Improving motor neuron-like cell differentiation of hEnSCs by the combination of epothilone B loaded PCL microspheres in optimized 3D collagen hydrogel

Narges Mahmoodi,Zahra Hassannejad,Vafa Rahimi-Movaghar,Houra Nekounam,Jafar Ai,Alexander R Vaccaro,Elham Hasanzadeh,Somayeh Ebrahimi-Barough

doi:10.1038/s41598-021-01071-2

Abstract

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.

Highlights

Spinal cord injury (SCI) is a very common traumatic event and is mainly caused by falls and motor vehicle accidents (MVAs)[1,2]
The Human endometrial stem cells (hEnSCs) were positive for mesenchymal stem cell markers CD44 (98.5%), CD90 (98.4%) and CD105 (98.4%), endometrial stem cell marker CD146 (89.1%). hEnSCs were negative for hematopoietic CD45, CD34, and endothelial CD31 markers (Fig. 2a)
While understanding the exact mechanism of epothilone B (EpoB) and adapting it to the complex mechanism of spinal cord injury (SCI) yet need to achieve a clear vision, today its role in stem cell differentiation and SCI has garnered attention, and our study demonstrated the enhanced motor neuron (MN) differentiation efficiency of hEnSCs and neurite growth by 3D crosslinked collagen hydrogel containing EpoB-loaded microspheres which can be used as a combinatorial approach for MN-diseases modeling and a future aid in SCI repair

Summary

Introduction

Spinal cord injury (SCI) is a very common traumatic event and is mainly caused by falls and motor vehicle accidents (MVAs)[1,2]. As a growing research area, using various stem cells has been garnered great attention for nerve tissue regeneration through cell differentiation for lost cell replacement, anti-inflammatory effects, and neurotrophic factor s ecretion[4,10]. Human endometrial stem cells (hEnSCs) as a MSCs have considered attention for cell therapy and tissue e ngineering[16,17,18,19,20]. In a recent study, a comparison between hEnSCs and human bone marrow stem cells (hBM-MSCs) showed a better motor neuron (MN) differentiation potential for h EnSCs21. Improvement in differentiating stem cells into specialized neurons is one solution for replacing damaged or lost cells, thereby leading to potential functional recovery[22]

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