Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system

Sun, Min; Yuan, Quan; Niu, Minghui; Wang, Hong; Wen, Lei; Yao, Chencheng; Hou, Jingmei; Chen, Zheng; Fu, Han; Zhou, Fan; Li, Chong; Gao, Shaorong; Gao, Wei‐Qiang; Li, Zheng; He, Zuping

doi:10.1038/s41418-017-0015-1

Cited by 73 publications

(62 citation statements)

References 52 publications

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“…Besides FSH addition in both media at a concentration of 5 UI/L, in one medium (M1) some molecules known to play a role in GC survival and differentiation were added, like hCG, which stimulates the production of testosterone, known as a differentiation and survival factor for GC ( Sharpe, 1987 ). Moreover, M1 was also supplemented with retinoic acid (RA), which has been exploited in culture of human adult tissue with achievement of meiosis ( Yang et al, 2014 ; Perrard et al, 2016 ; Sun et al, 2018 ). However, while addition of RA to the culture media did not allow GC differentiation in our previous work despite SCs maturation ( de Michele et al, 2017a ), GC differentiation was obtained in this work in M2 in the absence of RA.…”

Section: Discussionmentioning

confidence: 99%

“…It is however worth noting that spermatozoa-like cells were obtained after culture in chitosan bioreactors of seminiferous tubules retrieved from transsexual men which had impaired spermatogenesis leaving spermatogonia as residual germ cells in the tissue after hormonal therapy ( Perrard et al, 2016 ). More recently, Sun et al, reported the differentiation of SSCs retrieved from azoospermic adult patients and cultured in a Matrigel system up to haploid cells able to fecundate mouse oocytes ( Sun et al, 2018 ). All other attempts to in vitro differentiate human germ cells (GCs) exploited mature testicular tissue and only allowed the achievement of some steps of GC differentiation in organotypic ( Steinberger et al, 1964 ; Tesarik et al, 2000b ; Roulet et al, 2006 ), or 3D ( Lee et al, 2006 , 2007 ; Yang et al, 2014 ) culture.…”

Section: Introductionmentioning

confidence: 99%

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Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys

et al. 2018

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While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen–thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by the increase in STAR expression (p ≤ 0.007) and a peak in testosterone secretion, followed by a reduction (p ≤ 0.001) with stabilization. According to our knowledge, this is the first report of generation of haploid cells in human ITT. Differentiating germ cells have to be further evaluated for their ability to complete differentiation, their fecundability and epigenetic characteristics.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys

et al. 2018

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“…First, an efficient approach is required to be developed to differentiate SSCLCs into haploid cells. Sun et al reported that only 6.1% of haploid cells were generated from hSSCs derived OA patients by two-dimensional-induced (2D-I) differentiation system, but 17.9% of haploid cells were produced from hSSCs by three-dimensional-induced system (3D-I) [ 48 ]. In our study, we used 2D-I system to differentiate SSCLCs into haploid cells, thus, in the further research, 3D-I system is needed for the differentiation of SSCLCs into haploid cells.…”

Section: Discussionmentioning

confidence: 99%

Derivation and propagation of spermatogonial stem cells from human pluripotent cells

Yang

Tian

et al. 2020

Stem Cell Res Ther

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Objectives This study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs). Methods hPSCs were differentiated into SSC-like cells (SSCLCs) by a three-step strategy. The biological characteristics of SSCLCs were detected by immunostaining with antibodies against SSC markers. The ability of self-renewal was measured by propagating for a long time and still maintaining SSCs morphological property. The differentiation potential of SSCLCs was determined by the generation of spermatocytes and haploid cells, which were identified by immunostaining and flow cytometry. The transcriptome analysis of SSCLCs was performed by RNA sequencing. The biological function of SSCLCs was assessed by xeno-transplantation into busulfan-treated mouse testes. Results SSCLCs were efficiently generated by a 3-step strategy. The SSCLCs displayed a grape-like morphology and expressed SSC markers. Moreover, SSCLCs could be propagated for approximately 4 months and still maintained their morphological properties. Furthermore, SSCLCs could differentiate into spermatocytes and haploid cells. In addition, SSCLCs displayed a similar gene expression pattern as human GPR125+ spermatogonia derived from human testicular tissues. And more, SSCLCs could survive and home at the base membrane of seminiferous tubules. Conclusion SSCLCs were successfully derived from hPSCs and propagated for a long time. The SSCLCs resembled their counterpart human GPR125+ spermatogonia, as evidenced by the grape-like morphology, transcriptome, homing, and functional characteristics. Therefore, hPSC-derived SSCLCs may provide a reliable cell source for studying human SSCs biological properties, disease modeling, and drug toxicity screening.

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“…Other research groups revealed similar germ cell development using Vero cells to support the differentiation of spermatocytes in vitro [36]. More recently, in vitro generation of human haploid spermatids, able to develop into embryos after ROSI in murine oocytes, were obtained from SSCs of adult patients with cryptorchidism [37] and obstructive azoospermia [38].…”

Section: Perspectives and Challenges Of Fertility Restoration Strategiesmentioning

confidence: 95%

Fertility sparing strategies for pre- and peripubertal male cancer patients

Stukenborg

Wyns

2020

ecancer

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Genetic parenthood following cancer therapy is considered to be a major factor of quality of life. Given the rising proportion of patients surviving cancer due to improved therapeutic protocols, it is an issue of growing importance. Hence, the efforts to preserve fertility have motivated researchers to develop options for the paediatric population facing fertility-threatening cancer therapies. In prepubertal boys who do not yet produce sperm, cryo-banking of testicular tissue containing spermatogonial stem cells (SSCs) is the only viable option for future fertility preservation. While proposed in a number of clinics worldwide, however, this strategy remains still experimental. Transplanting the SSCs, or testicular tissue containing SSCs, back to the cured patient appears the most promising strategy. However, experiments performed with human testicular tissue in mice models reveal spermatogonial loss after transplantation, indicating the need for further optimisation of the transplantation procedure. The approach further poses the risk of reintroducing tumour cells back to the patient. In cases of haematological and blood-metastasising malignancies, in vitro generation of sperm combined with assisted reproductive technologies (ART), is the only possibility, avoiding reintroducing cancer cells. Although xenotransplantation would allow to recover sperm cells for ART being thus on the safe side with regard to cancer cells, the risk of infections with xeno-microbiological agents makes this option incompatible with clinical application. So far, offspring from in vitro matured sperm has only been achieved in mice. While human haploid germ cells, showing specific morphological features, expression of post-meiotic markers, as well as DNA and chromosome content, as well as fertilisation and development capacity, have been obtained by culturing spermatogonia or immature testicular tissue, the functionality of these cells still needs to be demonstrated. Despite the promising results obtained in recent years, further research is urgently warranted to establish a clinical tool offering these boys a fertility restoration option in the future. This minireview will focus on current achievements and future challenges of fertility preservation in young boys and underscore the next steps required to translate experimental strategies into clinical practice.

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Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system

Cited by 73 publications

References 52 publications

Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys

Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue From Prepubertal Boys

Derivation and propagation of spermatogonial stem cells from human pluripotent cells

Fertility sparing strategies for pre- and peripubertal male cancer patients

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