Three-Dimensional Culture of Mouse Spermatogonial Stem Cells Using A Decellularised Testicular Scaffold
Journal Title: Cell Journal(Yakhteh) - Year 2020, Vol 21, Issue 4
Abstract
Objective: Applications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells. Materials and Methods: In this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson’s trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC. Results: Haematoxylin-eosin (H&E) staining showed that the cells were successfully removed by SDS and Triton X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This confirmed that our decellularisation protocol was efficient. Masson’s trichrome and alcian blue staining respectively showed that glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous tubule. Analyses of gene expression using real-time PCR indicated that expression of the Plzf gene was unchanged over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture, suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules. Conclusion: Spermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.
Authors and Affiliations
Nasrin Majidi Gharenaz, Mansoureh Movahedin, Zohreh Mazaheri
Keywords
Related Articles
- EP ID EP624551
- DOI 10.22074/cellj.2020.6304
- Views 186
- Downloads 0
Orexin-A Modulates Firing Of Rat Rostral Ventromedial Medulla Neurons: An In Vitro Study
The rostral ventromedial medulla (RVM) acts a key role in the descending inhibitory pain modulation. Neuropeptide orexin-A (ORXA) is confined to thousands of neurons in the lateral hypothalamus (LH). While RVM gets the o...
Percutaneous Autologous Bone Marrow-Derived Mesenchymal Stromal Cell Implantation Is Safe for Reconstruction of Human Lower Limb Long Bone Atrophic Nonunion
Objective Nonunion is defined as a minimum of a 9-month period of time since an injury with no visibly progressive signs of healing for 3 months. Recent studies show that application of mesenchymal stromal cells (MSCs) i...
Histone Acylation Beyond Acetylation: Terra Incognita In Chromatin Biology
Histone acetylation, one of the first and best studied histone post-translational modifications (PTMs), as well as the factors involved in its deposition (writers), binding (readers) and removal (erasers), have been show...
Differential Proliferation Effects after Short-Term Cultivation of Mouse Spermatogonial Stem Cells on Different Feeder Layers
Objective: Spermatogonial stem cells (SSCs) provide the cellular basis for sperm production transforming the male’s genetic information to the next generation. We aimed to examine the effect of different feeder layer on...
Optimization of Porous Silicon Conditions for DNA-based Biosensing via Reflectometric Interference Spectroscopy
Objective: Substantial effort has been put into designing DNA-based biosensors, which are commonly used to detect presence of known sequences including the quantification of gene expression. Porous silicon (PSi), as a na...