Genetically Engineered Stem Cells to Treat Osteoporosis in Mice


Osteoporosis is a nasty condition characterized by weak and brittle bones often leading to devastating bone fractures and other injuries. Unfortunately, millions of people worldwide have been diagnosed with osteoporosis.

Osteoporosis

Contrary to popular belief, out bones are dynamic organs that undergo constant remodeling consisting of bone resorption and renewal. However, once bone resorption rates outpace bone renewal, bone densities decrease, which puts bones at risk of fractures. Medical researchers are would like to find new ways to not only discourage bone resorption, but generate new bone material to replace demineralized bone. Ideally, therapies would rejuvenate bone growth so that it the bone reverts back to its original density levels.

Now a promising strategy to accomplish this goal is relies on stem cell therapy. A collaborative study by Xiao-Bing Zhang and his colleagues from Loma Linda University and Jerry L. Pettis from the Memorial VA Medical Center has built on their prior work with genetically modified hematopoietic stem cells (HSCs) that identified a growth factor that caused a 45% increase in bone strength in mouse models. This work was published in the journal Proceedings of the National Academy of Sciences, USA.

Zhang and his coworkers wanted to find a gene therapy that promotes bone growth while minimizing side effects. To that end, Zhang’s group focused on a growth factor called PGDFB or “platelet-derived growth factor, subunit B.” The properties of this growth factor make it a promising candidate, since it is already FDA approved for treating bone defects in the jaw and mouth.

platelet-derived growth factor, subunit B
platelet-derived growth factor, subunit B

First, Zhang and others isolated HSCs from the bone marrow of donor mice. HSCs were chosen because they can be given intravenously, after which they will home in to one of the major sites of bone loss (the endosteal bone surface). The isolated HSCs were then genetically engineered to overexpress the growth factor PGDFB. Experimental mice were then irradiated to wipe out their own HSCs, and then these same mice were transplanted with the modified HSCs.

After four weeks, the upper leg bones of the mice (femur) were tested. Zhang and his colleagues found that PGDFB promoted new trabecular bone formation, but because the PGDFB was expressed at high levels, it negatively affected bone mineral density. Zhang and others then used weaker promoters to optimize the dosage of PGDFB expression in the HSCs. They discovered that the phosphoglycerate kinase promoter (PGK) worked well to mitigate the amount of PGDFB that is expressed in cells. When these HSCs were transplanted into irradiated mice, they observed increases in trabecular bone volume, thickness, and number as well as increases in connectivity density. Additionally, cortical bone volume increased by 20-30% while cortical porosity was reduced by 40%. Importantly, the lower dosage of PGDFB resulted in no observed decreases in bone mineral density due to osteomalacia or hyperparathyroidism.

These treated femurs and a control sample underwent three-point mechanical testing to test the integrity of the new bone. The PGK-PGDFB-treated femur displayed a 45% increase in maximum load-to-failure in the midshaft of the femur and a 46% increase in stiffness, indicating quality bone formation. Thus the new bone that is deposited it also of high quality.

The next step in this work would like to determine why this combination of a PGK promotor and PDGFB worked so well. Zhang and others have discovered that PDGFB promotes bone marrow mesenchymal stem cell formation and angiogenesis, which are two important factors in bone growth. They also found that optimizing the dosage of PDGFB is quite important for promoting osteoblast (bone-forming) cell formation.

Finally Zhang’s group investigated how osteoclastogenesis, or the creation of cells that reabsorb bone (osteoclasts) is affected by PDGFB with a PGK promotor. The treated femurs also had an increase in biomarkers for osteoclasts. This increase in both osteoblasts and osteoclasts indicates that the treated bones undergo the normal bone rebuilding and remodeling cycle.

Overall, this research provides a compelling investigational pathway for future cell therapies to treat osteoporosis. Mouse models show a fast-acting technique that result in bone formation and increasing bone strength.

Converting Mesenchymal Stem Cells to Bone Makers


Within human bone, cells called osteoblasts make new bone and without the constant activity of osteoblasts, bone becomes thin and fragile. Osteoblasts are derived from mesenchymal stem cells in the bone marrow. When bones break, orthopedic surgeons try to use growth factors to push more mesenchymal stems and their progeny to become osteoblasts. The growth factor in question is bone morphogen protein (BMP). BMP, however, does not work consistently, and it has some rather nasty side effects (cancer, The specific complications that are drawing the most concern include swelling in the neck and throat, radiating leg pain, and male sterility). Therefore, an alternative method for converting mesenchymal stem cells into osteoblasts is highly desirable.

Kurt Hankenson from the University of Pennsylvania School of Veterinary Medicine has worked on this very problem and described the situation this way, “In the field, we’re always searching for new ways for progenitor cells to become osteoblasts so we became interested in the Notch signaling pathway.” When it comes to BMP, Hankenson said, “it has become clear that BMPs have some issues with safety and efficacy.”

Is there a better way to make bone? There seems to be. A protein called Jagged-1 has been shown by Hankenson’s team to be highly expressed in bone. Jagged-1 is a component of the widely used Notch signaling pathway, which is found in the nervous system and in many other cells as well.

In mouse stem cells, introducing Jagged-1 blocks the progression of mesenchymal stem cells to osteoblasts. This finding has actually hampered osteoblast research for the last two years. Hankenson again, “That had been our operating dogma for a year or two.”

However, as is so often the case in science, you never truly know the result of an experiment until you actually do it. When Jagged-1 was added to human mesenchymal stem cells, the results were very different. Hankenson said, “It was remarkable to find that just putting the cells onto the Jagged-1 ligand seemed sufficient for driving the formation of bone-producing cells.”

From a developmental genetics perspective, this makes perfect sense, since mutations in the Jagged-1 gene cause an inherited disease known as Alagille syndrome which causes liver problems, abnormal metabolisms, and fragile bones that break easily. Also, genome-wide association studies have shown that particular versions of the Jagged-1 gene cause low bone density.

Hankenson and his collaborators are examining ways to manipulate the levels of the Jagged-1 protein in patients with bone problems. To that end, Hankenson is collaborating with Kathleen Loomes of Penn’s Perelman School of Medicine and the Children’s Hospital of Pennsylvania to study pediatric patients with Alagille syndrome to determine if bone abnormalities in these patients are indeed connected to Jagged-1 malfunctions.

Hankenson and his former graduate student Mike Dishowitz started a company called Skelegen through the University of Pennsylvania’s Center for Technology Transfer (CTT) UPstart program. The goal of Skelegen is to develop and improve a system for delivering Jagged-1 to sites that require new bone growth in the hopes of treating bone fractures and other skeletal problems.

See Fengchang Zhu et al., “Pkcdelta is required for Jagged-1 induction of hMSC osteogenic differentiation.” Stem Cells 2013; DOI 10.1002/stem.1353.

A New Hybrid Molecule Directs Mesenchymal Stem Cells To Increase Bone Formation and Bone Strength


Osteoporosis is a disease that affects bone and results from aging or a lack of estrogen. Osteoporotic bone is less dense than normal bone, and the loss of bone density leads a tendency for bones to fracture easily. In particular, the bones of the wrist, hip, or back can fracture and fortunately, bone scans can help diagnose osteoporosis earlier and earlier.

Typically, osteoporosis is treated by prescribing a group of drugs collectively known as the “bisphosphonates.” These drugs have a common mode of action that includes one of the two cells involved in bone remodeling and healing. Cells called “osteoblasts” act as bone-building cells. Osteoblasts come from bone marrow (the squishy stuff inside your long bones), and they make new bone called “osteoid” that consists of a protein called “collagen” and a few other proteins. Then they deposit calcium and other minerals onto the protein matrix. After filling a cavity with bone, the osteoblasts flatten and line the cavity where they regulate the movement of calcium into and from the bone. Some of the osteoblasts become trapped in the bone while it is being deposited and they extend long extensions and become known as “osteocytes.” Osteocytes monitor the bone health and signal when there are breaks in the bone.

Osteoblasts

The second cell involved in bone remodeling is the osteoclast. Osteoclasts are large cells with many nuclei that dissolve existing bone. When a bone is broken, the osteocytes signal to each other and recruit osteoclasts to the site of the bone break. Osteoclasts dissolve the broken bone, and this gives room to the osteoblasts so that they can deposit new bone. The activities of both cell types are essential for bone healing and remodeling. The activities of these two cell types are also very carefully regulated.

When osteoblast activity is too high, a disease called “osteopetrosis” ensues, and this disease squeezes out the bone marrow and prevents the synthesis of enough blood cells. When osteoclast activity is too high, osteoporosis ensues, and bone density decreases so that fractures are a genuine possibility. Bisphosphonates bind to the surface of osteoclasts and prevent them from destroying bone. However, since both osteoclasts and osteoblasts are required for proper bone health, bisphosphonates essentially cause bone deposition to come to a stand-still. For this reason, some people experience increased fractures on bisphosphonates. What is needed is a treatment that can reverse the thinning of the bones and increase bone density.

A very interesting study led by scientists at the UC Davis Heath System examined a mouse model of osteoporosis to test the efficacy of a new treatment that can potentially increase bone density. If the results of this study are confirmed by further work, it could revolutionize osteoporosis treatments. The UC Davis team developed a novel technique to enhance bone growth by injecting a specific molecule into the bloodstream that guides mesenchymal stem cells to bone surfaces. Once there, these stem cells differentiate into osteoblasts, which promote bone growth.

Wei Yao, the principal investigator and lead author of the study said: “There are many stem cells, even in elderly people, but they do not readily migrate to bone. Finding a molecule that attaches to stem cells and guides them to the targets we need is a real breakthrough.”

Even though there is a great deal of research to develop stem cell-based treatments for many conditions and injuries that range from peripheral artery disease and macular degeneration to blood disorders, skin wounds and diseased organs, directing stem cells to travel and adhere to the surface of bone for bone formation has been among the elusive goals in regenerative medicine. To accomplish this, Yao and others used a unique hybrid molecule, LLP2A-alendronate that consists of two parts: the LLP2A part that attaches to mesenchymal stem cells in the bone marrow, and a second part that consists of the bone-homing bisphosphonate-class drug, alendronate (trade name – Fosamax). LLP2A-alendronate was injected into the bloodstream, and it bound to the cell surfaces of mesenchymal stem cells in the bone marrow and directed those cells to the surfaces of bone, where the stem cells carried out their natural bone-formation and repair functions.

The study shows that stem-cell-binding molecules can be exploited to direct stem cells to therapeutic sites inside an animal. One author even said. It represents a very important step in making this type of stem cell therapy a reality.

Twelve weeks after the LLP2A-alendronate was injected into mice, bone mass in the femur (thigh bone) and vertebrae (in the spine) increased and bone strength improved compared to control mice who did not receive LLP2A-alendronate. The treated mice were older mice that normally showed a particular degree of bone loss, but with this treatment, they had improved bone formation, as did those that were models for menopause.

Even though alendronate is commonly prescribed to women with osteoporosis to reduce the risk of fracture, it was used in this study because it goes directly to the bone surface, where it slows the rate of bone breakdown. The alendronate dose in this experiment was very low and was, therefore, unlikely to have inhibited LLP2A’s therapeutic effect.

Co-investigator on the study and director of the UC Davis Musculoskeletal Diseases of Aging Research Group, Nancy Lane, noted: “For the first time, we may have potentially found a way to direct a person’s own stem cells to the bone surface where they can regenerate bone. This technique could become a revolutionary new therapy for osteoporosis as well as for other conditions that require new bone formation.”

Mesenchymal stem cells from bone marrow induce new bone remodeling, which thicken and strengthen bone. The potential use of this stem cell therapy is not limited to treating osteoporosis, since it may prove invaluable for other disorders and conditions that could benefit from enhanced bone rebuilding, which includes bone fractures, bone infections or cancer treatments.

Jan Nolta, professor of internal medicine, an author of the study and director of the UC Davis Institute for Regenerative Cures opined, “These results are very promising for translating into human therapy. We have shown this potential therapy is effective in rodents, and our goal now is to move it into clinical trials.”

Paper citation: “Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass;” Min Guan, Wei Yao, Nancy E Lane et al.; Nature Medicine, 2012; DOI: 10.1038/nm.2665.