POC (poly(octanediol-co-citrate). These cell-filled molds were used to create a patch graft that was transplanted into rat bladders. This type of surgery is not that unlike the bladder augmentation surgery used for spina bifida patients.

Next, the Cheng lab workers determined if the human tissue survived in the implanted patch. In those cases where both human cell types (MSCs and CD34+) were combined, over half the implanted patch was covered with muscle tissue, four weeks after the implantation. However, if only CD34+ cells were used, only a quarter of the patch was covered with muscle tissue. Interestingly, the implanted patch also showed evidence of some peripheral nerve growth and blood vessel formation, both of which are found in healthy, normal bladder walls. These experiments demonstrate suggest that a patient’s own bone marrow stem cells can be used to help construct a tissue patch that can potentially act as a graft patch for bladder augmentation surgeries. Also, since some nerve growth in the implanted patch was observed, and this definitely an exciting result. Could it be possible to re-connect the reconstructed bladder tissue with the main nervous system? Possibly, but the most severe cases of neurogenic bladder are almost certainly more difficult cases to treat successfully.

bladder_wall_pic

Despite the exciting possibilities in this study, there are some caveats. First of all, was the muscle formed from the stem cells or the surrounding tissue? It is not clear from this paper, and since implantation of an empty POC scaffold without any human stem cells results in 20% coverage with muscle tissue, at least some of the newly formed muscle tissue is actually derived from the host rat and not from human stem cells. Secondly, how do these patch grafts compare to the intestinal patches? This was not assessed. Finally, rats with neurogenic bladder were not implanted, and this is an important control, since it is at least possible, that the muscle growth would be less robust in an animal with neurogenic bladder. Thus, despite this great potential shown in this paper, several questions remain.

A second paper used a very different approach. Debra Franck and others in Joshua R. Mauney’s lab at Harvard Medical School coated a silk thread scaffold with extracellular matrix proteins and coated with smooth muscle cells that were made from induced pluripotent stem cells into the smooth muscle cells. Unfortunately, Franck and colleagues did not evaluate the newly created patch in a living animal. Also, this paper used mouse induce pluripotent stem cells, and it is not clear that human induced pluripotent stem cells would be able to do the same thing.

While these two studies are strictly experimental, they might provide some new avenues of research for new treatments for neurogenic bladder.