Autophagy-induction to augment MSCs function and promote healing of critical-sized bone defects
Summary
Reconstruction of critical-sized bone defects (CSDs) remains an important orthopedic challenge. Unlike fracture healing, in CSDs, the intrinsic capacity of self-regeneration is not sufficient to repair the defect. One approach for the reconstruction of CSDs is the use of mesenchymal stromal/stem cell (MSC)-based therapies. However, a major impediment to the development of MSC-based therapies has been poor cell survival and engraftment at the site of injury. In particular, the microenvironment found in such large defects (i.e., ischemic, hypoxic, nutrient-deprived, low pH) is prone to disrupt cell metabolism thereby compromising the function and survival of implanted MSCs. Therefore, approaches that extend the lifespan of MSC at the implantation site would improve their therapeutic potential. Modulation of MSC metabolism is a recent approach for adapting cells to microenvironment stressors within large bone defects.
Autophagy is a cellular stress response pathway that directs lysosomal degradation of redundant or damaged proteins and organelles in response to starvation, oxidative or hypoxic stress. Autophagy of cytoplasmic contents not only recycles building blocks and energy for the cell, but also clears damaged or aggregated proteins and organelles preventing further damage to the cell, and thereby promotes cell survival. Previous studies in rats have shown that pharmacological induction of autophagy by rapamycin promotes fracture healing. The beneficial effect of rapamycin treatment was associated with increased production of angiogenic factors and increased osteogenesis. Other studies have shown that rapamycin promotes MSCs secretion angiogenic factors and that rapamycin-induced autophagy plays a protective role in hypoxia and serum deprivation-induced apoptosis of MSCs in vitro. However, no studies have attempted to modulate autophagy to improve MSC survival or osteogenesis in large bone defects.
Based on these observations, we hypothesize that “Autophagy-induction will improve MSC-based reconstruction of the critical sized-defects (CSDs)”. To test our hypothesis, we propose using a genetic and a pharmacological approach.
Approach 1: Genetic induction of autophagy in MSCs will promote the healing of CSDs.
To increase autophagy of human bone-marrow-derived MSCs (hBMSCs), we will use CRISPR activation (CRISPRa) to overexpress a transcriptional regulator of autophagy and lysosomal biogenesis, namely Transcription Factor EB (TFEB). Human MSCs expressing luciferase and overexpress TFEB will be implanted within femoral CSDs made in immunocompromised rats.
Approach 2: Pharmaceutical induction of autophagy in vivo will promote the healing of CSDs.
Human MSCs expressing luciferase will be implanted within femoral CSDs made in immunocompromised rats. Starting the day after the implantation, these rats will be injected daily with rapamycin (1 mg/kg body weight/dose).
In both approaches, the efficacy of autophagy activation for CSD repair will be tested using dual-energy X-Ray absorptiometry, micro-computed tomography, and histology. Bioluminescence imaging of luciferase will be used to assess the survival of implanted hMSCs. Moreover, the direct versus the indirect contribution of MSCs to healing will be determined using immunohistochemistry for human mitochondria.
Keywords:
- bone
- Mesenchymal Stem Cells