Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems

Gholami, Kheirollah; Pourmand, Gholamreza; Koruji, Morteza; Sadighigilani, Mohammadali; Navid, Shadan; Izadyar, F.; Abbasi, Mehdi

doi:10.1016/j.repbio.2018.09.006

Cited by 34 publications

(21 citation statements)

References 36 publications

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“…Agar is a biocompatible, complex polysaccharide extracted from red sea weeds and was first used by Lin and colleagues (Lin et al 1975) to establish a 3D cell culture model (i.e., SACS) for characterizing clonal expansion of bone marrow cells. Since then several research groups have utilized SACS to investigate proliferation and differentiation of male germ cells in vitro (Stukenborg et al 2008(Stukenborg et al , 2009; Abu Elhija et al 2012; Reda et al 2016;Navid et al 2017;Gholami et al 2018aGholami et al , 2018bMohammadzadeh et al 2019). Application of SACS for in vitro spermatogenesis, however, was not devoid of pitfalls.…”

Section: Discussionmentioning

confidence: 99%

“…In order to provide an appropriate condition for the ex vivo proliferation of SSCs, it is essential to develop convenient cell culture systems that can effectively imitate not only the compositional and physiological properties but also the structural and architectural characteristics of the native SSC microenvironment (Dan et al 2006;Shakeri et al 2013). Indeed, recent investigations on the development of SSC culture systems have chiefly been focused on the preparation of new physicochemical (Lee et al 2013;Antoni et al 2015) and physiological (Kubota et al 2004a;Gholami et al 2018b) microenvironments that can adequately promote self-renewal and differentiation of stem cells. However, no satisfactorily efficient culture model has so far been identified by these studies (Park et al 2017).…”

Section: Introductionmentioning

confidence: 99%

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Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold

Kashani

Bagher

Asgari

et al. 2020

Systems Biology in Reproductive Medicine

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Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/−GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/−GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of premeiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold

Kashani

Bagher

Asgari

et al. 2020

Systems Biology in Reproductive Medicine

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“…Long-term SCCs culturing under laboratory conditions can lead to the loss of unique properties of these cells. Therefore, achieving a culturing system, which can create the [17][18][19][20][21][22][23][24]. The results confirmed the importance of 3D substrate in cell differentiation.…”

Section: Discussionmentioning

confidence: 68%

In Vitro Spermatogenesis by Three-dimensional Culture of Spermatogonial Stem Cells on Decellularized Testicular Matrix

Movassagh

Dehkordi

Koruji

et al. 2019

GMJ

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Background: In the males, Spermatogonial Stem Cells (SSCs) contribute to the production of sex cells and fertility. In vitro SSCs culture can operate as an effective strategy for studies on spermatogenesis and male infertility treatment. Cell culture in a three-dimensional (3D) substrate, relative to a two-dimensional substrate (2D), creates better conditions for cell interaction and is closer to in vivo conditions. In the present study, in order to create a 3D matrix substrate, decellularized testicular matrix (DTM) was used to engender optimal conditions for SSCs culture and differentiation. Materials and Methods: After, testicular cells enzymatic extraction from testes of brain-dead donors, the SSCs were proliferated in a specific culture medium for four weeks, and after confirming the identity of the colonies derived from the growth of these cells, they were cultured on a layer of DTM as well as in 2D condition with a differentiated culture medium. In the Sixth week since the initiation of the differentiation culture, the expression of pre meiotic (OCT4 & PLZF), meiotic (SCP3 & BOULE) and post meiotic (CREM & Protamine-2) genes were measured in both groups. Results: The results indicated that the expression of pre meiotic, meiotic and post meiotic genes was significantly higher in the cells cultured on DTM (P ≤ 0.001). Conclusion: SSCs culture in DTM with the creation of ECM and similar conditions with in vivo can be regarded as a way of demonstrating spermatogenesis in vitro, which can be adopted as a treatment modality for male infertility. [GMJ.2019;8:e1565]

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“…Alternatively, the use of DMEM-F12 medium over StemPro-34 medium might be beneficial for spermatogonial growth [68]. Other advances have been made with regards to replacing the feeder layer with a cell-free structure, by exploiting hydrogel forms of human testis derived extracellular matrix [69], or three-dimensional agar culture in combination with knockout serum replacement [70]. In addition, changing temperature may be useful to further improve propagation of SSCs in culture, as this was also beneficial in an organotypic testicular organ culture [71].…”

Section: Discussionmentioning

confidence: 99%

ITGA6+ Human Testicular Cell Populations Acquire a Mesenchymal Rather than Germ Cell Transcriptional Signature during Long-Term Culture

Struijk

Mulder

Daalen

et al. 2020

IJMS

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Autologous spermatogonial stem cell transplantation is an experimental technique aimed at restoring fertility in infertile men. Although effective in animal models, in vitro propagation of human spermatogonia prior to transplantation has proven to be difficult. A major limiting factor is endogenous somatic testicular cell overgrowth during long-term culture. This makes the culture both inefficient and necessitates highly specific cell sorting strategies in order to enrich cultured germ cell fractions prior to transplantation. Here, we employed RNA-Seq to determine cell type composition in sorted integrin alpha-6 (ITGA6+) primary human testicular cells (n = 4 donors) cultured for up to two months, using differential gene expression and cell deconvolution analyses. Our data and analyses reveal that long-term cultured ITGA6+ testicular cells are composed mainly of cells expressing markers of peritubular myoid cells, (progenitor) Leydig cells, fibroblasts and mesenchymal stromal cells and only a limited percentage of spermatogonial cells as compared to their uncultured counterparts. These findings provide valuable insights into the cell type composition of cultured human ITGA6+ testicular cells during in vitro propagation and may serve as a basis for optimizing future cell sorting strategies as well as optimizing the current human testicular cell culture system for clinical use.

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Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems

Cited by 34 publications

References 36 publications

Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold

Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold

In Vitro Spermatogenesis by Three-dimensional Culture of Spermatogonial Stem Cells on Decellularized Testicular Matrix

ITGA6+ Human Testicular Cell Populations Acquire a Mesenchymal Rather than Germ Cell Transcriptional Signature during Long-Term Culture

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