Propagation Of Spermatogonial Stem Cells Research Articles

Optimal isolation, culture, and invitro propagation of spermatogonial stem cells in Huaixiang chicken.

Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs invitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.

Fathering a child after childhood cancer treatments

AbstractAmong many deleterious ramifications of oncological treatments, there is permanent male infertility due to the damage to spermatogonial stem cells (SSC) in the testes after chemotherapy or irradiation. For those patients that cannot produce sperm before cancer treatment, because of prepubertal age, there are no clinical options available to father a child. To preserve fertility in childhood cancer patients, freezing of a testis biopsy is already offered before cancer treatment, while fertility treatment options using this biopsy are still under development, including spermatogonial stem cell transplantation (SSCT). SSCT requires isolation and in vitro propagation of spermatogonial stem cells from the cryopreserved biopsy, followed by autologous transplantation back to the adult cancer survivor. Given the implications of this potential stem cell therapy to recipients, their partners, and future offspring, we here aim to thoroughly appraise the state-of-the-art of SSCT focussing on safety for both patient and his future children.

Derivation and propagation of spermatogonial stem cells from human pluripotent cells

ObjectivesThis study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs).MethodshPSCs 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.ResultsSSCLCs 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.ConclusionSSCLCs 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.

Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys.

Spermatogonial stem cell (SSC) transplantation therapy is a promising strategy to renew spermatogenesis for prepubertal boys whose fertility is compromised. However, propagation of SSCs is required due to a limited number of SSCs in cryopreserved testicular tissue. This propagation must be done under xeno-free conditions for clinical application. SSCs were propagated from infant testicular tissue (7 mg and 10 mg) from two boys under xeno-free conditions using human platelet lysate and nutrient source. We verified SSC-like cell clusters (SSCLCs) by quantitative real-time polymerase chain reaction (PCR) and immune-reaction assay using the SSC markers undifferentiated embryonic cell transcription factor 1 (UTF1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), GDNF receptor alpha-1 (GFRα-1) Fα and promyelocytic leukaemia zinc finger protein (PLZF). The functionality of the propagated SSCs was investigated by pre-labelling using green fluorescent Cell Linker PKH67 and xeno-transplantation of the SSCLCs into busulfan-treated, therefore sterile, immunodeficient mice. SSC-like cell clusters (SSCLCs) appeared after 2 weeks in primary passage. The SSCLCs were SSC-like as the UTF1, UCHL1, GFRα1 and PLZF were all positive. After 2.5 months’ culture period, a total of 13 million cells from one sample were harvested for xenotransplantation. Labelled human propagated SSCs were identified and verified in mouse seminiferous tubules at 3–6 weeks, confirming that the transplanted cells contain SSCLCs. The present xeno-free clinical culture protocol allows propagation of SSCs from infant boys.

The optimized condition for the isolation and in vitro propagation of mouse spermatogonial stem cells.

The ability for isolation and in vitro propagation of spermatogonial stem cells (SSCs) offer a base for studies on spermatogenesis, and also contribute to the development of new methods for the preservation of livestock and animal genetic modification. The aim of this study was to find the optimal isolation and culture condition for efficient propagation of SSCs. Three different isolation methods (mechanical, one-, and two-step enzymatic digestion) were compared to find the optimal isolation method. To find the best culture conditions for in vitro propagation, isolated SSCs were cultured for 7 days in three different culture conditions supplemented with 10% FBS, 0.25% BSA, and 10% KSR, respectively. The result showed that two-step enzymatic digestion produced a significant high fraction of live cells compared the other two. Non-adhering cells collected after 48 hr and cultured in BSA- and KSR-supplemented medium had a significantly high number of SSCs clump formation compared to FBS-supplemented group. The expression of CD9 confirmed that cell clumps were SSCs clumps. Spermatogonial stem cells cultured in BSA-supplemented medium were positive for NGN3 and PLZF expressions, whereas negative for Stra8 (a meiotic-specific gene) expression, suggesting that most of the cells were undifferentiated SSCs in BSA culture system. In contrast, in FBS- and KSR-supplemented groups, the SSCs were positive forNGN3, PLZF, and Stra8. These data revealed that two-step enzymatic digestion is the best method for the isolation, and 0.25% BSA-supplemented culture condition is effective for optimal in vitro propagation of SSCs.

Bisphenol A induced apoptosis and transcriptome differences of spermatogonial stem cells in vitro.

Bisphenol A (BPA) is widely used as an industrial plasticizer, which is also an endocrine disruptor and considered to have adverse effects on reproduction. In male mammals, the long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). However, little is known about the effects of BPA on the viability of SSCs. To investigate the influence of BPA exposure on SSCs in vitro, we isolated SSCs from mouse and successfully established in vitro propagation of SSCs. After BPA treatment, we found that BPA reduced the viability of SSCs and induced SSC apoptosis. For revealing the transcriptome differences of the BPA-treated SSCs, we performed high-throughput RNA sequencing and found that 860 genes were differentially expressed among 18,272 observed genes. The gene ontology (GO) terms, regulation of programmed cell death and apoptotic process, were enriched in the differentially expressed genes (DEGs). Among the cluster of DEGs associated with the kyoto encyclopedia of genes and genomes (KEGG) apoptosis pathway, activating transcription factor 4 (Atf4) and DNA damage inducible transcript 3 (Ddit3) genes were significantly up-regulated in BPA-treated SSCs, which were proved by qPCR. Taken together, these findings suggest that BPA can increase the mRNA expression of pro-apoptosis genes and reduce the viability of SSCs by inducing apoptosis.

In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells

The invitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells invitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the invitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation.

Eliminating acute lymphoblastic leukemia cells from human testicular cell cultures: a pilot study

To study whether acute lymphoblastic leukemia (ALL) cells survive in a human testicular cell culture system. Experimental laboratory study. Reproductive biology laboratory, academic medical center. Acute lymphoblastic leukemia cells from three patients and testicular cells from three other patients. Acute lymphoblastic leukemia cells were cultured alone or in combination with testicular cells, at various concentrations, in a system that has recently been developed to propagate human spermatogonial stem cells. Viability of ALL and testicular cells during culture was evaluated by flow cytometry using markers for live/dead cells. Furthermore, the presence of ALL cells among testicular cells was determined by highly sensitive (1:10,000 to 1:100,000 cells) patient-specific antigen-receptor minimal residual disease polymerase chain reaction. The presence of spermatogonia at the end of culture was determined by reverse transcription-polymerase chain reaction for ZBTB16, UCHL1, and GPR125. The ALL cells cultured separately did not survive beyond 14 days of culture. When cultured together with testicular cells, even at extremely high initial concentrations (40% ALL cells), ALL cells were undetectable beyond 26 days of culture. Reverse transcription-polymerase chain reaction confirmed the presence of spermatogonia at the end of the culture period. Our pilot study shows that the described testicular cell culture system not only allows for efficient propagation of spermatogonial stem cells but also eliminates contaminating ALL cells.

New advances on the expansion and storage of human spermatogonial stem cells

Fertility in adult life can be severely impaired by gonadotoxic therapies and with remarkable advancements in the treatment of childhood cancers there is a growing population of adult survivors of childhood malignancies. The aim of the study is to review the developments that have been made in spermatogonial stem cell research and potential future utility in fertility preservation. Whereas intense interest and subsequent research surrounds the regenerative potential of spermatogonial stem cells, a recent article highlights the in-vitro propagation of human spermatogonial stem cells from testicular biopsies for future transplantation and restoration of fertility. Whereas in-vitro propagation of spermatogonial stem cells has been established in animal models this is the first study in humans. Spermatogonial stem cell transplantation began as a theoretical approach that currently is studied ardently by several research groups to make this a valid clinical option. Restoration of fertility following spermatogonial stem cell transplantation in animals suggests therapeutic potential for the technique in humans, and further research is proceeding to address the safety and efficacy of this technique.

Serum- and Feeder-Free Culture of Mouse Germline Stem Cells1

Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis. Although several in vitro SSC culture systems have been developed, these systems include serum or fibroblast feeders, which complicate SSC self-renewal analyses. Here, we developed a serum- and feeder-free culture system for long-term propagation of SSCs. In addition to the SSC self-renewal factors, including glial cell line-derived neurotrophic factor, supplementation with fetuin and lipid-associated molecules was required to drive SSC proliferation in vitro. Cultured cells proliferated for at least 6 mo at a rate comparable to that of serum-supplemented cultured cells. However, germline potential was reduced under serum- and feeder-free conditions, as indicated by a lower SSC frequency after germ cell transplantation. Nevertheless, the cultured cells completed spermatogenesis and produced offspring following spermatogonial transplantation into seminiferous tubules of infertile mice. This culture system provides a basic platform for understanding the regulation of SSC fate commitment in vitro and for improving SSC culture medium.

Fertility preservation for boys with cancer.

Childhood cancer is a curable disease due to the development of chemo- and radiation therapies, but long-term survivors suffer late side-effects including infertility. Cytotoxic agents and radiation impair spermatogenesis and cause oligospermia or azoospermia as well as genetic damage in sperm. To date, the only established option to preserve fertility is cryopreservation of sperm before treatment and artificial reproduction techniques, if men with cancer can ejaculate, but only a quarter of men have banked sperm. Lack of information is the most common reason for failing to bank sperm. However, prepubertal patients who have only spermatogonia and spermatocytes in their testes do not benefit from cryopreservation of their sperm and assisted reproductive techniques. Thus, the only available option is to harvest testicular tissues before treatment for cryopreservation, from which immature germ cells can somehow be maturated. Autotransplantation of germ cells into the testis holds promise for fertility restoration, but contamination by malignant cells may induce relapse. Fluorescence-activated cell sorting (FACS) with two surface markers could exclude contaminated leukemic cells from murine germ cells, and transplantation of sorted germ cells successfully restored fertility without transmission of leukemia. Human germ cells could be also isolated from human leukemia and lymphoma cell lines by FACS using surface markers. Before autotransplantation can be applied clinically, some issues, including the risk of contamination by malignant cells and in vitro propagation of spermatogonial stem cells, should be resolved.

Long‐term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non‐obstructive azoospermia under exogenous feeder‐free culture conditions

The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non-obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology. We have applied a cell-sorting method, using collagen and magnetic activated cell separation to overcome obstacles, developing a collection system, and simple long-term proliferation system, that yields large numbers of high-purity SSCs from obstructive OA and NOA patients. SSCs derived from OA and NOA patients proliferated and maintained their characteristics for more than 12 passages (>6 months) in vitro. Moreover, the population of cells positive for the SSC-specific markers GFRalpha-1 and integrin alpha6, increased to more than 80% at passage 8. These finding may support the idea that in vitro propagation of SSCs could be a useful tool for infertility treatment and study of reproductive biology.

In vitro propagation of spermatogonial stem cells from KM mice

Spermatogonial stem cells (SSCs) are a unique type of stem cells in that they transmit genetic information to the next generation by producing sperms. Studies of SSC proliferation and differentiation have been hampered by the inability of reconstructing these processes in vitro, particularly in a serum-free culture system. Several groups have reported the long term culture of SSCs during which SSCs self-renew and restore spermatogenesis when transplanted back to recipient testes. However, different protocols and mice with particular genetic background have been used by different laboratories, and the techniques have not been adopted widely. In the present study, we first established a SSC isolation and culture system composed of differential adherence selection of SSCs, serum-free medium and mouse embryonic fibroblast (MEF) feeder cells. SSCs from KM pups could be cultured on MEF feeders in StemPro-34 SFM Medium supplemented with glial cell line-derived neurotrophic factor (GDNF), soluble GDNF family receptor alpha-1 (GFRa1) and basic fibroblast growth factor (bFGF) for 1 month. These SSCs were characterized morphologically and by examining the expression of marker genes. Expression of Oct4 and Sox2, which are crucial factors in embryonic stem cell (ESC) self-renewal, were detected in our cultured SSCs, suggesting that SSCs may share with ESCs some common mechanisms in self-renewal regulation. We also found that LIF had no effect on the proliferation of cultured SSCs derived from KM mice.