Transcriptional profiling of β-2M−SPα-6+THY1+ spermatogonial stem cells in human spermatogenesis

Givelet, Maëlle; Firlej, Virginie; Lassalle, Bruno; Gille, Anne Sophie; Lapoujade, Clémentine; Holtzman, Isabelle; Jarysta, Amandine; Haghighirad, Farahd; Dumont, Florent; Jacques, Sébastien; Letourneur, Franck; Pflumio, Françoise; Allemand, Isabelle; Patrat, Catherine; Thiounn, Nicolas; Wolf, Jean Philippe; Riou, Lydia; Barraud-Lange, Virginie; Fouchet, Pierre

doi:10.1016/j.stemcr.2022.02.017

Cited by 3 publications

(12 citation statements)

References 49 publications

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“…We studied the effects of O 2 tension on murine SSCs, the best-characterized model in mammals to date. To establish these SSCs cultures, adult α6 + c-Kit - β2M − and prepubertal EpCAM + β2M − germinal cell fractions were flow sorted and seeded on mitomycin-C-treated MEFs in low-serum medium in the presence of GDNF, FGF2 and GFRA1 at 21% O 2 as previously described ( Barroca et al, 2009 ; Corbineau et al, 2017 ; Dura et al, 2022 ; Givelet et al, 2022 ). The culture of murine SSCs is relatively heterogeneous, containing self-renewing undifferentiated spermatogonia and their immediate differentiating progeny.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, we investigated whether human SSC culture at 3.5% O 2 could improve the in vitro maintenance and propagation of human SSCs. Indeed, prepubertal and adult human SSCs show only a limited potential of maintenance in vitro up to 2–3 weeks of culture ( Wu et al, 2009 ; Medrano et al, 2016 ; Givelet et al, 2022 ). Thus, we tested the ability of the spermatogonial population expressing the markers α-6 integrin and THY1, and negative for the β-2 microglobulin, which we identified as highly enriched in SSCs, to form in vitro germinal clusters in medium supplemented with GDNF and FGF2 factors ( Givelet et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, prepubertal and adult human SSCs show only a limited potential of maintenance in vitro up to 2–3 weeks of culture ( Wu et al, 2009 ; Medrano et al, 2016 ; Givelet et al, 2022 ). Thus, we tested the ability of the spermatogonial population expressing the markers α-6 integrin and THY1, and negative for the β-2 microglobulin, which we identified as highly enriched in SSCs, to form in vitro germinal clusters in medium supplemented with GDNF and FGF2 factors ( Givelet et al, 2022 ). We previously demonstrated that PIWIL4, C19orf84, TSPAN33, PLPPR3, FGFR3, and UTF1 primitive spermatogonial markers were expressed in this population, and that it did not contain somatic cells ( Givelet et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

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Impact of the hypoxic microenvironment on spermatogonial stem cells in culture

Gille,

Givelet,

Pehlic

et al. 2024

Front. Cell Dev. Biol.

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The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Impact of the hypoxic microenvironment on spermatogonial stem cells in culture

Gille,

Givelet,

Pehlic

et al. 2024

Front. Cell Dev. Biol.

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“…We recently identified a spermatogonial subpopulation highly enriched in SSCs with the phenotype Side population(SP)β2M − ITGA6 + THY1 + population using a 5-color panel 12 . Based on this set of markers, we aimed to increase the panel to apply a multi-parameter flow cytometry strategy to further characterize human spermatogenesis and improve the delineation of SSCs identity.…”

Section: Resultsmentioning

confidence: 99%

“…We recently identified a spermatogonial subpopulation highly enriched in SSCs with the phenotype Side population(SP)b2M -ITGA6 + THY1 + population using a 5-color panel 12 .…”

Section: Development Of a 16-c Panel To Characterize Human Spermatoge...mentioning

confidence: 99%

Characterization and hierarchy of the spermatogonial stem cell compartment in human spermatogenesis by spectral cytometry using a 16-colors panel

Lapoujade,

Blanco,

Givelet

et al. 2023

Preprint

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About one in six couples experience fertility problems, and male infertility accounts for about half of these cases. Spermatogenesis originates from a small pool of spermatogonial stem cells (SSCs), which are of interest for the treatment of infertility but remain poorly characterized in humans. Using multiparametric spectral flow cytometric analysis with a 16-colours (16-C) panel of cell markers, we identify novel markers of SSCs and provide insights into unravelling and resolving the heterogeneity of the human spermatogonial cells. This 16-C panel of markers allowed the identification of a primitive SSCs state with the β-2M−CD51/61−ITGA6+SSEA4+TSPAN33+THY1+CD9+EPCAMmedCD155+CD148+CD47highCD7highphenotype, with a profile close to the most primitive SSCs states 0 and SSC1-B previously defined by sc-RNAseq approach. The hierarchy of events in the spermatogonial stem cell and progenitor compartment of human spermatogenesis has been delineated. This highlights the importance of a multi-parametric and spectral cytometry approach. The in-depth characterization of testicular cells should help to overcome the lack of stem cell knowledge, that hinders the understanding of the regenerative potential of SSCs, and is a critical parameter for the successful development of new SSCs-based cell therapies.

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Postnatal development of mouse spermatogonial stem cells as determined by immunophenotype, regenerative capacity, and long-term culture-initiating ability: a model for practical applications

Song,

Zhang,

Desmarais

et al. 2024

Sci Rep

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Spermatogonial stem cells (SSCs) are the foundation of life-long spermatogenesis. While SSC research has advanced greatly over the past two decades, characterization of SSCs during postnatal development has not been well documented. Using the mouse as a model, in this study, we defined the immunophenotypic profiles of testis cells during the course of postnatal development using multi-parameter flow cytometry with up to five cell-surface antigens. We found that the profiles progress over time in a manner specific to developmental stages. We then isolated multiple cell fractions at different developmental stages using fluorescent-activated cell sorting (FACS) and identified specific cell populations with prominent capacities to regenerate spermatogenesis upon transplantation and to initiate long-term SSC culture. The data indicated that the cell fraction with the highest level of regeneration capacity exhibited the most prominent potential to initiate SSC culture, regardless of age. Interestingly, refinement of cell fractionation using GFRA1 and KIT did not lead to further enrichment of regenerative and culture-initiating stem cells, suggesting that when a high degree of SSC enrichment is achieved, standard markers of SSC self-renewal or commitment may lose their effectiveness to distinguish cells at the stem cell state from committed progenitors. This study provides a significant information resource for future studies and practical applications of mammalian SSCs.

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Transcriptional profiling of β-2M−SPα-6+THY1+ spermatogonial stem cells in human spermatogenesis

Cited by 3 publications

References 49 publications

Impact of the hypoxic microenvironment on spermatogonial stem cells in culture

Impact of the hypoxic microenvironment on spermatogonial stem cells in culture

Characterization and hierarchy of the spermatogonial stem cell compartment in human spermatogenesis by spectral cytometry using a 16-colors panel

Postnatal development of mouse spermatogonial stem cells as determined by immunophenotype, regenerative capacity, and long-term culture-initiating ability: a model for practical applications

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