Gelatin Electrospun Mat as a Potential Co-culture System for In Vitro Production of Sperm Cells from Embryonic Stem Cells

Abstract

Engineering of 3D substrates with maximum similarity to seminiferous tubules would help to produce functional sperm cells in vitro from stem cells. Here, we present a 3D electrospun gelatin (EG) substrate seeded with Sertoli cells and determine its potential for guided differentiation of embryonic stem cells (ESCs) toward germline cells. The EG was fabricated by electrospinning, and its morphology under SEM, as well as cytobiocompatibility for sertoli cells and ESCs were confirmed by MTT and cell attachment assay. Embryoid bodies (EBs) were formed from ESCs and co-cultured with Sertoli cells, induced with BMP4 for 3 and 7 consecutive days to induce the differentiation of EBs toward germline cells. The differentiation was investigated by immunocytochemistry (ICC), flow cytometry and RT-PCR in four experimental groups of EB (EBs cultured in gelatin-coated cell culture plate); Scaffold/EB (EBs cultured on EG); ESCs/Ser (EBs and Sertoli cells co-cultured on gelatin-coated cell culture plate without EG); and Scaffold/EB/Ser (EBs and Sertoli cells co-cultured on EG). All experimental groups exhibited a significantly increased MVH (germline specific marker) and decreased c-KIT (stemness marker) expression when compared with EB group. ICC and flow cytometry revealed that Scaffold/EB/Ser had the highest level of MVH and the lowest c-KIT expression at both 3 and 7 days postdifferentiation compared with other groups. RT-PCR results showed a significant increase in germline marker (Dazl), and a significant decrease in the ESCs stemness marker (Nanog) in the Scaffold/EB compared to the EB group. The germline markers Gcna, Stella, Mvh, Stra8, Piwil2and Dazl were significantly increased in the Scaffold/EB/Ser compared to the Scaffold/EB group.Our findings revealed that the EG scaffold can provide an excellent substrate bio-mimicking the micro/nanostructure of native seminiferous tubules, and a platform for Sertoli cells-EBs communication required for growth and differentiation of ESCs into germline cells.

Self-Assembled Fibrinogen Scaffolds Support Cocultivation of Human Dermal Fibroblasts and HaCaT Keratinocytes

“…Of the three fibrinogen topographies studied in this work, only fibrinogen nanofibers will be able to facilitate nutrient and oxygen supply to cells due to their porous architecture, which is another important requirement for wound healing. , More importantly, in contrast to planar fibrinogen, self-assembled fibrinogen nanofibers have a decisive technical advantage because they can be detached from the underlying substrate in aqueous solution to obtain free-standing scaffolds with tailored thickness. , Due to their flexible nature when rehydrated, free-standing fibrinogen nanofibers could also become a very interesting scaffold type for other coculture systems and could for instance serve as innervated skin models or could be developed into tubular constructs for artificial vascular grafts, to replace seminiferous tubules or to support peripheral nerve regeneration . Combined with their good biocompatibility with fibroblast–keratinocyte cocultures, the possibility to use fibrinogen nanofibers as free-standing scaffolds will open the opportunity in the future to grow keratinocytes at an air–liquid interface (ALI) to promote cell differentiation and stratification, both being important features for successful wound closure .…”

Differentiation of human spermatogonial stem cells using a human decellularized testicular scaffold supplemented by platelet‐rich plasma

“…In recent years, several studies have evaluated different scaffolds for in vitro spermatogenesis of spermatogonial, embryonic, and adult stem cells 14–17 . In this line, Vardiani et al suggested that the gelatin electrospun scaffold can provide a microenvironment for the adhesion, proliferation, and differentiation of embryonic stem cells to sperm‐generating cells 15,17 .…”

Advanced bioengineering of male germ stem cells to preserve fertility

“…Electrospun 3D scaffolds are known to be highly favorable for cell seeding, because they are thought to be more morphologically and structurally similar to ECM, compared to those synthesized by other approaches. 123 , 127 , 128 , 144 The effect of surface topography of these scaffolds on expansion of testicular cells has thus far not been reported. Fibrillar electrospun scaffolds have been reported to provide appropriate interactions mimicking the ECM and promoting appropriate migration and morphological alterations.…”

“… 144 , 145 Such nano-surfaces have improved the paracrine secretions from Sertoli cells, modulated the expression of genes participating in ECM formation, promoted the functionality of signaling molecules, and maintained the stemness of spermatogonial stem-like cells. 125 , 128 , 129 , 144 …”