Diba Ghasemi
1 
, Somayeh Ebrahimi-Barough
1, Mohammad Hossein Nekoofar
2,3, Abdolreza Mohamadnia
4,5, Nasrin Lotfibakhshaiesh
1, Naghmeh Bahrami
1,6, Roya Karimi
1, Vajihe Taghdiri Nooshabadi
7, Mahmoud Azami
1, Elham Hasanzadeh
8, Jafar Ai
1* 1 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Endodontics, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
3 School of Dentistry, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK
4 Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
6 Craniomaxillofacial Research Center, Tehran University of Medical Sciences, Tehran, Iran
7 Department of Tissue Engineering and Applied Cell Sciences, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran
8 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran
Abstract
Introduction: Human endometrial mesenchymal stem cells (hEnMSCs) are a rich source of mesenchymal stem cells (MSCs) with multi-lineage differentiation potential, making them an intriguing tool in regenerative medicine, particularly for the treatment of reproductive and infertility issues. The specific process of germline cell-derived stem cell differentiation remains unknown, the aim is to study novel ways to achieve an effective differentiation method that produces adequate and functioning human gamete cells.
Methods: We adjusted the optimum retinoic acid (RA) concentration for enhancement of germ cell-derived hEnSCs generation in 2D cell culture after 7 days in this study. Subsequently, we developed a suitable oocyte-like cell induction media including RA and bone morphogenetic protein 4 (BMP4), and studied their effects on oocyte-like cell differentiation in 2D and 3D cell culture media utilizing cells encapsulated in alginate hydrogel.
Results: Our results from microscopy analysis, real-time PCR, and immunofluorescence tests revealed that 10 µM RA concentration was the optimal dose for inducing germ-like cells after 7 days. We examined the alginate hydrogel structural characteristics and integrity by rheology analysis and SEM microscope. We also demonstrated encapsulated cell viability and adhesion in the manufactured hydrogel. We propose that in 3D cell cultures in alginate hydrogel, an induction medium containing 10 µM RA and 50 ng/mL BMP4 can enhance hEnSC differentiation into oocyte-like cells.
Conclusion: The production of oocyte-like cells using 3D alginate hydrogel may be viable in vitro approach for replacing gonad tissues and cells.