Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold

Izanlou S. Afshar A. Zare A. Zhilisbayeva K.R. Bakhshalizadeh S. Safaei Z. Sehat-Bakhsh S. Khaledi S. Asgari H.-R. Kazemnejad S. Ajami M. Ajami M. Dehghan Tarzjani M. Najafzadeh V. Kouchakian M.R. Mussin N.M. Kaliyev A.A. Aringazina R.A. Mahdipour M. Shirazi R. Tamadon A.
December 2023 Elsevier Ltd

Tissue and Cell
2023 #85

Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.

Amniochorionic membrane , Co-culture , Female germ cells , Fibroin , Granulosa cells , Infertility , Menstrual blood stem cells , Scaffold , Silk

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Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
Student Research Committee, Bushehr University of Medical Sciences, Bushehr, Iran
PerciaVista R&D Co., Shiraz, Iran
Department of Scientific Work, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
Reproductive Development, Murdoch Childrens Research Institute, Melbourne, VIC, Australia
Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia
Center for Embryonic Cell and Gene Therapy, Oregon Health and Science University, Portland, 97239, OR, United States
Department of Anatomical Sciences, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran
Department of Hematology, Faculty of Paramedical Sciences, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran
Department of Gynecology and Obstetrics, Imam Khomeinin Hospital, Tehran University of Medical Sciences, Tehran, Iran
Queensland Fertility Group (QFG), Mackay, QLD, Australia
General Surgery, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
Department of Internal Medicine No. 1, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
Department of Anatomy, School of Biomedical Sciences, Medicine & Health, UNSW Sydney, Sydney, Australia

Department of Anatomical Sciences
Student Research Committee
PerciaVista R&D Co.
Department of Scientific Work
Reproductive Development
Department of Paediatrics
Center for Embryonic Cell and Gene Therapy
Department of Anatomical Sciences
Reproductive Biotechnology Research Center
Department of Medical Laboratory Sciences
Department of Hematology
Department of Gynecology and Obstetrics
Queensland Fertility Group (QFG)
General Surgery
Department of Internal Medicine No. 1
Stem Cell Research Center
Department of Reproductive Biology
Department of Anatomy

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