Enrichment Of Spermatogonial Stem Cells Research Articles

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

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.

Characterization and enrichment of spermatogonial stem cells of common carp (Cyprinus carpio)

Spermatogenesis is a systematically organized process that ensures uninterrupted sperm production in which the spermatogonial stem cells (SSCs) play a crucial role. However, the existing absence of teleost-specific molecular markers for SSCs presents a notable challenge. Herein we characterized phenotypically the spermatogonial stem cells using specific molecular markers and transmission electron microscopy. Moreover, we also describe a simple method to suppress common carp spermatogenesis using the combination of Busulfan and thermo-chemical treatment, and finally, we isolate and enrich the undifferentiated spermatogonial fraction. Our results showed that C-kit, GFRα1, and POU2 proteins were expressed by germ cells, meanwhile, undifferentiated spermatogonial populations preferentially expressed GFRα1 and POU2. Moreover, the combination of high temperature (35 °C) and Busulfan (40 mg/kg/BW) effectively suppressed the spermatogenesis of common carp males. Additionally, the amh expression analysis showed differences between the control (26 °C) when compared to 35 °C with a single or two Busulfan doses, confirming that the testes were depleted by the association of Busulfan at high temperatures. In an attempt to isolate the undifferentiated spermatogonial fraction, we used the Percoll discontinuous density gradient. Thus, we successfully dissociated the carp whole testes in different cellular fractions; subsequently, we isolated and enriched the undifferentiated spermatogonial population. Therefore, our results suggest that probably both GFRα-1 and POU2 are highly conserved factors expressed in common carp germinative epithelium and that these molecules were well conserved along the evolutionary process. Furthermore, the enriched undifferentiated spermatogonial population developed here can be used in further germ cell transplantation experiments to preserve and propagate valued and endangered fish species.

P-798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis

Abstract Study question Can a three-dimensional (3D) prepubertal testicular organoid be formed and provide an in vitro microenvironment for spermatogonial stem cells (SSCs) maintenance and future spermatogenesis? Summary answer Primary cells extracted from immature testicular tissue (ITT) or SSCs can be grown long-term as 3D organoids, providing the potential for in vitro study. What is known already Aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Such patients are recommended to undergo the cryopreservation of testicular material to protect future fertility. Within the testes, the specialized 3D structure and direct cell-to-cell interactions play a critical role in the proliferation and development of SSCs. Over recent decades, 3D culture systems and organoids have been used to culture cells in vitro, however, a system that allows investigations into testicular organogenesis in vitro, and its impact on the SSC niche, has yet to be developed. Study design, size, duration This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated by in vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements. Participants/materials, setting, methods Post-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP-9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα-1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Main results and the role of chance The viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (<10% SSCs) and enriched SSCs(>50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit either a grape-like structure and a round-shape structure. Cytoplasmic extensions of spermatogonia/Sertoli cells were in contact with each other within a forming colony. Organoids were formed faster and larger when seeded at a final concentration of 1.5 × 106 cells/ml, compared to 5 × 105 cells/ml and 1.5 × 105 cells/ml. Organoids grew to a diameter of 400 μm within 10 - 15 days and were passaged by mechanical disruption at a ratio of 1:3 every 7 - 10 days. Immunocytochemistry results showed that clusters of PGP9.5 and PLZF-positive cells were present within the organoids. The expression of selected germ cell and spermatogenesis markers in the testicular organoids closely resembled that of primary testicular cells for up to 20 days of culture. Limitations, reasons for caution We used calves (2 weeks-of-age) as an animal model to study testicular organoids. This tissue may act differently than human tissues and may not fully represent prepuberty. Furthermore, we only evaluated gene expression levels for selected markers that may not represent the full functional capability of germ cells. Wider implications of the findings Testicular organoids, as an in vitro bioengineering testicular model, could potentially be used to study testicular tissue development, cellular interactions, endocrinology, and spermatogenesis, in the laboratory but may also be applied for clinical purposes in the future. Trial registration number University of Oxford

P–798 Fertility preservation in pre-pubertal boys with cancer: A three-dimensional prepubertal testicular organoid for in vitro spermatogonial stem cell propagation and spermatogenesis

Abstract Study question Can a three-dimensional (3D) prepubertal testicular organoid be formed and provide an in vitro microenvironment for spermatogonial stem cells (SSCs) maintenance and future spermatogenesis? Summary answer Primary cells extracted from immature testicular tissue (ITT) or SSCs can be grown long-term as 3D organoids, providing the potential for in vitro study. What is known already Aggressive cancer treatments, such as chemo- or radiotherapy, can leave young prepubertal boys infertile. Such patients are recommended to undergo the cryopreservation of testicular material to protect future fertility. Within the testes, the specialized 3D structure and direct cell-to-cell interactions play a critical role in the proliferation and development of SSCs. Over recent decades, 3D culture systems and organoids have been used to culture cells in vitro, however, a system that allows investigations into testicular organogenesis in vitro, and its impact on the SSC niche, has yet to be developed. Study design, size, duration This study aims to develop a 3D organoid culture system to support the proliferation of SSCs and spermatogenesis. Primary bovine ITT cells and enriched SSCs were isolated and 3D organoids were generated by in vitro culture for up to 40 days. Organoid formation was observed after using different foundation cells seeded at different densities and cultured in medium containing gonadotropic supplements. Participants/materials, setting, methods Post-thaw bovine ITTs (2 weeks-of-age) were dissociated using two-step enzymatic digestion. Enriched SSCs were selected by Percoll gradients and differential plating. Viability and apoptosis were evaluated by trypan blue staining and TUNEL assays, respectively. SSCs were evaluated immunocytochemically for germ-cell markers (PGP–9.5, PLZF) and Sertoli cell markers (Vimentin, Sox9). Expression levels of SSCs and spermatogenesis-related genes (Plzf, Gfrα–1, Nanog, Oct4, Stra8, Thy1) were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Main results and the role of chance The viability of digested cells from thawed ITTs was 78.667% ± 2.03. Total testicular cells (<10% SSCs) and enriched SSCs(>50% SSCs) were observed to self-assemble into structurally complex organoids recapitulating the cell type compartmentalization of the testis, in a 3D Matrigel-based culture system with 10% knockout serum replacement (KSR) culture medium, but not with 10% fetal bovine serum(FBS) medium. Testicular organoids were found to exhibit either a grape-like structure and a round-shape structure. Cytoplasmic extensions of spermatogonia/Sertoli cells were in contact with each other within a forming colony. Organoids were formed faster and larger when seeded at a final concentration of 1.5 × 106 cells/ml, compared to 5 × 105 cells/ml and 1.5 × 105 cells/ml. Organoids grew to a diameter of 400 μm within 10 - 15 days and were passaged by mechanical disruption at a ratio of 1:3 every 7 - 10 days. Immunocytochemistry results showed that clusters of PGP9.5 and PLZF-positive cells were present within the organoids. The expression of selected germ cell and spermatogenesis markers in the testicular organoids closely resembled that of primary testicular cells for up to 20 days of culture. Limitations, reasons for caution We used calves (2 weeks-of-age) as an animal model to study testicular organoids. This tissue may act differently than human tissues and may not fully represent prepuberty. Furthermore, we only evaluated gene expression levels for selected markers that may not represent the full functional capability of germ cells. Wider implications of the findings: Testicular organoids, as an in vitro bioengineering testicular model, could potentially be used to study testicular tissue development, cellular interactions, endocrinology, and spermatogenesis, in the laboratory but may also be applied for clinical purposes in the future. Trial registration number University of Oxford

Expression profile of spermatogenesis associated genes in male germ cells during postnatal development in mice

Spermatogonial stem cells are self-renewal and differentiate into sperm in post-pubertal mammals. There exists a balance between the self-renewal and differentiation in the testes. Spermatogonial stem cells make up only 0.03% of testicular cells in adult mice. These cells maintain sperm production by differentiating after puberty. Therefore, analyzing the expression of genes associated with spermatogenesis is critical for understanding differentiation. The present study aimed to establish the postnatal period of cells in relation to spermatogenesis. To study the expression of differentiated and undifferentiated marker genes in enriched spermatogonial stem cells, in vitro culture was performed and cells from pup (6–8-day-old) and adult (4-months-old) testicular tissues were isolated. As a result, undifferentiated genes, Pax7, Plzf, GFRa1, Etv5 and Bcl6b , were highly increased in cultured spermaotogonial stem cells compared with pup and adult testicular cells. On the other hands, differentiated gene, c-kit was highly increased in adult testicular cells, Also Stra8 gene was highly increased in pup and adult testicular cells. This study provides a better understanding of spermatogenesis-associated gene expression during postnatal periods.

Homologous transplantation of fluorescently labelled enriched buffalo (Bubalus bubalis) spermatogonial stem cells to prepubertal recipients

Spermatogonial stem cell transplantation provides a unique opportunity to study the biology of spermatogenesis and also offers an alternative approach for genetic modification in large animals. The present study aimed to extend this technique to the water buffalo. Spermatogonial stem cells (SSCs) were isolated from prepubertal buffalo testes (3-6 months of age) using two-step enzymatic digestion method and enriched by differential plating and Percoll density gradient centrifugation. The enriched SSCs expressed numerous spermatogonial transcriptional markers, viz. ID4, THY1, BCL6B, UCHL1, ETV5 and REX1 which confirmed their bonafide SSC identity. Subsequently, the enriched SSCs were labelled with a fluorescent dye PKH26 and transplanted into buffalo calves under ultrasound guidance. The recipient testes were recovered after 7-8 weeks by castration and their fluorescence microscopebased examination exhibited the persistence and localization of the fluorescent donor cells within the recipient seminiferous tubules. Further validation was done by the flow cytometric evaluation of PKH26 labeled donor cells among those isolated by two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated the feasibility of SSC transplantation technique in the water buffalo.

Stage-specific embryonic antigen 4 is a membrane marker for enrichment of porcine spermatogonial stem cells.

Spermatogonial stem cells (SSCs), as tissue-specific stem cells, are capable of both self-renewal and differentiation and supporting the continual and robust spermatogenesis for male fertility. As a rare sub-fraction of undifferentiated spermatogonia, SSCs share most molecular markers with the progenitor spermatogonia. Thus, the heterogeneity of the progenitor cells often obscures the characteristics of stem cells. Distinguishing SSCs from the progenitors is of paramount importance to understand the regulatory mechanisms governing their actions. The present study was designed to reveal that SSEA4 can be a marker for putative porcine SSCs that distinguished it from the progenitors and to build a sorting program for efficient enrichment of porcine SSCs. To explore expression of SSEA4 within the undifferentiated spermatogonial population, we performed co-immunofluorescent staining for SSEA4 and common molecular markers (VASA, DBA, PLZF, c-KIT, and SOX9) in the 7-, 90-, and 150-day-old porcine testicular tissues. SSEA4-positive cells were isolated from the 90-day-old porcine testes by flow cytometry. Immunofluorescent, RNA-sequencing, and transplantation analysis were used to reveal that SSEA4-positive fraction holds the stem cell capacity. We found that SSEA4 was expressed in a rare sub-fraction of porcine undifferentiated spermatogonia, and RNA-sequencing analysis revealed that the genes for stem cell maintenance and SSC-specific markers (ID4 and PAX7) were up-regulated in the SSEA4-sorted fraction, compared with undifferentiated spermatogonia. In addition, germ cell transplantation assay demonstrated that SSEA4-positive spermatogonia colonized in the recipient testicular tubules. Sorting of the undifferentiated spermatogonia with anti-SSEA4 antibody resulted in a 2.5-fold enrichment of SSCs compared with the germ cell gate group, and 21-fold enrichment of SSCs compared with the SSEA4-negative spermatogonia group. Our findings revealed that SSEA4 is a new surface marker for porcine undifferentiated spermatogonia. This finding helps to elucidate the characteristics of porcine SSCs and facilitates the culture and manipulation of SSCs.

Transcriptome profiling reveals signaling conditions dictating human spermatogonia fate in vitro

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-β, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.

Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions.

In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.

Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture

The present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9<sup>th</sup>, 30<sup>th</sup> and 65<sup>th</sup> day) for the development of long-term culture method. The testes from 7–15-day old piglets were collected and two-step enzymatic digestion was used to isolate SSCs. Before in vitro culture of SSC-like cells on the Sertoli cell feeder layer, the cells were enriched by differential plating and Percoll density gradient centrifugation. The isolated SSCs were characterised by alkaline phosphatase and immunofluorescence staining and qPCR analysis of SSC specific marker genes. Stemness was compared based on the expression of different SSC specific marker genes. The putative spermatogonial stem cells (PSSCs) from all the days of culture were found to be positive for alkaline phosphatase and immunofluorescence staining. The results from qPCR analysis showed that PSSCs were positive for SSC marker genes, though their expression decreased gradually from day 9 to day 65 of culture. The shape of the cells was found to change from compact round or oval to amorphous shape on day 65 of culture. Colony diameter ranged from 68.92 ± 1.20 µm (day 9) to 213.53 ± 12.52 µm (day 65) and differed significantly from each other. The number of colonies on day 65 of culture was significantly lower than on days 9 and 30. These results suggest that the enriched SSCs from Doom pigs can be maintained up to two months in vitro in the present culture system.

The Progresses of Spermatogonial Stem Cells Sorting Using Fluorescence-Activated Cell Sorting.

In recent years, the research on stem cells has been more and more in-depth, and many achievements have been made in application. However, due to the small number of spermatogonial stem cells (SSCs) and deficiency of efficient markers, it is difficult to obtain very pure SSCs, which results in the research on them being hindered. In fact, many methods have been developed to isolate and purify SSCs, but these methods have their shortcomings. Fluorescence-activated cell sorting (FACS), as a method to enrich SSCs with the help of specific surface markers, has the characteristics of high efficiency and accuracy in enrichment of SSCs, thus it is widely accepted as an effective method for purification of SSCs. This review summarizes the recent studies on the application of FACS in SSCs, and introduces some commonly used markers of effective SSCs sorting, aiming to further optimize the FACS sorting method for SSCs, so as to promote the research of germline stem cells and provide new ideas for the research of reproductive biology.

Successful transplantation of transfected enriched buffalo (Bubalus bubalis) spermatogonial stem cells to homologous recipients

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ± 4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ± 0.25%) and lipofectamine 3000 (15.57 ± 0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.

Colonization of Mouse Spermatogonial Cells in Modified Soft Agar Culture System Utilizing Nanofibrous Scaffold: A New Approach.

Background: Spermatogonial stem cells (SSCs) are considered in fertility management approaches of prepubertal boys facing cancer therapies. However, in vitro propagation has become an important issue due to a small number of SSCs in testicular tissue. The present study aimed to investigate a modified soft agar culture system by using a nanofibrous scaffold as a new approach to mimic in vivo conditions of SSCs development. Materials and Methods: The SSCs were isolated from neonate mouse testes, cultured on polycaprolactone scaffold, and covered by a layer of soft agar for 2 weeks. Then, the number and diameter of colonies formed in experimental groups were measured and spermatogonial markers (i.e., Plzf, Gfrα1, Id4, and c-Kit) in SSCs colonies were evaluated by a real-time polymerase chain reaction and immunostaining. Results: Our results indicated that the colonization rate of SSCs was significantly higher in the present modified soft agar culture system (P<0.05). Only Plzf indicated a significant increased at the levels (P<0.05), the gene expression levels of Id4, Plzf, and Gfrα1 were higher in the present culture system. In addition, the expression of the c-Kit gene as a differentiating spermatogonia marker was higher in presence of scaffold and soft agar compared with the amount of other experimental groups (P<0.05). Conclusion: The culture system by using nanofibrous scaffold and soft agar as a new culture method suggests the potential of this approach in SSCs enrichment and differentiation strategies for male infertility treatments, as well as in vitro spermatogenesis.

Colonization of Mouse Spermatogonial Cells in Modified Soft Agar Culture System Utilizing Nanofibrous Scaffold: A New Approach

Background: Spermatogonial stem cells (SSCs) are considered in fertility management approaches of prepubertal boys facing cancer therapies. However, in vitro propagation has become an important issue due to a small number of SSCs in testicular tissue. The present study aimed to investigate a modified soft agar culture system by using a nanofibrous scaffold as a new approach to mimic in vivo conditions of SSCs development. Materials and Methods: The SSCs were isolated from neonate mouse testes, cultured on polycaprolactone scaffold, and covered by a layer of soft agar for 2 weeks. Then, the number and diameter of colonies formed in experimental groups were measured and spermatogonial markers (i.e., Plzf, Gfrα1, Id4, and c-Kit) in SSCs colonies were evaluated by a real-time polymerase chain reaction and immunostaining. Results: Our results indicated that the colonization rate of SSCs was significantly higher in the present modified soft agar culture system (P&lt;0.05). Only Plzf indicated a significant increased at the levels (P&lt;0.05), the gene expression levels of Id4, Plzf, and Gfrα1 were higher in the present culture system. In addition, the expression of the c-Kit gene as a differentiating spermatogonia marker was higher in presence of scaffold and soft agar compared with the amount of other experimental groups (P&lt;0.05). Conclusion: The culture system by using nanofibrous scaffold and soft agar as a new culture method suggests the potential of this approach in SSCs enrichment and differentiation strategies for male infertility treatments, as well as in vitro spermatogenesis. [GMJ.2019;8:e1319]

A novel undifferentiated spermatogonia-specific surface protein 1 (USSP1) in neonatal mice

Mammalian spermatogenesis is maintained by a rare population of spermatogonial stem cells (SSCs), which are important for male fertility. SSCs remain a subset of undifferentiated spermatogonia, which can be isolated by a combination of surface markers. Specific markers to identify and isolate undifferentiated spermatogonia are lacking. Ussp1, a transcript previously annotated as long noncoding RNA (RIKEN cDNA 4933427D06, Gene ID: 232217), virtually encodes a membrane protein, USSP1, in a highly testis-specific manner in mouse. We demonstrate its expression on the membrane of undifferentiated spermatogonia by a homemade polyclonal rabbit antibody against the protein. In vivo, USSP1+ clusters consist mainly of As, Apr (GFRα1+) and Aal (PLZF+) cells. USSP1+ cells exhibit enrichment of undifferentiated spermatogonia, as shown by increased expression of SSC self-renewal molecular markers and the potential to form SSC clones in vitro and in vivo. However, Ussp1 knockout did not affect the number of SSCs or spermatogenesis in mice. Thy1+ cells from Ussp1 null mice did not show any defect in the SSC colony formation capacity, indicating that USSP1 is not essential for SSC self-renewal. Our data demonstrate that Ussp1 is specifically expressed in undifferentiated murine spermatogonia, indicating the potential to sort undifferentiated spermatogonia with USSP1 antibodies. Ussp1 might be a good maker for SSC enrichment in neonatal mice.

Isolation and Enrichment of Spermatogonial Stem Cells From Human Testis Tissues.

This article describes a protocol for isolating human spermatogonial stem cells (hSSCs) from human testis tissues, which can be utilized for further characterization. Human testis tissues are first subjected to mechanical separation, enzymatic digestion, and physical filtering, yielding a single testicular cell suspension. SSCs or other cell types of interest can be sorted out via different enrichment approaches using selected cell surface markers (e.g., magnetic-activated cell sorting, MACS). Those enriched cells can further be used for different purposes, including cell culture, RNA sequencing, and other genomics-based profiling, which allow for further characterization of their properties. © 2019 by John Wiley & Sons, Inc.

Sendai virus-mediated transduction of mammalian spermatogonial stem cells†.

Spermatogonial stem cells (SSCs) provide the foundation of spermatogenesis. However, because of their small number and slow self-renewal, transfection of SSCs has met with limited success. Although several viral vectors can infect SSCs, genome integration and an inability to maintain long-term gene expression have hampered studies on SSCs. Here we report successful SSC infection by Sendai virus (SV), an RNA virus in the Paramyxoviridae. The SV efficiently transduced germline stem (GS) cells, cultured spermatogonia with enriched SSC activity, and maintained gene expression for at least 5 months. It also infected freshly isolated SSCs from adult testes. The transfected GS cells reinitiated spermatogenesis following spermatogonial transplantation into seminiferous tubules of infertile mice, suggesting that SV transfection does not interfere with spermatogenesis progression. On the other hand, microinjection of SV into the seminiferous tubules of immature mice transduced SSCs and Sertoli cells, but did not transduce Leydig or peritubular cells by interstitial virus injection. SV-infected hamster GS cells, and freshly isolated rabbit or monkey SSC-like cells were identified following xenogeneic spermatogonial transplantation, suggesting that SV transduces SSCs from several mammalian species. Thus, SV is a useful vector that can transduce both SSCs and Sertoli cells and overcome problems associated with other viral vectors.

Phospholipase D Family Member 6 Is a Surface Marker for Enrichment of Undifferentiated Spermatogonia in Prepubertal Boars.

Spermatogonial stem cells (SSCs) have a functional ability to maintain self-renewal and sustain production of spermatozoa throughout the reproductive lifespan of a male. Studies on SSCs can thus better the understandings of spermatogenesis and unravel the mechanisms for self-renewal and differentiation of male germline stem cells. However, the rarity of SSCs in the testis and the lack of reliable surface markers obstruct the related study and further application of SSCs. This is especially the case in livestock animals. In this study, we identified that phospholipase D family member 6 (PLD6) is a surface marker for undifferentiated spermatogonia in boar testes. By magnetic-activated cell sorting, PLD6+ cell fraction comprises 84.45% ± 0.35% of undifferentiated spermatogonia (marked by PLZF). Xenotransplantation of PLD6+ cells into the recipient mouse testis revealed a ninefold increase of donor cell-derived colony formation compared with that in the unselected cell group, indicating the significant enrichment of SSCs. Furthermore, based on the sorted PLD6+ cells with a high SSC content, we established a feeder-free culture system that could maintain porcine undifferentiated spermatogonia for 4 weeks in vitro with the expression of typical markers throughout the culture period. In conclusion, this study demonstrates that PLD6 is a surface marker of undifferentiated spermatogonia in testes of prepubertal boars and could be utilized to unprecedentedly enrich porcine undifferentiated spermatogonia. These data provide the basis for future studies on the refinement of germ cell culture and manipulation of porcine undifferentiated spermatogonia.

Embryonic stem cell derived germ cells induce spermatogenesis after transplantation into the testes of an adult mouse azoospermia model.

The present study aimed to: (i) identify the exogenous factors that allow in vitro differentiation of mouse spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs); (ii) evaluate the effects of Sertoli cells in SSC enrichment; and (iii) assess the success of transplantation using in vitro differentiated SSCs in a mouse busulfan-treated azoospermia model. A 1-day-old embryoid body (EB) received 5 ng/ml of bone morphogenetic protein 4 (BMP4) for 4 days, 3 µM retinoic acid (RA) in a SIM mouse embryo-derived thioguanine and ouabain resistant (STO) co-culture system for 7 days, and was subsequently co-cultured for 2 days with Sertoli cells in the presence or absence of a leukaemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and RA composition, and in the presence of these factors in simple culture medium. Higher viability, proliferation and germ cell gene expression were seen in the presence of the LIF, bFGF and RA composition, on top of Sertoli cells. Immunocytochemistry results showed higher CDH1 expression in this group. Sertoli co-culture had no effects on SSC proliferation. Eight weeks after transplantation, injected cells were observed at the base of the seminiferous tubules and in the recipient testes. The number of spermatogonia and the mass of the testes were higher in transplanted testes relative to the control group. It seems that transplantation of these cells can be useful in infertility treatment.

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.