M3 - Isolation and implantation of dental pulp stem cells for spinal tissue regeneration in mice with spinal muscular atrophy

Introduction: : Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease caused by the deletion of the survival motor neuron 1 (SMN1) gene. Due to a mutation, a homologous flanking gene, called SMN2, can only make around 10% of the protein the body needs. A chronic deficiency of the SMN protein results in progressive degeneration of motor neurons within the lower spinal cord. The severity of SMA is inversely related to the number of copies of SMN2 an individual has. SMA is classified into five types, corresponding to disease severity and age. Type I SMA is the most common and, unfortunately, the most severe. Before disease-modifying therapies became available, type I patients typically had a lifespan of less than two years. Patients suffer from difficulties in crawling, sitting, and walking and are more likely to have other comorbidities, including orthopedic and pulmonary complications.
Recent roll-outs of disease-modifying gene therapies have remarkably changed the grim outlook these patients once faced. However, these therapies are associated with risky side effects and do not reverse the neurodegeneration that is present. An alternative to the current treatments is stem cell therapy. Dental pulp stem cells (DPSCs) have proliferative and neurogenic differentiation potential. They also excrete neurotrophic factors, and can be autologously transplanted for patient-specific care. We aim to transplant DPSCs into the spinal cord of SMA-afflicted mice to investigate the potential for neuromuscular tissue regeneration, improved motor ability, and increased life expectancy.

Materials and

Methods: : DPSCs will be isolated from a control line of SMNdelta7 mice by dissecting the mandible and removing the inner pulp from the incisors and molars. The pulp will be digested with collagenase and filtered through 70 mm nylon cell strainers. Newly isolated DPSCs will be resuspended in DPSC basal media supplemented with FBS, ascorbic acid, L-glutamine, and gentamicin from Lonza Biosciences. Clones will be isolated to begin a new clonal culture for implantation and fluorescently labeled for tracking purposes.
Heterozygous SMNdelta7 mice, obtained from Jackson Laboratories, will be bred together to create null SMN1 offspring. These mice are smaller in size, have progressive muscle weakness, and a lifespan of ~2 weeks. Post-natal day 1 mice will be intrathecally injected with 15-25,000 DPSCs. Beginning on post-natal day 10, mice will be taken for two behavioral tests every three days. A grip strength test will be performed by allowing the mice to grasp onto a metal bar suspended slightly in the air, and recording the time until the mouse releases the bar. Additionally, an open field test will assess the willingness and ability of the mice to move and explore in a wooden box. When mice present with signs of severe clinical illness, they will be sacrificed. The mice will be imaged using IVIS spectrum imaging to assess cell migration. Spinal cords and hind limbs will be fixed and embedded in paraffin for sectioning. H&E and cresyl violet staining will be used to visualize muscle tissue and motor neuron counts.

Results, Conclusions, and Discussions:: We expect SMA mice treated with DPSCs will have a higher average weight and longer lifespan than non-treated SMA mice. In addition, treated SMA mice will have longer hang times on the grip strength test, and an increased time and distance moved during the open field test. H&E staining performed on the treated SMA mice's hind legs is expected to show an increase in tissue mass. Cresyl violet staining on spinal cord sections is anticipated to show more motor neurons and longer axons than non-treated mice.
At the end of this study, we hope to confirm the feasibility of using DPSCs to treat neurodegeneration due to SMA. Intrathecal transplantation will deliver the cells directly to the target area and hopefully integrate with the native tissue. DPSCs have neuronal differentiation potential, making them an appealing candidate for stem cell therapies for neurodegenerative diseases like SMA. A successful engraftment of DPSCs into the spinal cord will potentially have numerous positive effects, including increased muscle mass and strength, a normalized growth rate, and improved motor ability. Ultimately, we plan to harvest DPSCs from patient premolars or wisdom teeth for autologous transplantation into the spinal cord. Our use of stem cell engineering using DPSCs has promising applications for regenerative medicine focused on patient-specific care.

Acknowledgements and/or References (Optional):: Thank you to Clemson University Creative Inquiry and SCTRIMH for funding and research support for this work.