Adipose-Derived Mesenchymal Stem Cells From a Hypoxic Culture Improve Neuronal Differentiation and Nerve Repair.

Szu-Hsien Wu,Yu-Ting Liao,Ting-Chen Tseng,Hui-Kuang Huang,Shan-Hui Hsu,Tung-Ming Chen,Jung-Pan Wang,Kuang-Kai Hsueh,En-Rung Chiang

doi:10.3389/fcell.2021.658099

Abstract

Hypoxic expansion has been demonstrated to enhance in vitro neuronal differentiation of bone-marrow derived mesenchymal stem cells (BMSCs). Whether adipose-derived mesenchymal stem cells (ADSCs) increase their neuronal differentiation potential following hypoxic expansion has been examined in the study. Real-time quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were employed to detect the expression of neuronal markers and compare the differentiation efficiency of hypoxic and normoxic ADSCs. A sciatic nerve injury animal model was used to analyze the gastrocnemius muscle weights as the outcomes of hypoxic and normoxic ADSC treatments, and sections of the regenerated nerve fibers taken from the conduits were analyzed by histological staining and immunohistochemical staining. Comparisons of the treatment effects of ADSCs and BMSCs following hypoxic expansion were also conducted in vitro and in vivo. Hypoxic expansion prior to the differentiation procedure promoted the expression of the neuronal markers in ADSC differentiated neuron-like cells. Moreover, the conduit connecting the sciatic nerve gap injected with hypoxic ADSCs showed the highest recovery rate of the gastrocnemius muscle weights in the animal model, suggesting a conceivable treatment for hypoxic ADSCs. The percentages of the regenerated myelinated fibers from the hypoxic ADSCs detected by toluidine blue staining and myelin basic protein (MBP) immunostaining were higher than those of the normoxic ones. On the other hand, hypoxic expansion increased the neuronal differentiation potential of ADSCs compared with that of the hypoxic BMSCs in vitro. The outcomes of animals treated with hypoxic ADSCs and hypoxic BMSCs showed similar results, confirming that hypoxic expansion enhances the neuronal differentiation potential of ADSCs in vitro and improves in vivo therapeutic potential.

Highlights

Nerve grafts, which are composed of natural or artificial tubes, are considered ideal treatments to guide nerve regeneration across the injury gaps (Faroni et al, 2013, 2014)
RT-qPCR was used to confirm the neuronal markers of the normoxic and hypoxic adiposederived mesenchymal stem cells (ADSCs) differentiated neuron-like cells by monitoring the gene expressions of TUBB3 (Figure 2I), microtubule-associated protein 2 (MAP2) (Figure 2J), neurofilament medium polypeptide (NEFM) (Figure 2K), and NEFH (Figure 2L) through the time course
The NEFM gene were highly expressed in the hypoxic ADSC differentiated neuron-like cells on day 1 (P < 0.05), and NEFH was significantly enhanced on days 3 and 7 (P < 0.05)

Summary

Introduction

Nerve grafts, which are composed of natural or artificial tubes, are considered ideal treatments to guide nerve regeneration across the injury gaps (Faroni et al, 2013, 2014). Utilization of ADSCs to treat and improve nerve regeneration in animal models with peripheral nerve defects has been well reported (Di Summa et al, 2010; Erba et al, 2010; Liu et al, 2011; Scholz et al, 2011; Marconi et al, 2012; Orbay et al, 2012; Sowa et al, 2016). ADSCs can differentiate into Schwann cells (SCs)-like (Kingham et al, 2007; Di Summa et al, 2014) and neuron-like (Scholz et al, 2011; New et al, 2015) phenotypes in vitro and in vivo to promote nerve regeneration. There is no difference in terms of transplantation with undifferentiated ADSCs or differentiated ones in nerve regeneration (Orbay et al, 2012), suggesting the treatment time can be shortened by direct ADSC transplantation

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