SMN1 transcript enhanced dental pulp stem cells for motor neuron regeneration

Introduction:: Spinal Muscular Atrophy (SMA) is a neurodegenerative disease caused by the loss of motor neurons in the spinal cord due to a mutation in the survival motor neuron gene (SMN1), resulting in a nonfunctional protein. The complete loss of function is characteristic of SMA type 1, however in SMA type 2 the duplicated SNM2 gene is partially functional in a truncated form but does not restore full functionality. Patients with SMA suffer from atrophy of the skeletal muscle, eventually leading to paralysis and early death. SMA is the leading cause of hereditary infant mortality, but there are currently no affordable treatment options available.

Stem cell therapy has gained much attention in recent years due to its self-renewal and differentiation capabilities. This method is promising for many untreatable diseases, including SMA. Current in vitro models of SMA include fibroblasts derived from SMA patients and neural stem cells. Pluripotent dental pulp stem cells (DPSCs), which can easily be obtained from wisdom teeth or premolars, have neural crest origin and tissue regeneration capabilities that can be developed into potential SMA therapies¹. DPSCs can be autologously transplanted to reduce the possibility of immune system rejection and have few ethical concerns.

We explore the feasibility of improving SMA symptoms using SMN1 transcript enhanced DPSCs. Ultimately, we hope to achieve autologous transplantation of DPSCs that carry SMN1 transcripts that code for functional SMN1 protein for motor neuron regeneration.


Materials and Methods:: A pET30b plasmid with the SMN1 coding sequence under the direction of the T7 promoter was transformed into DH5α E.coli. AMP resistance allowed for the selection of bacteria containing the plasmid. The plasmid was extracted from AMP resistant E.coli and linearized by SCA1 restriction digest. The DNA was purified using Monarch PCR Cleanup Kit (#T1030S) and was used for in vitro mRNA synthesis. Poly-A tails were added to the end of the transcript. Dental pulp stem cells were obtained from adult human wisdom teeth and cultured according to Lonza Poietics DPSC protocol. DPSCs were transfected with the SMN1 transcript using Invitrogen Lipofectamine 3000, omitting the reagent P3000 as it is specified for use in DNA transfection. DPSCs transfected with only reagents and nuclease-free water were used as controls. Three days post-transfection, DPSCs were transferred to Gibco Neurobasal Media supplemented with anti-anti, N2 and B27 (Gibco). 7 days post-transfection, fluorescence imaging, flow cytometry, and RT-qPCR were completed for analysis of transfected cells. An additional enhancement of DPSCs using Invitrogen Lipofectamine P3000 to transfect eGFP (GenScript) was conducted to investigate a practical tracking method for in vivo mice studies.


Results, Conclusions, and Discussions:: Microscopic images of SMN1 (+) DPSCs 7 days post-transcript delivery revealed the cells had become elongated, resembling features of a typical motor neuron (Figure 1A, 1C). Flow cytometer reading of the neuronal stem cell marker A2B5 indicated a higher surface presence on the SMN1 (+) cells as compared to the SMN1 (-) control (Figure 1B, 1D). RT-qPCR analysis using a primer set encompassing the clinically typical deletion of exon 7 showed a significant increase in Log₂ differential SMN1 transcript abundance for the cells enhanced with the SMN1 transcript. Specifically, cells receiving no P3000 reagent had a 263-fold increase in the amount of SMN1 abundance. DPSCs transfected with eGFP were examined using fluorescence microscopy to observe luminescent intensity. Under GFP filter, the cells luminesced green indicating a practical way to track cells in in vivo mice studies.

We have shown through RT-qPCR that SMN1 transcript abundance is significantly higher in the DPSCs delivered with SMN1 transcript. Culturing of the SMN1 enhanced cells in neural induction media induced a motor neuron like morphological change in addition to displaying neural surface markers recognized by the anti-A2B5 antibody. Our data suggests the potential of DPSCs as a route for motor neuron regeneration. DPSCs can be obtained from a patient’s premolars, transfected with the wild-type SMN1 transcript and be autologously transplanted back into the patient to enhance a personalized treatment plan. To investigate clinical translatability, the use of an SMA mice model would greatly increase our understanding of how the proposed treatment would interact in vivo. DPSCs enhanced with SMN1 and eGFP will be used for transplantation into a mice model with SMA-like symptoms. Success will be determined by behavioral and survival assays to assess the effectiveness of improving SMA symptoms. More research is needed to assess the motor neuron properties of the SMN1 enhanced DPSCs. Our use of stem cell engineering using DPSCs reveals a promising avenue for tissue regeneration applications.


Acknowledgements (Optional): : The authors are partially supported by institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers [P20GM121342 and P20GM139769]; Prisma Health Research Seed Grant, Clemson University’s Vice President for Research; Clemson University’s College of Science; Clemson University’s Creative Inquiry, and the South Carolina Governor & Joint Bond Review Committee.


References (Optional): : 1. Ebert AD, Svendsen CN. Stem cell model of spinal muscular atrophy. Arch Neurol. 2010 Jun;67(6):665-9. doi: 10.1001/archneurol.2010.89. PMID: 20558385; PMCID: PMC3140872.