Endogenous Expression Levels of Repressor Element-1 Silencing Transcription Factor (REST) in Peripheral Multipotent Progenitor Cells
Marvin E Dingle, MD1; Stephen Fernicola, MD1; Alexandra Yaszemski, MBA2; Sonia Zicaria, PhD2; Edmund C Nesti, PhD3; Leon J. Nesti, MD, PhD4
1Walter Reed National Military Medical Center, Bethesda, MD, 2Uniformed Services University of Health Sciences, Bethesda, MD, 3Alcamena Stem Cell Therapeutics, Spencerville, MD, 4Orthopaedic Surgery, Walter Reed National Military Medical Center, Bethesda, MD
INTRODUCTION: Peripheral nerve injury continues to present a challenging clinical problem, with limited regeneration preventing full functional recovery, resulting in poor quality of life. The capacity for the peripheral nerves to regenerate is multifactorial and relies,in part, on trophic support received from surrounding cells to include previously reported trauma-induced mesenchymal progenitor cells (MPCs). The repressor element-1 silencing transcription factor (REST) is a nuclear transcription factor that acts as a master regulator of neuronal gene expression. Previous reports have shown an increase in REST following central nervous system insult, but the role that REST plays in peripheral nerve injury, the local tissues, and subsequent regeneration is still unknown. Our previous work has demonstrated the ability of trauma-induced MPCs to secrete neurotrophic factors, many of which are controlled by REST. Here we describe the expression profiles of REST, Brain-derived Neurotrophic Factor (BDNF) – a direct target of REST – and C-terminal Domain Small Phosphatase-1 (CTDSP-1) – a key regulator of REST activity – in MPCs isolated from peripheral nerve injuries.
METHODS: MPCs were seeded at a density of 1000 cells per square centimeter. After 4 days in growth media, cells were treated using our previously described neuroinduction media for a total of 14 days. REST, BDNF and CTDSP-1 expression levels were analyzed throughout the treatment process by RT-PCR and Western Blot. Additionally, REST and CTDSP-1 siRNAs were used to further characterize the expression of REST, BDNF and CTDSP-1.
RESULTS: We found that MPCs expressed REST in their undifferentiated (stem) state. Following treatment with the neuroinduction media, and siRNA treatment, REST and CTDSP-1 protein expression was decreased and the BDNF message was increased.
CONCLUSION: REST is a transcriptional regulator of many neural genes, including BDNF, and its function is controlled by CTDSP-1. Here we show that induction of these MPCs into a neuronal support cell phenotype is regulated by REST expression. Additional work is being done to characterize expression levels of other neurotrophic factors that are downstream targets of REST in these MPCs. The ability to increase neurotrophic factor expression in these endogenous progenitor cells at the site of injury is a novel approach to augment peripheral nerve regeneration and improve the treatment and functional outcomes of peripheral nerve injury.
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