Wound Healing Therapy That Combines Gene and Stem Cell Therapy

Researchers from Johns Hopkins University have examined wound healing in older mice and discovered that increasing blood flow to the wound can increase the rate of wound healing. Increasing blood flow to the wound requires a combination of gene therapy and the same stem cells the body already uses to heal itself.

John W. Harmon is professor of surgery at Johns Hopkins School of Medicine, and in a presentation to the American College of Surgeons’ Surgical Club, made the case that harnessing the power of bone marrow stem cells can increase the rate at which older people heal.

As we age, our wounds do not heal as fast. However, Harmon thinks that harnessing the power of bone marrow stem cells can remedy this disparity in healing rates.

To heal burns or other wounds, stem cells from the one marrow rush into action and home to the wound where they can differentiate into blood vessels, skin, and other reparative tissues. Stem cell homing is mediated by a protein called Hypoxia-Inducible-Factor-1 (HIF-1). According to Harmon, in older patients, few of these stem cells are released from the bone marrow and there is a deficiency of HIF-1. HIF-1 was actually discovered about 15 years ago by one of Harmon’s collaborators, a Johns Hopkins scientist named Gregg J. Semenza.


Harmon’s first strategy was to boost HIF-1 levels by means of gene therapy. This simply consisted of injecting the rodents with a copy of the HIF-1 gene that yielded higher levels of HIF-1 expression.

Even though higher levels of HIF-1 improved wound healing rates, burns were another story. To accelerate burn healing, Harmon and his co-workers used bone marrow stem cells from younger mice combined with the increased levels of HIF-1. This combination of HIF-1 and bone marrow stem cells from younger mice led to accelerated healing of burns so that after 17 days, almost all the mice had completely healed burns. These animals that healed so fast showed better blood flow to the wound and more blood vessels supplying the wound.

Harmon said that while this strategy is promising, he think that a procedure that uses a patient’s own bone marrow cells would work better since such cells would have a much lower chance of being rejected by the patient’s immune system. In the meantime, HIF-1 gene therapy has been successfully used in humans with a sudden lack of blood flow to a limb (see Rajagopalan S., et al., Circulation. 2007 Mar 13;115(10):1234-43). Harmon postulated that “it’s not a stretch of the imagination to think this could someday be used in elderly people with burns or other difficult wounds.”

First Patient Treated in Study that Tests Stem Cell-Gene Combo to Repair Heart Damage

The first patient has been treated in a groundbreaking medical trial in Ottawa, Canada, that uses a combination of stem cells and genes to repair tissue damaged by a heart attack. The first test subject is a woman who suffered a severe heart attack in July and was treated by the research team at the Ottawa Hospital Research Institute (OHRI). Her heart had stopped beating before she was resuscitated, which caused major damage to her cardiac muscle.

The therapy involves injecting a patient’s own stem cells into their heart to help fix damaged areas. However, the OHRI team, led by cardiologist Duncan Stewart, M.D., took the technique one step further by combining the stem cell treatment with gene therapy.

“Stem cells are stimulating the repair. That’s what they’re there to do,” Dr. Stewart said in an interview. “But what we’ve learned is that the regenerative activity of the stem cells in these patients with heart disease is very low, compared to younger, healthy patients.”

Stewart and his colleagues will supply the stem cells with extra copies of a particular gene in an attempt to restore some of that regenerative capacity. The gene in question encodes an enzyme called endothelial nitric oxide synthase (eNOS). Nitric oxide is a small, gaseous molecule that is made from the amino acid arginine by the enzyme nitric oxide synthase. Nitric oxide or NO signals to smooth muscle cells that surround blood vessels to relax, which causes blood vessels to dilate and this increases blood flow. In the damaged heart, NO also helps build up new blood vessels, which increase healing of the cardiac muscle. Steward added, “That, we think, is the key element. We really think it’s the genetically enhanced cells that will provide the advantage.”

Nitric oxide synthesis

The study will eventually involve 100 patients who have suffered severe heart attacks in Ottawa, Toronto and Montreal.

Stem Cell Gene Therapy for Sickle Cell Disease Moves Toward Clinical Trials

UCLA stem cell researchers and “gene jockeys” have successfully proven the efficacy of using genetically engineered hematopoietic (blood cell-making) stem cells from a patient’s own bone marrow to treat sickle-cell disease (SCD).

In a study led by Donald Kohn, professor of pediatrics and microbiology at the UCLA Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, an “anti-sickling” gene was introduced into the hematopoietic stem cells (HSCs) from patients with SCD. Because the HSCs divide continuously throughout the life of the individual, all the blood cells they make will possess the anti-sickling gene and will therefore no sickle. This breakthrough gene therapy technique is scheduled to begin clinical trials by early 2014.

SCD results from a specific mutation in the beta-globin gene. Beta-globin is one of the two proteins that makes the multisubunit protein hemoglobin. Hemoglobin is a four-subunit protein that ferries oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs. It is tightly packed into each red blood cell.

The structure of hemoglobin, each subunit is in a different color.
The structure of hemoglobin, each subunit is in a different color.

Hemoglobin has a very high affinity for oxygen when oxygen concentrations are high, but a low affinity for oxygen when oxygen concentrations are low. Therefore, hemoglobin does a very good job of binding oxygen when it is in the lungs, where oxygen is plentiful, and a very good job of releasing oxygen in the tissues, where oxygen is not nearly as plentiful. This adaptive ability displayed by hemoglobin is the result of cooperativity between the four polypeptide chains that compose hemoglobin. Two of these polypeptide chains are alpha-globin proteins and the other two a beta globin proteins. Hemoglobin acts as though it is an alpha-beta dimer, or as though it is composed of two copies of an alpha-globin.beta-globin pair. The interactions between these polypeptide chains and the movement of the hemoglobin subunits relative to each other creates the biochemical properties of hemoglobin that are so remarkable.

A mutation in the beta-globin gene that substitutes a valine residue where there should be a glutamic acid residue (position number 6), creates a surface on the outside of the beta-globin subunit that does not like water, and when oxygen concentrations drops, the mutant hemoglobin molecule changes shape and this new water-hating surface becomes a site for protein polymerization.

Sickle cell hemoglobin

The mutant hemoglobin molecules for long, stiff chains that deform the red blood cells into a quarter moon-shaped structure that clogs capillaries.

Sickle cell RBCs


This new therapy seeks to correct the genetic mutation by inserting into the genome of the HSC that makes the abnormal red blood cells a gene for beta-globin that encodes a normal version of beta globin rather than a version of it that causes sickle-cell disease.  By introducing those engineered HSCs back into the bone marrow of the SCD patient, the engineered HSCs will make normal red blood cells that do not undergo sickling under conditions of low oxygen concentration.

Dr. Kohn noted that the results from his research group “demonstrate that our technique of lentiviral transduction is capable of efficient transfer and consistent expression of an effective anti-sickling beta-globnin gene in human SCD bone marrow progenitor cells, which improved the physiologic parameters of the resulting red blood cells.” Dr. Kohn’s statement may lead the reader to believe that this was done in a human patient, but that is not the case.  All this work was done in culture and in laboratory animals.

Kohn and his co-workers showed that in laboratory experiments, genetically engineered HSCs from SCD patients produced new non-sickled blood cells at a rate that would effectively allow SCD patients to show significant clinical improvement.  These new red blood cells also survived longer than those made by the nonengineered SCD HSCs.  The in vitro success of this technique has convinced the US Food and Drug Administration to grant Kohn the right to conduct clinical trials in SCD patients by early next year.

SCD affects more than 90,000 patients in the US, but it most affects people of sub-saharan African descent.  As stated before, the mutation that causes SCD produces red blood cells that are stiff, long, and get stuck in the tiny blood vessels known as capillaries that feed organs.  SCD causes multi-organ dysfunction and failure and can lead to death.


Treatment of SCD include bone marrow transplants, but immunological rejection of such transplants remains a perennial problem.   The success rate of bone marrow transplants is low and it is typically restricted to those patients with very severe disease who are on the verge of dying.

If Kohn’s clinical trials are successful, this stem cell-based treatment will hopefully become the gold standard for treatment of patients with SCD.  One potential problem with this technique is the use of lentiviral vectors to introduce a new gene into the HSCs.  Because lentiviruses are retroviruses, they insert their DNA into the genome of the host cells.  Such insertions can produce mutations, and it will be incumbent on Kohn and his colleagues to carefully screen each transformed HSC line to ensure that the insertion is not problematic and that the transformed cells are not sick or potentially tumorous.  However, such a vector is necessary in order to ensure permanent residence of the newly-introduced gene.

Even with these caveats, Kohn’s SCD treatment should go forward, and we wish all the best to Dr. Kohn, his team, and to the patients treated in this trial.

Researchers from the University of Minnesota Use Genetically Corrected Stem Cells To Repair Muscles

University of Minnesota researchers from the Lillehei Heart Institute have combined genetic engineering techniques to repair mutations in abnormal muscle cells with cellular reprogramming to generate stem cells that can repair and regenerate muscle regeneration in a mouse model for Duchenne Muscular Dystrophy (DMD). This research is a proof-of-principle experiment that determines the feasibility of combining induced pluripotent stem cell technology and genetic engineering techniques that correct mutations to treat muscular dystrophy. Experimental strategies such as these could represent a major step forward in autologous cell-based therapies for DMD. Furthermore, it might pave the way for clinical trials to test this approach in reprogrammed human pluripotent cells from muscular dystrophy patients.

University of Minnesota researchers combined three groundbreaking technologies to achieve effective muscular dystrophy therapy in a mouse model of DMD. First, researchers reprogrammed skin cells into induced pluripotent stem cells (iPSCs). iPSCs are capable of differentiating into any of the mature cell types within an adult organism. In this case, the University of Minnesota researchers generated pluripotent cells from the skin of mice that carry mutations in two genes; the dystrophin and utrophin genes. Mice with mutations in both the dystrophin and utrophin genes develop a severe case of muscular dystrophy that resembles the type of disease observed in human DMD patients. This provided a model system platform that successfully mimicked what would theoretically occur in humans.

The second technology employed is a genetic correction tool developed at the University of Minnesota. In this case, they used a transposon, which is a segment of DNA that can jump from one location to another within the genome. The specific transposon used is the “Sleeping Beauty Transposon.” The use of this transposon allowed them to transport genes into cells in a convenient manner. The Lillehei Heart Institute researchers used the Sleeping Beauty transposon to deliver a gene called “micro-utrophin” into the iPSCs made from the DMD mice.

Sleeping Beauty Transposon

Human micro-utrophin can support muscle fiber strength and prevent muscle fiber injury throughout the body. However, there is one essential difference micro-utrophin and dystrophin: dystrophin is absent in muscular dystrophy patients, but if it is introduced into the bodies of DMD patients, their immune system will initiate a devastating immune response against it. However, in those same patients, utrophin is active and functional, which makes it essentially “invisible” to the immune system. This invisibility allows the micro-utrophin to replace dystrophin build and repair muscle fibers within the body.


The third technology utilized is a method to produce skeletal muscle stem cells from pluripotent cells. This procedure was developed in the laboratory of Rita Perlingeiro, who is also the principal investigator of this latest study.

Rita Perlingeiro Ph.D.
Rita Perlingeiro Ph.D.

Perlingeiro’s technology gives pluripotent cells a short pulse of a muscle stem cell protein called Pax3, which nudges the pluripotent cells to become skeletal muscle stem cells, which can then be exponentially expanded in culture. These Pax3-induced muscle stem cells were then transplanted back into the same strain of DMD mice from which the pluripotent stem cells were originally derived.

Pax3 and 7

When combined, these platforms created muscle-generating stem cells that would not be rejected by the body’s immune system. According to Perlingeiro, the transplanted cells performed very well in the dystrophic mice, and they generated functional muscle and responded to muscle fiber injury.

“We were pleased to find the newly formed myofibers expressed the markers of the correction, including utrophin,” said Perlingeiro, a Lillehei endowed scholar within the Lillehei Heart Institute and an associate professor in the University of Minnesota Medical School. “However, a very important question following transplantation is if these corrected cells would self-renew, and produce new muscle stem cells in addition to the new muscle fibers.”

By injuring the transplanted muscle and watching it repair itself, the researchers demonstrated that the transplanted muscle stem cells endowed the recipient mice with fully functional muscle cells. This latest project provides the proof-of-principle for the feasibility of combining induced pluripotent stem cell technology and genetic correction to treat muscular dystrophy.

“Utilizing corrected induced pluripotent stem cells to target this specific genetic disease proved effective in restoring function,” said Antonio Filareto, Ph.D., a postdoctoral fellow in Perlingeiro’s laboratory and the lead author on the study. “These are very exciting times for research on muscular dystrophy therapies.”

These studies pave the way for testing this approach in a clinical trial that would use reprogrammed human pluripotent cells from muscular dystrophy patients.

According to Perlingeiro, “Developing methods to genetically repair muscular dystrophy in human cells, and demonstrating efficacy of muscle derived from these cells are critical near-term milestones, both for the field and for our laboratory. Testing in animal models is essential to developing effective technologies, but we remained focused on bringing these technologies into use in human cells and setting the stage for trials in human patients.”

This research was published in Nature Communications.