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Differentiation and Proliferation of Spermatogonial Stem Cells Using a Three-Dimensional Decellularized Testicular Scaffold: A New Method to Study the Testicular Microenvironment in Vitro Publisher Pubmed



Naeemi S1 ; Eidi A1 ; Khanbabaee R2 ; Sadriardekani H3 ; Kajbafzadeh AM4
Authors
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Authors Affiliations
  1. 1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
  2. 2. Department of Biology, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Iran
  3. 3. Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, 27157, NC, United States
  4. 4. Pediatric Urology and Regenerative Medicine Research Center, Section of Tissue Engineering and Stem Cells Therapy, Children’s Hospital Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Gharib’s Street, Keshavarz Boulevard, Tehran, 1419433151, Iran

Source: International Urology and Nephrology Published:2021


Abstract

Purpose: Successful in vitro transplantation of spermatogonial stem cells (SSCs) demands effective culture systems for SSCs proliferation and differentiation. Natural extracellular matrix (ECM) creates a microenvironment suitable for culture of stem cells. In the present study, we intended to assess the capability of the porous scaffold consisting of hyaluronic acid (HA), chitosan, and decellularized testicular matrix (DTM) as a proper niche for SSCs seeding. Methods: The testes of four NMRI mice were extracted for further detergent-based decellularization process. We isolated, cultured, and clarified neonate mouse SSC, and a three-dimensional scaffold was prepared for SSCs culture. The loaded SSCs and hydrogel-based scaffold were investigated by several studies including scanning electron microscopy (SEM), 4′,6-diamidino-2-phenylindole (DAPI), 3-[4, 5-dimethyl (thiazol-2yl)-3,5diphenyl] tetrazolium bromide (MTT), Acridine orange, and Immunohistochemistry (IHC) staining. Results: The efficiency of decellularization process was confirmed by DAPI, hematoxylin and eosin (H&E), and Masson’s Trichrome staining. Acridine orange also depicted SSCs proliferation and viability. SEM approved the preservation of ECM components and also showed complex, coiled, and tubular seminiferous tubules, with intact and condensed collagenous form of the tunica albuginea. MTT test also revealed the scaffold’s non-toxicity. Expression of PLZF, TP1, and TEKT1 markers also verified the capacity of SSCs proliferation on a cogel scaffold. Conclusion: In conclusion, cogel scaffold consisting of DTM, HA, and chitosan may provide the supporting layer for in vitro SSC differentiation and proliferation. © 2021, The Author(s), under exclusive licence to Springer Nature B.V.
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