Human Spermatogonial Stem Cells Scrutinized under the Single-Cell Magnifying Glass

Tan, Kun; Wilkinson, Miles

doi:10.1016/j.stem.2019.01.010

Cited by 30 publications

(27 citation statements)

References 11 publications

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“…The direct functional implications of this heterogeneity are beyond the scope of the present study, but may indicate there are differential roles for individual SNAIs in the 3‐10 clusters of SSC subpopulations, as defined by some of the studies above (reviewed in ref. 61). The tight regulation of SNAI transcription factors in spermatogenesis is an ongoing area of interest for our laboratory.…”

Section: Discussionmentioning

confidence: 99%

‘Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway’

et al. 2020

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Background Snail transcription factors mediate key cellular transitions in many developmental processes, including spermatogenesis, and their production can be regulated by TGF‐β superfamily signalling. SNAI1 and SNAI2 support many cancers of epithelial origin. Their functional relevance and potential regulation by TGF‐β superfamily ligands in germ cell neoplasia are unknown. Methods SNAI1, SNAI2 and importin 5 (IPO5; nuclear transporter that selectively mediates BMP signalling) cellular localization was examined in fixed normal adult human and/or neoplastic testes using in situ hybridization and/or immunohistochemistry. SNAI1 and SNAI2 functions were assessed using the well‐characterized human seminoma cell line, TCam‐2. Cell migration, adhesion/proliferation and survival were measured by scratch assay, xCELLigence and flow cytometry following siRNA‐induced reduction of SNAI1 and SNAI2 in TCam‐2 cells. The potential regulation of SNAI1 and SNAI2 in TCam‐2 cells by TGF‐β signalling ligands, activin A and BMP4 was evaluated following 48 hours culture, including with siRNA regulation of IPO5 to selectively restrict BMP4 signalling. Results In normal testes, SNAI1 transcript was identified in some spermatogonia and in spermatocytes, and SNAI2 protein localized to nuclei of spermatogonia, spermatocytes and round spermatids. In neoplastic testes, both SNAI1 and SNAI2 were detected in GCNIS and in seminoma cells. SNAI1 and SNAI2 reduction in TCam‐2 cells by siRNAs significantly inhibited migration and survival, respectively. Exposure to BMP4, but not activin A, significantly increased SNAI2 (~18‐fold). IPO5 inhibition by siRNAs decreased BMP4‐induced SNAI2 upregulation (~5‐fold). Additionally, SNAI2 reduction using siRNAs inhibited BMP4‐induced TCam‐2 cell survival. Conclusions This is the first evidence that SNAI1 and SNAI2 are involved in human spermatogenesis, with independent functions. These outcomes demonstrate that SNAI1 and SNAI2 inhibition leads to loss of migratory and viability capacities in seminoma cells. These findings show the potential for therapeutic treatments targeting SNAIL or BMP4 signalling for patients with metastatic testicular germ cell tumours.

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Section: Discussionmentioning

confidence: 99%

‘Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway’

et al. 2020

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“…The continuous production of a large number of sperm depends on the self‐renewal and differentiation of SSCs (Suzuki & Withers, 1978; K. Tan & Wilkinson, 2019). To ensure the stability of normal physiological processes of spermatogenesis, a large number of transcription factors are needed to regulate the dynamic balance of self‐renewal and differentiation of SSCs (K. Tan & Wilkinson, 2019). For the low expression of well‐known self‐renewal maintaining transcription factors, we concluded that CD49f − cells were differentiated spermatogonia cells.…”

Section: Discussionmentioning

confidence: 99%

“…They are also known as male germline stem cells (mGSCs), which are capable of differentiating into a series of spermatocytes and sperm cells (Phillips, Gassei, & Orwig, 2010). Scientists have been using a variety of tools and methods to explore gametogenesis and germline‐niche communication (K. Tan & Wilkinson, 2019). Large amounts of progressions on mice and human SSCs study were obtained through a wide variety of approaches (Garbuzov et al, 2018; Kaomei et al, 2006; Kubota, Avarbock, & Brinster, 2004; Law & Oatley, 2020; Legrand et al, 2019; L. Li et al, 2017; N. Li et al, 2019; Park et al, 2019; Pech et al, 2015; Sohni et al, 2019a; K. Tan & Wilkinson, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Scientists have been using a variety of tools and methods to explore gametogenesis and germline‐niche communication (K. Tan & Wilkinson, 2019). Large amounts of progressions on mice and human SSCs study were obtained through a wide variety of approaches (Garbuzov et al, 2018; Kaomei et al, 2006; Kubota, Avarbock, & Brinster, 2004; Law & Oatley, 2020; Legrand et al, 2019; L. Li et al, 2017; N. Li et al, 2019; Park et al, 2019; Pech et al, 2015; Sohni et al, 2019a; K. Tan & Wilkinson, 2019). However, a little is known about SSCs from ruminants (Bahadorani et al, 2012; Park et al, 2019; Shi, Duan, Yao, Song, & Ren, 2020).…”

Section: Introductionmentioning

confidence: 99%

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PAX7 promotes CD49f‐positive dairy goat spermatogonial stem cells' self‐renewal

Wei

et al. 2020

Journal Cellular Physiology

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Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA-sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f + spermatogonia, and PAX7 can maintain the selfrenewal of CD49f-positive SSCs.

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“…Single cell RNA sequencing and bioinformatic analyses have revealed four type of undifferentiated spermatogonia in adult testis and three germ cell subsets in neonatal testis. UCHL1, PLZF and LIN28 are expressed both undifferentiated and differentiated spermatogonia (Sohni et al, 2019; Tan and Wilkinson, 2019). Because these transcripts are not specific for any subpopulation of undifferentiated spermatogonia, it is not possible to determine which subtype of spermatogonia is proliferating in vitro .…”

Section: Discussionmentioning

confidence: 99%

Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys

et al. 2019

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BackgroundGonadotoxic treatment of malignant diseases as well as some non-malignant conditions such as cryptorchidism in young boys may result in infertility and failure to father children later in life. As a fertility preserving strategy, several centers collect testicular biopsies to cryopreserve spermatogonial stem cells (SSCs) world-wide. One of the most promising therapeutic strategies is to transplant SSCs back into the seminiferous tubules to initiate endogenous spermatogenesis. However, to obtain sufficient numbers of SSC to warrant transplantation, in vitro propagation of cells is needed together with proper validation of their stem cell identity.Materials and MethodsA minute amount of testicular biopsies (between 5 mg and 10 mg) were processed by mechanical and enzymatic digestion. SSCs were enriched by differential plating method in StemPro-34 medium supplemented with several growth factors. SSC-like cell clusters (SSCLCs) were passaged five times. SSCLCs were identified by immunohistochemical and immunofluorescence staining, using protein expression patterns in testis biopsies as reference. Quantitative polymerase chain reaction analysis of SSC markers LIN-28 homolog A (LIN28A), G antigen 1 (GAGE1), promyelocytic leukemia zinc finger protein (PLZF), integrin alpha 6 (ITGA6), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and integrin beta 1 (ITGB1) were also used to validate the SSC-like cell identity.ResultsProliferation of SSCLCs was achieved. The presence of SSCs in SSCLCs was confirmed by positive immunostaining of LIN28, UCHL1 and quantitative polymerase chain reaction for LIN28A, UCHL1, PLZF, ITGA6, and ITGB1, respectively.ConclusionThis study has demonstrated that SSCs from infant boys possess the capacity for in vitro proliferation and advance a fertility preservation strategy for pre-pubertal boys who may otherwise lose their fertility.

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Human Spermatogonial Stem Cells Scrutinized under the Single-Cell Magnifying Glass

Cited by 30 publications

References 11 publications

‘Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway’

‘Snail factors in testicular germ cell tumours and their regulation by the BMP4 signalling pathway’

PAX7 promotes CD49f‐positive dairy goat spermatogonial stem cells' self‐renewal

Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys

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