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.


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.

Stem Cell-Tweaking Drug Might Treat Osteoporosis

A research group from the Florida campus of The Scripps Research Institute (TSRI) has identified a new therapeutic approach that could promote the development of new bone-forming cells in patients suffering from bone loss.

The study was published in the journal Nature Communications, and it focused on a protein called PPARγ, which is a master regulator of fat, and the impact of this molecule on the fate of mesenchymal stem cells derived from bone marrow. Since these mesenchymal stem cells can differentiate into several different cell types, including fat, connective tissues, bone and cartilage. Consequently mesenchymal stem cells have a number of potentially important therapeutic applications.

A partial loss of PPARγ in a genetically modified mouse model led to increased bone formation. Could the use of drugs to inhibit PPARγ and potentially mimic that effect? This group combined a variety of structural biology approaches and then tried to design drugs that could fit PPARγ. This type of strategy is called “rational design,” and this yielded a new compound that could repress the biological activity of PPARγ.

The new drug, SR2595 (SR=Scripps Research), when applied to mesenchymal stem cells, significantly increased bone cell or osteoblast formation, a cell type known to form bone.

“These findings demonstrate for the first time a new therapeutic application for drugs targeting PPARy, which has been the focus of efforts to develop insulin sensitizers to treat type 2 diabetes,” said Patrick Griffin, chair of the Department of Molecular Therapeutics and director of the Translational Research Institute at Scripps Florida. “We have already demonstrated SR2595 has suitable properties for testing in mice; the next step is to perform an in-depth analysis of the drug’s efficacy in animal models of bone loss, aging, obesity and diabetes.”

In addition to identifying a new, potential therapeutic use for bone loss, this study may have even broader implications.

“Because PPARG is so closely related to several proteins with known roles in disease, we can potentially apply these structural insights to design new compounds for a variety of therapeutic applications,” said David P. Marciano, first author of the study, a recent graduate of TSRI’s PhD program and former member of the Griffin lab. “In addition, we now better understand how natural molecules in our bodies regulate metabolic and bone homeostasis, and how unwanted changes can underlie the pathogenesis of a disease.” Marciano will focus on this subject in his postdoctoral work in the Department of Genetics at Stanford University.

A Small RNA that Increases Bone Formation in Osteoporotic Bone-Making Cells

We normally think of bone as a very static tissue that does not change very much. However bone is actually a very dynamic tissue is constantly being remodeled in response to the needs of the organism. Bone remodeling is mediated by two different types of cells: osteoblasts that build bone and osteoclasts that resorb bone. Osteoblasts are derived from mesenchymal stem cells in the stroma of the bone marrow. The differentiation of mesenchymal stem cells into osteoblasts is mediated by molecules made by bone cells when bone is damaged. Osteoclasts come from pre-osteoclast cells that are monocyte-derived cells that fuse into multinucleate osteoclasts in response to the death of osteocytes (bone cells).

In healthy bone, osteocytes secrete a molecule called sclerostin, which prevents any new bone deposition. A break in bone causes the death of osteocytes near the site of the break, and the nearby osteocytes stop secreting sclerostin and start producing growth factors, nitric oxide and prostaglandins.

Bone deposition

The lining cells of the bone marrow cavity detach and fuse with blood vessels. The mesenchymal stromal cells, under influence from IL-1, become pre-osteoblasts, and they start to secrete M-CSF, which prepares the pre-osteoclasts to fuse and become multinucleate osteoclasts. Pre-osteoclasts then express a molecule called RANKL, which binds to the RANK receptor on the surface of pre-osteoclasts and this induces them to fuse, and become mature osteoclasts. The osteoclasts secrete acid and cathepsin K to dissolve the damaged bone. The osteoclasts stop eating bone when the pre-osteoblasts mature into full-fledged osteoblasts that stop making RANKL and start making OPG, which binds to RANK, but does not activate it. Without this stimulation, the osteoclasts die. Then the osteoblasts divide, fill the cavity made by the now-deceased osteoclasts, and remake the bone. Some of the osteoblasts become entrapped in the bone matrix and become osteocytes. The bone takes several months to remineralize and 3-4 years to completely remineralize.  See here for a video of this.

Bone resorption-deposition

If there is a relative increase in bone resportion relative to bone deposition, the result is fragile, poorly mineralized bones, and this condition is known as osteoporosis. Decreased bone mass and bone strength causes an increased incidence of bone fractures, which often leads to further disability and early mortality. Bone healing is also impaired.

To treat osteoporosis, clinicians usually prescribe anti-resorptive agents that exert their effect by decreasing the rate of bone resorption. This strategy, however, has drawbacks, since as noted above, bone deposition relies on bone resorption. Inhibition of bone resorption also inhibits bone deposition, and bone tends to remain static and heal poorly.

A new paper has examined osteoporosis from the perspective of osteoblasts. It has been well established that in osteoblasts function is diminished in osteoporotic patients. Therefore increase osteoblast function is of chief interest. Work from the laboratories of Jihua Chen and Yan Jin from the Fourth Medical University has shown that a miniature RNA molecule called miR-26a plays a critical role in modulating bone formation during osteoporosis. Chen and Jin and others discovered that miR-26a treatment of mesenchymal stem cells effectively improved the osteogenic differentiation capability of these mesenchymal stem cells. In these experiments, they isolated mesenchymal stem cells from female mice that had their ovaries removed. Such mice are prone to undergo osteoporosis because they lack the hormone estrogen that stimulates osteoblast function. When these stem cells were treated with MiR-26a, they increased their bone-making capacities by in culture and when injected into live mice.

Further work showed that MiR-26a directly targets a gene called Tob1. Tob1 negatively regulates the BMP/Smad signaling pathway, and MiR-26a binds to the rear mRNA (3′-untranslated region) of Tob1, and prevents Tob1 translation.

These findings indicate that miR-26a is a potentially promising therapeutic candidate to enhance bone formation in order to treat osteoporosis and to promote bone regeneration in osteoporotic fracture healing.

For the article, go here.