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.

Umbilical Cord Blood Stem Cells in a Biodegradable Scaffold Regenerate Full-Thickness Skin Defects


In a new study published in the ASAIO Journal by Reza Zeinali and others in the laboratory of Kamal Asadipour, specific stem cell from umbilical cord blood called unrestricted somatic stem cells (USSCs) have been grown on a biodegradable scaffold to promote skin regeneration and wound healing.

USSCs are considered by many stem cell scientists to be a type of mesenchymal stem cell, but USSCs can be grown in the laboratory and have the ability to differentiate into a wide variety of adult cell types.

Asadipour and others used a material called PHBV or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to make a skin-like scaffold upon which the USSCs were grown. They discovered that attaching a molecule called “chitosan” to the PHBV made it quite resilient and a very good substrate for growing cells. When grown on these scaffolds, the USSCs adhered nicely to them and grew robustly.

Then Zeinali and his colleagues used these cell-impregnated scaffolds to treat open surgical wounds in laboratory rodents. After three weeks, the group treated with the cell grown on the scaffolds healed significantly better than those animals treated with just cells, just scaffolds, or neither.

Thus it seems likely that tissue-engineered skin made from modified PHBV scaffolds and embedded umbilical cord blood-based stem cells might be a potent treatment for wound patients with large injuries that do heal slowly.  In the words of the abstract of this paper, “These data suggest that chitosan-modified PHBV scaffold loaded with CB-derived USSCs could significantly contribute to wound repair and be potentially used in the tissue engineering.”

Some larger animal studies should further test this protocol and if it can augment the healing of large animal wounds, then human clinical trials should be considered.

Increasing Engraftment Rates of Umbilical Cord Blood Transplantations


Harvard Stem Cell Institute (HSCI) researchers have published initial results of a Phase Ib human clinical trial of a therapeutic that has the potential to improve the success of blood stem cell transplantation. This publication marks a success for the HSCI and their ability to carry a discovery from the lab bench to the clinic. This was actually the mandate for the HSCI when it was founded.

This Phase 1b safety study was published in the journal Blood, and it included 12 adult patients who underwent umbilical cord blood transplantation for leukemia or lymphoma at the Dana Farber Cancer Institute and Massachusetts General Hospital. Each patient received two umbilical cord blood units; one of which was untreated and another that was treated with a small molecule called 16,16 dimethyl prostaglandin E2 (dmPGE2). The immune systems of all 12 patients were successfully reconstituted and their bone marrow tissues were able to make blood cells. However, 10 of the 12 patients had blood formation that was solely derived from those umbilical cord blood cells that had been treated with dmPGE2.

This clinical test is now entering Phase II, during which the HSCI scientists will determine the efficacy of this treatment in 60 patients at 8 different medical centers. They expect results from this trial within 18-24 months.

The success of the HSCI depended on collaborations with scientists at different Harvard-affiliated institutions. These collaborations included 1) Leonard Zon, chair of the HSCI Executive Committee and Professor of Stem Cell and Regenerative Biology at Harvard, and his colleagues, 2) Dana-Farber Cancer Institute and Massachusetts General Hospital, led by hematologic oncologist and HSCI Affiliated Faculty member Corey Cutler, and 3) Fate Therapeutics, Inc., a San Diego-based biopharmaceutical company of which Zon is a founder, sponsored the Investigational New Drug application, under which the clinical program was conducted, and translated the research findings from the laboratory into the clinical setting.

“The exciting part of this was the laboratory, industry, and clinical collaboration, because one would not expect that much close interplay in a very exploratory trial,” Cutler said. “The fact that we were able to translate someone’s scientific discovery from down the hall into a patient just a few hundred yards away is the beauty of working here.”

Gastroenterologists have been interested in dmPGE2 for decades, because it has the ability to protect the intestinal lining from stress. However, its ability to amplify stem cell populations was identified in 2005 during a chemical screen exposing 5,000 known drugs to zebrafish embryos. Wolfram Goessling, MD, PhD, and Trista North, PhD former Zon postdoctoral fellows, were involved in that work.

“We were interested in finding a chemical that could amplify blood stem cells and we realized looking at zebrafish embryos that you could actually see blood stem cells budding from the animal’s aorta,” Zon said. “So, we elected to add chemicals to the water of fish embryos, and when we took them out and stained the aortas for blood stem cells, there was one of the chemicals, which is this 16,16 dimethyl prostaglandin E2, that gave an incredible expansion of stem cells—about a 300 to 400 percent increase.”

The dramatic effects of this molecule on blood stem cells causes Zon, who practices as a pediatric hematologist, consider how this prostaglandin could be applied to bone marrow transplantation. Bone marrow transplantations are often used to treat blood cancers, including leukemia and lymphoma. Bone marrow contains the body’s most plentiful reservoir of blood stem cells, and so patients with these conditions may be given bone marrow transplants to reconstitute their immune systems after their cancer-ravaged bone marrow has been wiped out with chemotherapy and radiation.

Zon designed a preclinical experiment, similar to the one later done with cord blood patients, in which mice undergoing bone marrow transplants received two sets of competing bone marrow stem cells, one set treated with dmPGE2 and a second untreated set.

“What we found was the bone marrow stem cells that were treated with prostaglandin, even for just two hours, had a four times better chance of engrafting in the recipient’s marrow after transplant,” he said. “I was very excited to move this into the clinic because I knew it was an interesting molecule.”

Zon and his team’s then visited the Dana Farber Cancer Institute (DFCI). There, they presented the mouse research at bone marrow transplant rounds and found physicians interested in giving the prostaglandin to patients.

“We basically sat down in a room and we brainstormed a clinical trial based on their scientific discovery, right then and there,” said Farber oncologist Corey Cutler. “They knew that it was something they could bring to the clinic, but they just didn’t know where it would fit. We said, if this molecule does what you say it does, significant utility would lie in umbilical cord blood transplants.”

A cord blood transplant is similar to a bone marrow transplant, but the blood stem cells are not from an adult donor but from the umbilical cord blood of a newborn. The degree of tissue matching is less in an umbilical cord blood transplant than in a bone marrow transplant. The umbilical cord stem cells are young and incipient and the immune system simply does not recognize them as readily as adult cells. Therefore, potentially fatal graft-versus-host disease is less common with umbilical cord blood transplants. About 10-20 percent of stem cell transplantation procedures now use umbilical cord blood. However the main disadvantage of umbilical cord blood transplantations is that the cord blood contains uses smaller amounts of cells, which makes engraftment is more difficult.

Umbilical cord blood transplants fail about 10 percent of the time. Therefore, increasing the procedure’s success would significantly help patients who do not have adult bone marrow donors, including a disproportionate number of non-Caucasian patients in North America. Increasing the engraftment rate would also allow the use of smaller umbilical cord blood units that are potentially better matches to their recipients, increasing the number of donations that go on to help patients.

Fate Therapeutics received the first green light from the US Food and Drug Administration, and the DFCI Institutional Review Board for this clinical trial. Umbilical cord blood processing was done by Dana-Farber’s Cell Manipulation Core Facility, directed by HSCI Executive Committee member Jerome Ritz, MD. There was a stumbling block in that once the human trial was underway with the first nine patients in that the protocol in use, which was developed in mice, did not translate to improved engraftment in humans.

“The initial results were very disappointing,” Cutler said. “We went back to the drawing board and tried to figure out why, and it turned out some of the laboratory-based conditions were simply not optimized, and that was largely because when you do something in the lab, the conditions are a little bit different than when you do it in a human.”

Fate Therapeutics discovered that the human cord blood was being handled at temperatures that were too cold (4-degrees Celsius) for the prostaglandin to biologically activate the stem cells. Therefore even after prostaglandin treatment, the umbilical cord blood did not show enhanced engraftment rates. Fate further demonstrated that performing the incubation of the hematopoietic stem cells at 37-degrees Celsius and increasing the incubation time from 1 hour to 2 hours elicited a much stronger gene and protein expression response that correlated with improved engraftment in animal models.

In running a second cohort of the Phase Ib trial, which included 12 patients, dmPGE2 appeared to enhance the engraftment properties of the blood stem cells in humans and was deemed safe to continue into Phase II. “It’s probably the most exciting thing I’ve ever done,” Zon said. “Basically, to watch something come from your laboratory and then go all the way to a clinical trial is quite remarkable and very satisfying.”

Umbilical Cord Blood Stem Cells Revive Child From Persistent Vegetative State


Physicians from Ruhr-Universitaet-Bochum (RUB) have successfully treated cerebral palsy in a 2.5-year old boy with his own cord blood.

“Our findings, along with those from a Korean study, dispel the long-held doubts about the effectiveness of the new therapy,” says Dr. Arne Jensen of the Campus Clinic gynaecology. Jensen collaborated with his colleague Prof. Dr. Eckard Hamelmann of the Department of Pediatrics at the Catholic Hospital Bochum (University Clinic of the RUB). This case study was published in the journal Case Reports in Transplantation.

At the end of November 2008, a young child’s heart stopped (cardiac arrest), and his brain suffered oxygen deprivation, and, consequently, severe brain damage. He was in a persistent vegetative state, and his body was completely paralyzed. This condition, infantile cerebral palsy, until now, has no recognized treatment. Typically, the prognosis of children with infantile cerebral palsy is rather grim, since the chances of survival miniscule and months after suffering severe brain damage, the surviving children usually only exhibit minimal signs of consciousness. According to the physicians at RUB, “The prognosis for the little patient was threatening if not hopeless.”

However, this child’s persistent parents scoured the literature for alternative therapies to infantile cerebral palsy. Arne Jensen explains. “They contacted us and asked about the possibilities of using their son’s cord blood, frozen at his birth.”

Nine weeks after suffering brain damage, on 27 January 2009, Jensen and his colleagues administered the child’s prepared cord blood intravenously. They studied the child’s progressive recovery at 2, 5, 12, 24, 30, and 40 months after treatment.

After the cord blood therapy, the patient, however, recovered quickly. Within two months, the child’s spasms decreased significantly. He was able to see, sit, smile, and to speak simple words again. Forty months after treatment, the child was able to eat independently, walk with assistance, and form four-word sentences. “Of course, on the basis of these results, we cannot clearly say what the cause of the recovery is,” Jensen says. “It is, however, very difficult to explain these remarkable effects by purely symptomatic treatment during active rehabilitation.”

Just listen to the description of the child’s recovery from this paper:

After two years, there was independent eating and speech competence of eight words (pronunciation slurred, mimicking prosody) with broad understanding. The patient moved from a prone to a free sitting position and crawled without cross-pattern, but using the arms. Independent passive standing, walking with support, and independent locomotion in a gait trainer was possible (video S5). He played imaginative games, and recognized colours, animals, and objects, assigning them correctly. Fine motor control improved to such an extent that he managed to steer a remote control car (video S6). At 30 months, he formed two-word-sentences using 80 words.

After 40 months, there was further improvement in both receptive and expressive speech competence (four-word-sentences, 200 words), walking (Crocodile Retrowalker), crawling with cross-pattern, and getting into vertical position.

And this is from a child who was a in a persistent vegetative state, who could neither speak, nor eat on his own, nor talk.

In animal studies, scientists have examined the therapeutic potential of cord blood. In a previous study with rats, RUB researchers revealed that cord blood cells migrate to the damaged area of the brain in large numbers within 24 hours of administration.  Umbilical cord stem cells are also known to secrete gobs of neurotropic molecules that stimulate neuron growth and differentiation, promote neuron survival, quell inflammation, staunch star formation in the brain (gliosis), and stimulate the growth and formation of blood vessels.

In March 2013, in a controlled study of one hundred children, Korean doctors reported for the first time that they had successfully treated cerebral palsy with someone else’s cord blood.

These results show that cord blood has tremendous therapeutic potential for pediatric neurological conditions.  This remarkable recovery is seemingly miraculous.  Certainly this merits more work and excitement.

When To Use Umbilical Cord Blood Stem Cells


Umbilical cord blood stem cells (UCB-SCs) have been used in a variety of clinical trials and treatments. Their use in treatment bone marrow-based conditions is very well-known, but they have also been used in other experimental treatments as well.

Treatments with UCB-SCs suffer from inconsistent results that stem from a variable number of viable cells in UCB-SC samples. Establishing high numbers of viable cells in UCB-SC samples is not easy, and there is a great interest in being able to grow UCB-SCs in culture and expand them. However, even though UCB-SCs can be grown in culture, the effects of culturing UCB-SCs is presently unclear.

To address this question in a rigorous fashion, Miguel Alaminos at the University of Granada and his colleagues grew UCB-SCs in culture and analyzed cell viability and gene expression at every passage.

What they discovered was astounding. When UCB-SCs were passaged two or three times, the cells showed signs of cells death, and gene expression studies revealed that many of the cells expressed genes associated with programmed cell death. Cells passaged eight, nine, or ten times also showed extensive cell death. However, cells passaged five or six times showed the highest viability.

This suggests that different studied have used cells that were grown for different periods of time and probably had different viabilities. This explains why UCB-SCs have performed so variably in experiments and clinical trials. This suggests that therapies that utilize UCB-SCs should use them after they are passaged for the fifth or sixth time in order to ensue the highest levels of viability.

Umbilical Cord Stem Cells and Cancer


The umbilical cord blood stem cells have been used to treat cancer patients whose bone marrow tissues have been wiped out by radiation or chemotherapeutic treatments. Several clinical trials have addresses the capacity of umbilical cord blood to reconstitute the bone marrow of cancer patients.

The first set of clinical trials have examined the use of umbilical cord blood in children. Gluckman and her colleagues reported the use of umbilical cord blood to treat children who had suffered from a variety of blood maladies. 74 patients were treated with umbilical cord blood. 63% of the patients survived one year after the procedure, and the rate of graft-versus-host disease (GVHD) was only 9%. Now this study showed that umbilical cord blood could be used to reconstitute the bone marrow, but how well does it work compared to bone marrow transplants?

To answer this question, Rocha and his colleagues compared kids who had received umbilical cord blood transplants with those who had received bone marrow transplants. 113 cord blood transplant patients were compared to 2052 bone marrow transplant recipients. In this study, the umbilical cord blood recipients took longer to have their bone marrow reconstituted, but the rate of graft-versus-host disease was lower. The survival rate of the two groups three years after the procedure was also about the same (64% for the umbilical cord blood recipients and 66% for the bone marrow recipients). Thus, umbilical cord blood seemed to work as well as bone marrow when it came to reconstituting the bone marrow.

Since the rates of GVH disease were so low, could umbilical cord blood that was not properly tissue matched to the recipient also work? The answer was yes. Once again Gluckman and her colleagues showed that the rate of GVH disease was rather low, and the rate of recovery in a group of 65 patients was quite high (87%). Such a treatment with unmatched bone marrow would be a disaster, since GVH disease would almost certainly result from such a treatment. The results of Gluckman’s small study were confirmed by a much larger study by Rubinstein and others in 1998.

Can cord blood be used to treat adults with similar maladies? Clinical studies have confirmed that the answer is yes. Survival rates from a host of clinical trials have ranged from 15%-70%, but clearly adults can benefit from umbilical cord blood transplantations. Once again, the rates of GVH disease were lower in umbilical cord blood recipients when compared to bone marrow recipients, but once again, the time required for bone marrow recovery was greater.

In Minnesota, Wagner and his colleagues pioneered the use of “double umbilical cord blood grafts” in which umbilical cord blood is taken from two different babies to treat an adult patient. This overcomes the limited volume and cell numbers in an umbilical collection from a single donor. These are only used for patients who are very ill, but studies have shown that patients who have received double umbilical cord blood grafts have a ten-fold lower decrease in the risk of relapse of blood cancers.

Thus over the past two decades, umbilical cord blood transplants have become rather attractive sources of material to reconstitute bone marrow. Although low cell numbers are still a chronic problem with them, the ability to culture and expand these cells in culture may give a new life to this useful treatment.