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.

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.

Urinary Stem Cells and Their Therapeutic Potential


Yuanyuan Zhang, assistant professor of regenerative medicine at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine, has extended earlier work on stem cells from urine that suggests that these cells might be more therapeutically useful than previously thought.

These urinary stem cells can be isolated from a patient’s urine sample, and they can be induced, in the laboratory, to form bladder-type cells; smooth muscle and urothelial (bladder-lining) cells. Such stem cells could certainly be used to treat urinary tract problems, even though a good deal more work is required to confirm that they can do just that.

Nevertheless, Zhang and his co-workers have discovered that these urinary tract stem cells are much more plastic than previously thought. In the laboratory, Zhang and others have managed to differentiate urinary tract stem cells into bone, cartilage, fat, skeletal muscle, nerve, and endothelial cells (the cells that line blood vessels). This suggests that urine-derived stem cells could be used in a variety of therapies.

USCs undergo multipotential differentiation in vitro. (a-c) endothelial differentiation of USCs. USCs (p3) were induced to endothelial lineage by culture in EBM-2 medium containing VEGF 50 ng/ml for 14 days. (a) In vitro vessel formation. Endothelial differentiated USCs were cultured on Matrigel for 18h to form branched networks (angiogenesis) and tubular structures. Scale bar = 100μm. (b) Expression analysis of endothelial-specific transcripts by RT-PCR. (c) Immunofluorescence staining using endothelial-specific markers revealed enhanced staining of the markers with differentiation (middle row) compared to the non-treated control (top row). Scale bar = 50μm.
USCs undergo multipotential differentiation in vitro. (a-c) endothelial differentiation of
USCs. USCs (p3) were induced to endothelial lineage by culture in EBM-2 medium containing
VEGF 50 ng/ml for 14 days. (a) In vitro vessel formation. Endothelial differentiated USCs were
cultured on Matrigel for 18h to form branched networks (angiogenesis) and tubular structures. Scale
bar = 100μm. (b) Expression analysis of endothelial-specific transcripts by RT-PCR. (c)
Immunofluorescence staining using endothelial-specific markers revealed enhanced staining of the
markers with differentiation (middle row) compared to the non-treated control (top row). Scale bar =
50μm.

Zhang said that urinary tract stem cells could be used to treat urological disorders such a kidney disease, urinary incontinence, and erectile dysfunction. However, Zhang is optimistic that they can also be used to treat a wider variety of treatment options, such as making replacement bladders, urine tubes, and other urologic organs.

Since these stem cells come from the patient’s own body, they can have a low chance of being rejected by the immune system. Also, they do not cause tumors when implanted into laboratory animals.

In their latest work, Zhang and his colleagues obtained urine samples from 17 healthy individuals whose ages ranged from five to 75 years old. Even though these stem cells are only one of a large collection of cells in urine, isolating urinary stem cells from urine only requires minimal processing.

A single USC (inset) is followed through different passages (p0-p12). The cells were expanded to a colony were cultured in KSFM-EFM medium with 5% serum and images recorded with passage. Images shown at x100
A single USC (inset)
is followed through different passages (p0-p12). The cells were expanded to a colony were cultured in
KSFM-EFM medium with 5% serum and images recorded with passage. Images shown at x100

In the laboratory, Zhang and his team differentiated the cells into derivatives of all three embryological layers (endoderm – skin and nervous tissue; mesoderm – bone, muscle, glands, and blood vessels; and endoderm – digestive system).

Differentiation of one USC clone into UCs and SMCs. (a) USCs (p3) t were used to differentiate into two distinct lineages. Culture in SMCs-lineage differentiation (2.5 ng/ml TGF-􀈕1 and 5 ng/ml PDGF-BB) and UCs-lineage differentiation (30 ng/ml EGF) medium was used for 14 days.
Differentiation of one USC clone into UCs and SMCs. (a) USCs (p3) t were used to
differentiate into two distinct lineages. Culture in SMCs-lineage differentiation (2.5 ng/ml TGF-􀈕1 and
5 ng/ml PDGF-BB) and UCs-lineage differentiation (30 ng/ml EGF) medium was used for 14 days.

After showing the multipotent nature of urinary tract stem cells in the laboratory, Zhang and others took smooth muscle cells and urothelial cells made from urinary tract stem cells and transplanted them into mice with tissue scaffolds that had been made from decellularized pig intestine. The scaffolds only had extracellular molecules and not cells. After one month, the implanted cells had formed multi-layered, tissue-like structures.

USCs were infected with BMP9 or control GFP and were injected subcutaneously into nude mice. i) Bony masses were only observed in mice implanted with BMP-transduced USCs at week 4. ii) The harvested bony masses were subjected to microCT imaging revealing the isosurface (left) and density heat maps (right).
USCs were infected with BMP9 or control GFP and were
injected subcutaneously into nude mice. i) Bony masses were only observed in mice implanted with
BMP-transduced USCs at week 4. ii) The harvested bony masses were subjected to microCT imaging
revealing the isosurface (left) and density heat maps (right).

Urinary tract stem cells or as Zhang calls them, urine-derived stem cells or USCs, have many cell surface characteristics of mesenchymal stem cells from bone marrow, but they are also like pericytes, which are cells on the outside of small blood vessels. Zhang and others suspect that USCs come from the upper urinary tract, including the kidney. Patients who have had kidney transplants from male donors have USCs with a Y chromosome in them, which suggests that the kidney is a source or one of the sources of these cells.

Determination of USC source. Several clones of USCs (p3) were cultured and analyzed for expression of kidney-lineage marker. (a) FISH (left) and amelogenin gene PCR analysis (right) analysis of USCs isolated from urine obtained from a male donor-to-female recipient kidney transplant for presence of Y-chromosome (L: DNA ladder, M: male control, F: female control, A4: USC from male donor-to-female recipient urine sample, N: negative control).
Determination of USC source. Several clones of USCs (p3) were cultured and analyzed for
expression of kidney-lineage marker. (a) FISH (left) and amelogenin gene PCR analysis (right)
analysis of USCs isolated from urine obtained from a male donor-to-female recipient kidney transplant
for presence of Y-chromosome (L: DNA ladder, M: male control, F: female control, A4: USC from
male donor-to-female recipient urine sample, N: negative control).

Even more work needs to be done before we can truly become over-the-moon excited about these cells as a source of material for regenerative medicine, Zhang’s work is certainly an encouraging start.

See Shantaram Bharadwaj, et al., Multi-Potential Differentiation of Human Urine-Derived Stem Cells: Potential for Therapeutic Applications in Urology. Stem Cells 2013 DOI: 10.1002/stem.1424.

Long-Lasting Blood Vessels Regenerated from Reprogrammed Human Cells


Researchers from Massachusetts General Hospital (MGH) in Boston, MA have used human induced pluripotent stem cells to make vascular precursor cells to produce functional blood vessels that lasted as long as nine months.

Rakesh Jain, director of the Steele Laboratory for Tumor Biology at MGH and his team derived human induced pluripotent stem cells (iPSCs) from adult cells of two different groups of patients. One group of individuals were healthy and the second group had type 1 diabetes. Remember that iPSCs are derived from adult cells through the process of genetic engineering. By introducing specific genes into these adult cells, the adult cells are de-differentiated into an embryonic-like state. The embryonic-like cells can be cultured and grown into a cell line that can be differentiated into various cell types in the laboratory. These differentiated cells types can then be transplanted into laboratory animals for regenerative purposes.

“The discovery of ways to bring mature cells back to a ‘stem-like’ state that ca differentiate into many different types of tissue has brought enormous potential to the field of cell-based regenerative medicine, but the challenge of deriving functional cells from these iPSCs still remains,” said Rakesh. “our team has developed an efficient method to generate vascular precursor cells from human iPSCs and used them to create networks of engineered blood vessels in living mice.”

The ability to regenerate or repair blood vessels could be a coup for regenerative medicine. Cardiovascular disease, for example, continues to be the number one cause of death in the United States and other conditions caused by blood vessel damage (e.g., the vascular complications of diabetes) continue to cause a great deal of morbidity and mortality each year. Also, providing a blood supply to newly generated tissue remains one of the greatest barriers to building solid organs through tissue engineering.

Some studies have used iPSCs to build endothelial cells (the cells that line blood vessels), and connective tissue cells that provide structural support. These cells, unfortunately, tend to not produce long-lasting vessels once they are introduced into laboratory animals. A collaborator with Jain, Dai Fukumura, stated, “The biggest challenge we faced during the early phase of this project was establishing a reliable protocol to generate endothelial cell lines that produced great quantities or precursor cells that could generate durable blood vessels.”

Jain’s group adapted a protocol that was originally designed to derived endothelial cells from human embryonic stem cells. They isolated cells based on the presence of more than one cell surface protein that marked out vascular potential. Then they expanded this population of cells using a culture system developed with embryonic stem cells that had been differentiated into endothelial cells. Further experiments confirmed that only those iPSCs that expressed all three cell surface proteins on their surfaces had the potential to form blood vessels.

To test the capacity of those cells to generate blood vessels, they implanted them onto the surface of the brain of mice in combination with mesenchymal precursors that generate smooth muscles that surround blood vessels.

Within two weeks after transplantation, the implanted cells had formed entire networks of blood vessels with blood flowing through them that has also fused with the already existing blood vessels. These engineered blood vessels continued to function for as long as 280 days in the living animal. Implantation under the skin, however, was a different story. It took 5 times the number of cells to get them to form blood vessels and they were short-lived. This is similar to the results observed in other studies.

Because type 1 diabetes can ravage blood vessels, Jain’s team made iPSCs from patients with type 1 diabetes to determine if iPSCs from such patients would generate functional blood vessels. Similarly to the cells generated from healthy individuals, vascular precursors generated from type 1 diabetics were able to form long-lasting blood vessels. However, these same cell lines showed variability in their ability to form vascular precursors. The reason for this is uncertain.

Rekha Samuel, one of the lead authors of this paper, said “The potential applications of iPSC-generated blood vessels are broad – from repairing damaged vessels supplying the heart or brain to preventing the need to amputate limbs because of the vascular complications of diabetes. But first we need to deal with such challenges as the variability of iPSC lines and the long-term safety issues involved in the use of these cells, which are being addressed by researchers around the world. We need better ways of engineering the specific type of endothelial cell needed for specific organs and functions.”

Exporting Tissue Engineering


Professor György K.B. Sándor from the Finland Distinguished Professor Program or FiDiPro believes that tissue engineering has the ability to become a new global export item.

FiDiPro is a joint funding program of the Academy of Finland and Tekes (the Finnish Funding Agency for Technology and Innovation) that enables top researchers to do work in Finland for a fixed period of time.

Sándor is a Canadian professor who specializes in oral and maxillofacial surgery who has participated in FiDIPro. Sandor’s research program examines bone regeneration, hyperbaric oxygen, tissue regeneration, and stem cells. He works at the BioMediTech research institute which is run by the University of Tampere and the Tampere University of Technology. BioMediTech is an innovation center that combines biomedical research with new technologies.

The goal of Sándor’s research program is to produce bone and cartilage by means of tissue engineering techniques that grow tissue-derived stem cells. Some people are missing bone at birth as a result of a developmental disorder, or, in some cases, bone defects from accidents, and various inflammatory diseases can cause bone loss. Particular surgeries that require bone removal can also cause bone loss,

Tissue engineering can produce tailored, living spare parts for human beings. If the protocols and methods of tissue engineering can be up-scaled appropriately, it could become the third alternative form of treatment alongside more traditional forms of treatment for such conditions that include surgery and drug treatments.

“Tissue-derived stem cells can be isolated from the patient’s own tissue. In that way, they will not cause a rejection reaction. Compared to tissue stem cells, human embryonic stem cells have a greater ability to differentiate into different cell types, In practice, that means that all cell types can be used,” Sándor said.

Sandor noted that Finland is a forerunner in developing bone engineering techniques. “At the moment expertise in the field is concentrated in Finland, but it also generated global interest in other medically advanced countries,” said Sándor .

In the near future, large numbers of patients might travel to Finland to receive tissue engineering-based treatments. As such forms of treatment increase and are perfected, expertise in tissue engineering can be exported for use on a larger scale.

“We have proven with more than 20 clinically successful operations that tissue engineering works,” Sándor said.

Sándor considers the research community in Tampere to be unique when it comes to the way it is run and functions. One of the key reasons why Sandor decided to stay and continue his research in Finland even after his experience with FiDiPro came to an end.

“In the field, BioMediTech is a unique concentration of researchers and expertise. In the Pirkanmaa region, also the cooperation between research, industry, and administration works well. That enables efficient decision-making which, in turn, contributes to the creation of new innovations,” he said.

“Cooperation with colleagues is smooth too. That was the determining factor in my decision to stay in Finland. Each day is like a new adventure.”