Purpose

Upregulation of differentiation potential of mesenchymal stem cells contributes to the development of regenerative medicine. Nanosecond pulsed electric fields (nsPEFs) have been shown to influence intracellular organelles and trigger strong biological effects [1, 2]. In current study, we study the effects of nsPEFs on differentiation potential of pig MSCs (Mesenchymal stem cells) in vitro and explore the mechanisms behind the phenomenon.

Methods and Materials

The effects of nsPEFs-preconditioning on the expression levels of tri-lineage differentiation were evaluated by quantitative RT-PCR of marker genes and Histological staining. Mechanisms were explored with gene expression level of OCT4 and NANOG and the methylation level of their promoter. The expression of DNA methylation transferase and global DNA methylation level were performed to further study the mechanisms behind.

Results

nsPEFs-preconditioning with proper parameters (10 ns at 20 kV/cm, 100 ns at 10 kV/cm) significantly potentiated tri-lineage differentiation capacity of MSCs with upregulated genes expression(Fig.1). nsPEFs-preconditioning promotes the formation of glycosaminoglycan for chondrogenic differentiation, calcium nodule for osteogenic differentiation, lipid droplet for adipogenic differentiation through demethylation of OCT4 and NANOG promoter and upregulation of OCT4 and NANOG, followed by downregulation of DNMT1 and global DNA methylation level (Fig.2).

Conclusion

This study demonstrates a unique approach of nsPEFs-treatment to potentiate the tri-lineage differentiation potential of MSCs through demethylation of OCT4 and NANOG which has translational potential for MSCs-based regenerative medicine.

Reference

1. Zhang, K., et al., Nanosecond Pulsed Electric Fields (nsPEFs) Regulate Phenotypes of Chondrocytes through Wnt/β-catenin Signaling Pathway. Scientific Reports, 2014. 4: p. 5836.

2. Ning, T., et al., Nanosecond pulsed electric fields enhanced chondrogenic potential of mesenchymal stem cells via JNK/CREB-STAT3 signaling pathway. Stem cell research & therapy, 2019. 10(1): p. 45.

Purpose

Purpose：Mesenchymal stem cells (MSCs) gradually lose multilineage differentiation potentials when cultured in vitro, which hinders their subsequent applications. Though much efforts have been enforced to take this challenge, progresses are limited. In our previous research, nanosecond pulsed electrical fields (nsPEFs）enhanced multilineage differentiation potential of MSCs. We tried to explore if concomitant applications of nsPEFs could enhanced differentiation potentials of MSCs during prolonged in vitro culture.

Methods and Materials

Materials & Methods: Pig mesenchymal stem cells cultured on conductive films were treated with nanosecond pulsed electrical fields (nsPEFs). After optimizing parameters (100ns 10kv/cm, 10ns 20kv/cm, judged by gene expression of Oct4A, Nanog and Sox2) of nsPEFs on conductive film, concomitant applications of multiple nsPEFs treatment (four times) through conductive film were performed.

Results

Results: In this study, pre-conditioning of mesenchymal stem cells with nanosecond pulsed electrical fields (nsPEFs) through gas cuvettes significantly enhanced the expression of Oct4A (3.8 fold of 100ns 10kv/cm, 3.4 fold of 10ns 20kv/cm), Nanog (3.2 fold of 100ns 10kv/cm, 2.8 fold of 10ns 20kv/cm) and Sox2 (1.8 fold of 100ns 10kv/cm, 2.0 fold of 10ns 20kv/cm). Furthermore, after optimizing parameters of nsPEFs on conductive film (100ns 10kv/cm) and time interval (3d between each nsPEFs treatment), concomitant applications of multiple nsPEFs treatment (four times) enhanced gene expression of Oct4A (3.5~4.5fold), Nanog (3.5~4fold) and Sox2 (1.3~2fold) significantly while maintained this high level gene expression for nearly 30 days of MSCs, much longer than single nsPEFs treatment (normal 7 days). Differentiation potential was significantly increased in both single and multiple nsPEFs treatment, judged by chondrogenic (3-fold of Sox9), osteogenic (6-fold Runx2) and adipogenic (4-fold of PPAR-γ) marker gene.

Conclusion

Conclusion: Our study suggested that multiple nsPEFs treatment through conductive film could enhance the multilineage differentiation potential of MSC for long time scale.