Lipoxin A4 and Resolvin D1 Preserve Neural Inductive Capacity of Dental Pulp Stem Cells Cultured under Inflammatory Conditionsïºï Implications for SCI Stem Cell Therapy
1Morse L, 1Nguyen N, 1Xu Y, 1Hasturk H, 1Kantarci A, 2Battaglino R
1Craig Hospital, Englewood, CO, United states; 2UC Denver, Aurora, CO, 80113
Few therapies exist for neuro-trauma. Dental pulp is a rich source of neural crest stems cells that can differentiate into functionally active neurons in vitro. Transplantation of human tooth-derived stem cells enhances neuro-recovery in rodent models of spinal cord injury. However, secondary inflammation may blunt the neuro-protective effect of stem cells in vivo. We previously reported that the inflammatory cytokine TNF- suppresses neural differentiation of dental pulp stem cells. We therefore sought to determine if 2 anti-inflammatory agents (lipoxin A4 and resolvin D1) mitigate TNF--induced suppression of neural differentiation of dental pulp stem cells.
Human DPSC were isolated from molars extracted for dental care and cultured in basal media. Neural differentiation was induced with neural differentiation media and cells were cultured for up to 21 days. Varying concentrations of TNF-α (1, 10, 25, and 100 ng/mL, PeproTech, Rocky Hill, NJ, USA), lipoxin A4 (10 and 100 nM, Cayman Chemical, Ann Arbor, MI, USA) or resolvin D1 (10 and 100 nM, Cayman Chemical, Ann Arbor, MI, USA) were added to the culture of both differentiated and non-differentiated cells starting 48 hours after seeding. In some experiments cells were treated with both TNF- α and lipoxin A4 or resolvin D1. Stem cell and neuronal cell marker expression was determined by flow cytometry. ANOVA tests were used for all statistical analyses; p<0.05 was considered significant.
At 21 days of neuronal induction, there was a significant reduction in the percentage of cells expressing the stem cell markers CD44 (57% reduction, p<0.05) and OCT 3/4 (42% reduction, p<0.005). Concomitantly, significantly more cells expressed the early neuronal markers nestin (300% increase, p<0.005) and doublecortin (62% increase, p<0.05), the intermediate neuronal marker β III-tubulin (228% increase, p<0.005), and the late neuronal marker NeuN (418% increase, p<0.005). 25ng/ml TNF- significantly reduced β III-tubulin expression (59% decrease, p=0.0002) consistent with failure to progress to late neuronal differentiation under inflammatory conditions. This reduction was partially blocked by treatment with either resolvin D1 (100 nM) or lipoxin A4 (10 nM). We found a significant 78% increase in β III-tubulin expression in cells undergoing neural induction in the presence of both TNF- and 100 ng/ml resolvin D1 (32% for 25ng/ml TNF- vs 57% for 25ng/ml TNF- + resolvin D1, p=0.02). Similarly, we found a significant 84% increase in β III-tubulin expression in cells undergoing neural induction in the presence of both TNF- and 10 nM lipoxin A4 (32% for 25ng/ml TNF- vs 59% for 25ng/ml TNF- + lipoxin A4, p=0.02).
We report lipoxin A4 and resolvin D1 both preserve dental pulp stem cell neural inductive capacity under inflammatory conditions. This finding suggests that combination therapy with anti-inflammatories and dental pulp stem cell transplantation may improve motor recovery following acute neurotrauma.
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