Abstract
The skeletal system is vitally important for supporting the body, protecting internal organs, and facilitating locomotion. Bone undergoes remodeling in response to different stimuli. This dynamic process is regulated by the interaction of two key regulators: the bone-building osteoblasts and the bone-resorbing osteoclasts. Under homeostasis, the activity of osteoblasts and osteoclasts is intimately balanced.
Interestingly, while osteoclasts and osteoblasts carry its function on the same tissue, they are derived from two distinct lineages. Osteoclasts are derived from hematopoietic stem cells, while osteoblasts are derived from skeletal stem and progenitor cells (SSPCs). Characterization of SSPCs performed in Dr. Fernando Fierro’s lab reveal unique properties, in which intravenously infused SSPCs can home to the bone and bone marrow and also participate in the repair of a monocortical fracture by mineralizing new bone. Therefore, with the potential to regenerate bone, SSPCs presents tremendous promise as a cellular therapy to address unmet medical needs in orthopaedics, including treatment of segmental bone defects, osteoporosis, and rare genetic disorders such as Snyder-Robinson syndrome. In addition, SSPCs are likely to have a prognostic value for diseases associated with SSPCs, in which either the quantity or quality of these cells may serve as biomarkers for disease progression.
Osteoporosis is characterized by a reduction in bone density that causes the bone to become brittle. The disease affects over half of the population that are over 50 years old, with approximately 80% of those affected being post-menopausal women. It is estimated that the disease is associated with over two million broken bones every year. For many patients suffering from osteoporosis-related fractures, their quality of life diminishes significantly since fractures can occur from simple movements such as stretching and coughing.
Current treatments for osteoporosis focus on limiting the progression of the disease by prescribing osteoclast inhibitors such as bisphosphonates. However, an anabolic intervention is required to regenerate the skeleton. To test the potential of SSPCs to treat osteoporosis, we first generated an osteoporotic mouse model by subjecting immune deficient NOD/SCID IL2Rγ-/- mice to surgical ovariectomy. We then isolated human SSPCs from effluent of autologous bone graft preparations and injected the SSPCs via tail vein. After 8 weeks, microcomputed tomography (microCT) analysis revealed a significant reduction in bone density in the vertebrae but not the femur. Ovariectomized mice treated with SSPCs showed a trend towards increasing bone density in vertebrae.
Snyder-Robinson Syndrome (SRS) is a rare X-linked disorder caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Amongst other symptoms, SRS patients develop musculoskeletal impairments including osteoporosis and low weight gain. Although the pathogenesis of SRS remains unclear, previous studies from the Fierro lab found that silencing SMS expression in mesenchymal stromal cells inhibited cell proliferation and reduced bone formation both in vitro and in vivo. This suggests that osteoporosis observed in SRS patients is attributed to impaired differentiation of SSPCs into osteoblasts.
Therefore, SRS presents as a good candidate to be treated using cellular therapy with SSPCs. To test this, we developed a mouse model for SRS using the cre-lox system in transgenic mice. Like SRS patients, the SMS conditional knockout (cKO) mice exhibited similar features as demonstrated by reduced body weight and impaired improvement on the rotarod. In a preliminary study, we isolated and injected 1000 murine SSPCs and subjected the vertebrae and the femur to microCT analysis. We did not find statistically significant differences in bone density in between groups, but we observed a small decrease in bone density in the femur of SMS cKO mice as compared to controls. The study is currently being repeated.
While SRS is characterized by a loss-of-function mutation in SMS, Fibrous dysplasia/McCune-Albright Syndrome (FD/MAS) is characterized by a gain-of-function mutation in the guanidine nucleotide alpha stimulating gene. FD/MAS patients develop fibrotic bone tissue instead of normal bone tissue. Due to its clinical presentation, FD/MAS can be often misdiagnosed as osteosarcomas. To improve the diagnosis of FD/MAS, we isolated SSPCs from FD/MAS patients and subjected the cells to single cell RNA sequencing to identify putative biomarkers. While we consistently isolated SSPCs and endothelial cells, we also identified a cell population that so far, we have been unable to associate with any particular cell type. The transcriptome of this cell cluster is consistent with cells undergoing impaired osteogenesis, although a more thorough bioinformatic analysis is still pending.
Although some of our studies have been inconclusive, they shed light toward different pathologies and encouraged the use of SSPCs for therapeutic and diagnostic purposes. This work is therefore a proof of principle that SSPCs can address many unmet medical needs in orthopaedic medicine.