scholarly journals In VitroCulture-Induced Pluripotency of Human Spermatogonial Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Jung Jin Lim ◽  
Hyung Joon Kim ◽  
Kye-Seong Kim ◽  
Jae Yup Hong ◽  
Dong Ryul Lee
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Embryoid Bodies ◽  
Pluripotent Cells ◽  
Induced Pluripotency ◽  
Derivatives Of ◽  
Direct Induction

Unipotent spermatogonial stem cells (SSCs) can be transformed into ESC-like cells that exhibit pluripotencyin vitro. However, except for mouse models, their characterization and their origins have remained controversies in other models including humans. This controversy has arisen primarily from the lack of the direct induction of ESC-like cells from well-characterized SSCs. Thus, the aim of the present study was to find and characterize pluripotent human SSCs inin vitrocultures of characterized SSCs. Human testicular tissues were dissociated and plated onto gelatin/laminin-coated dishes to isolate SSCs. In the presence of growth factors SSCs formed multicellular clumps after 2–4 weeks of culture. At passages 1 and 5, the clumps were dissociated and were then analyzed using markers of pluripotent cells. The number of SSEA-4-positive cells was extremely low but increased gradually up to ~ 10% in the SSC clumps during culture. Most of the SSEA-4-negative cells expressed markers for SSCs, and some cells coexpressed markers of both pluripotent and germ cells. The pluripotent cells formed embryoid bodies and teratomas that contained derivatives of the three germ layers in SCID mice. These results suggest that the pluripotent cells present within the clumps were derived directly from SSCs duringin vitroculture.

229 LONG-TERM PROPAGATION AND CRYOPRESERVATION OF CAT SPERMATOGONIAL STEM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 246
Author(s):  
L. M. Vansandt ◽  
M. Dickson ◽  
R. Zhou ◽  
L. Li ◽  
B. S. Pukazhenthi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Domestic Cat ◽  
Feeder Cell ◽  
Cell Clusters ◽  
Fetal Fibroblasts

Spermatogonial stem cells (SSC) are unique adult stem cells that reside within the seminiferous tubules of the testis. As stem cells, SSC maintain the ability to self-replicate, providing a potentially unlimited supply of cells and an alternate source for preservation of the male genome. While self-renewing, long-term SSC culture has been achieved in mice, there is virtually no information regarding culture requirements of felid SSC. Therefore, the objectives of this study were to (1) evaluate the ability of 3 feeder cell lines to support germ cell colony establishment in domestic cats (Felis catus), and (2) assess long-term culture using the best feeder(s). Cells isolated enzymatically from peripubertal cat testes (n = 4) and enriched by differential plating were cultured on mouse embryonic fibroblasts (STO line), mouse-derived C166 endothelial cells, and primary cat fetal fibroblasts (cFF). Colony morphology was assessed every other day and immunocytochemistry (ICC) was performed to investigate expression of SSC markers. At 5 days in vitro (DIV), a cluster forming activity assay was used to estimate the number of SSC supported by each feeder cell line. Differences among treatments were compared using Tukey-Kramer adjustment for pair-wise mean comparisons. Data were expressed as mean cluster number ± SE per 105 cells input. When cultured on STO feeders, cat germ cells were distributed as individual cells. On both C166 cells and cFF feeders, germ cell clumps (morphologically consistent with SSC colonies in other species) were observed. Immunocytochemistry revealed that the single germ cells present on STO feeders were positive for UCHL1 and weakly expressed PLZF and OCT4. Cells within the germ cell clumps on C166 cells and cFF co-expressed all 3 SSC markers. The C166 cells supported a higher number of germ cell clusters (77.4 ± 13.8) compared with STO (3.5 ± 1.1, P = 0.0003) or cFF (22.7 ± 1.0, P = 0.0024). Therefore, subsequent subculture experiments were performed exclusively with C166 feeder layers. Cultures from 2 donors were passaged at 12 DIV and periodically as needed thereafter. Germ cell clumps consistently reestablished following each subculture and immunocytochemistry analysis confirmed maintenance of all 3 SSC markers. Cells were also positive for alkaline phosphatase activity. Cells that had been cryopreserved in culture medium with 5% (vol/vol) dimethyl sulphoxide after144 DIV (7 passages) were thawed and cultured for an additional 18 days. These cells continued to express SSC markers and form germ cell clusters. Taken together, these data demonstrate that C166 feeder cells can facilitate colony establishment and in vitro propagation of germ cell clumps in the domestic cat. This represents an important first step towards attainment and optimization of a long-term SSC culture system in the cat. This system would provide a mechanism to explore regulation of spermatogenesis, test species-specific drugs, and produce transgenic biomedical models.

Germ cells and pluripotent stem cells in the mouse

2001 ◽  
Vol 13 (8) ◽  
pp. 661 ◽  
Author(s):  
Anne McLaren ◽  
Gabriela Durcova-Hills
Keyword(s):  
Growth Factors ◽  
Cell Lines ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Individual Growth ◽  
Developmental Potential

For many years, attempts to achieve long-term culture of mouse primordial germ cells (PGCs) proved unsuccessful, even when feeder layers were used and individual growth factors were added to the medium. However, when three growth factors were added simultaneously to the medium, some of the cells continued to proliferate indefinitely. Similar to embryonic stem cell lines, these embryonic germ (EG) cell lines were capable of giving rise to embryoid bodies in vitro, and colonizing all cell lineages in chimeras, including the germline. Initially, EG cells were made from PGCs before migration, 8.5 days post coitum (dpc), and after entry into the genital ridge, 11.5 and 12.5 dpc. New EG cell lines from 9.5 dpc (migrating) and 11.5 dpc PGCs, carrying either a LacZ or GFP transgene, are described here. The developmental potential of the new EG cell lines in vitro, in vivoin chimeras, and in tissue aggregates in organ culture was studied. The EG cells were compared with PGCs at the stage from which the EG cells were derived. The two cell types show several similarities, but also some differences in gene expression and cell behaviour, which require further exploration.

scholarly journals In vivo and in vitro differentiation of male germ cells in the mouse

Reproduction ◽  
2004 ◽  
Vol 128 (2) ◽  
pp. 147-152 ◽  
Author(s):  
Orly Lacham-Kaplan
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cellular Interactions ◽  
Germ Cell Differentiation

Primordial germ cells appear in the embryo at about day 7 after coitum. They proliferate and migrate towards the genital ridge. Once there, they undergo differentiation into germ stem cells, known as ‘A spermatogonia’. These cells are the foundation of spermatogenesis. A spermatogonia commit to spermatogenesis, stay undifferentiated or degenerate. The differentiation of primordial germ cells to migratory, postmigratory and germ stem cells is dependent on gene expression and cellular interactions. Some of the genes that play a crucial role in germ cell differentiation are Steel, c-Kit, VASA, DAZL, fragilis, miwi, mili, mil1 and mil2. Their expression is stage specific, therefore allowing solid identification of germ cells at different developmental phases. In addition to the expression of these genes, other markers associated with germ cell development are nonspecific alkaline phosphatase activity, the stage specific embryonic antigen, the transcription factor Oct3/4 and β1- and α6-integrins. Commitment of cells to primordial germ cells and to A spermatogonia is also dependent on induction by the bone morphogenetic protein (BMP)-4. With this knowledge, researchers were able to isolate germ stem cells from embryonic stem cell-derived embryoid bodies, and drive these into gametes either in vivo or in vitro. Although no viable embryos were obtained from these gametes, the prospects are that this goal is not too far from being accomplished.

288 THE EXPRESSION PATTERN OF ACETYLATED ALPHA-TUBULIN IS CONSERVED IN PORCINE AND MURINE SPERMATOGONIAL STEM CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 223
Author(s):  
J. Luo ◽  
S. Megee ◽  
I. Dobrinski
Keyword(s):  
Stem Cells ◽  
Basement Membrane ◽  
Expression Pattern ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Histone Deacetylase 6 ◽  
Pgp 9.5

During mammalian spermatogenesis, spermatogonial stem cells (SSCs) reside in the stem cell niche on the basement membrane where they undergo self-renewing divisions. Differentiating daughter cells are located progressively more toward the tubular lumen where they ultimately form spermatozoa. The mechanisms responsible for maintenance of SSCs at the basement membrane are unclear. Microtubules consisting of α/β-tubulin heterodimers are associated with many cellular functions. Reversible acetylation of α-tubulin at Lys40 has been implicated in regulating microtubule stability and function. Acetylation of α-tubulin is abundant in stable microtubules but absent from dynamic cellular structures. Deacetylation of α-tubulin is controlled by histone deacetylase 6 which is predominantly expressed in mouse testis. Here, we tested the hypothesis that differential acetylation of α-tubulin might be involved in maintenance of SSCs. Immunohistochemistry for acetylated α-tubulin (Ac-α-Tu) and the spermatogonia specific proteins PGP 9.5, DAZL, and PLZF were used to characterize the expression pattern of Ac-α-Tu in porcine and murine germ cells at different stages of testis development. In immature boar testes, Ac-α-Tu was present exclusively in gonocytes but not in other testicular cells at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating to the basement membrane of the seminiferous tubules, and Ac-α-Tu appeared to be polarized toward the basement membrane. In immature mouse testes, Ac-α-Tu was present in germ cells and Sertoli cells at 6 days of age, whereas at 2 weeks of age, Ac-α-Tu expression was stronger in spermatogonia co-expressing PGP 9.5 and in spermatocytes than in Sertoli cells or PGP 9.5-negative spermatogonia. In adult boar and mouse testes, Ac-α-Tu was detected in a few single or paired spermatogonia expressing PGP 9.5 localized on the basement membrane as well as in spermatocytes, spermatids, and spermatozoa. Spermatogonia with high levels of Ac-α-Tu expressed PLZF but did not express DAZL, suggesting that only undifferentiated spermatogonia maintain a high level of Ac-α-Tu. When seminiferous tubules from 1-week-old and adult boar testes were maintained in vitro for 1–2 days, high levels of Ac-α-Tu were detected in single or paired round spermatogonia with a large nucleus, compared to low levels in elongated paired and aligned spermatogonia. The unique expression pattern of Ac-α-Tu in undifferentiated germ cells during postnatal development appears to be conserved in mammalian testes. Since Ac-α-Tu is a component of long-lived stable microtubules and reducing acetylation of α-tubulin enhances cell motility, these results suggest that stabilization of microtubules might contribute to the maintenance of spermatogonial stem cells. This work was supported by 1R01 RR 17359-05.

Testicular endothelial cells promote self-renewal of spermatogonial stem cells in rats†

2019 ◽  
Vol 101 (2) ◽  
pp. 360-367
Author(s):  
Yong-Hee Kim ◽  
Myeong-Geun Oh ◽  
Dong Ha Bhang ◽  
Bang-Jin Kim ◽  
Sang-Eun Jung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Germ Cells ◽  
Cell Biology ◽  
Spermatogonial Stem Cells ◽  
Feeder Layer ◽  
In Vitro Proliferation ◽  
Cell Potential ◽  
Self Renewal

Abstract Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male due to their capability to multiply in numbers by self-renewal and subsequent meiotic processes. However, as SSCs are present in a very small proportion in the testis, in vitro proliferation of undifferentiated SSCs will facilitate the study of germ cell biology. In this study, we investigated the effectiveness of various cell lines as a feeder layer for rat SSCs. Germ cells enriched for SSCs were cultured on feeder layers including SIM mouse embryo-derived thioguanine and ouabain-resistant cells, C166 cells, and mouse and rat testicular endothelial cells (TECs) and their stem cell potential for generating donor-derived colonies and offspring was assessed by transplantation into recipient testes. Rat germ cells cultured on TECs showed increased mRNA and protein levels of undifferentiated spermatogonial markers. Rat SSCs derived from these germ cells underwent spermatogenesis and generated offspring when transplanted into recipients. Collectively, TECs can serve as an effective feeder layer that enhances the proliferative and self-renewal capacity of cultured rat SSCs while preserving their stemness properties.

scholarly journals Bisphenol A Affects on the Functional Properties and Proteome of Testicular Germ Cells and Spermatogonial Stem Cells in vitro Culture Model

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Polash Chandra Karmakar ◽  
Hyun-Gu Kang ◽  
Yong-Hee Kim ◽  
Sang-Eun Jung ◽  
Md. Saidur Rahman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bisphenol A ◽  
In Vitro Culture ◽  
Functional Properties ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Culture Model

scholarly journals Preservation of fertility in nature and ART

Reproduction ◽  
2002 ◽  
pp. 3-11 ◽  
Author(s):  
R Gosden ◽  
M Nagano
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Reproductive Technologies ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Natural Fertility ◽  
Fertilization In Vitro ◽  
Gonadal Failure

Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation.

189 Isolation and purification of rhinoceros and horse spermatogonial stem cells

2019 ◽  
Vol 31 (1) ◽  
pp. 219
Author(s):  
C. Yu-Su ◽  
T. Jensen ◽  
B. Durrant ◽  
M. C. Gómez
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Reproductive Age ◽  
Culture Methods ◽  
Isolation And Purification ◽  
Ceratotherium Simum ◽  
White Rhinoceros

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is critically endangered. Testicular tissue (TT) of an NWR that died at 46 years was cryopreserved and stored. Spermatogonial stem cells (SSC) have been isolated from TT and, following transplantation or in vitro culture, differentiated into mature spermatozoa. These SSC have been phenotypically characterised by the expression of markers specific for SSC, and in vitro culture methods optimized to isolate, purify, and enrich populations of SSC from mixed germ cells. The reproductive age may affect the percent of SSC in TT, affecting recovery from older animals. We previously identified in TT of NWR the surface markers GFRa1 and GPR125, both of which were expressed at various stages of spermatogenesis. However, characterisation, isolation, and purification of rhinoceros SSC have not been studied. Because of the limited availability of rhinoceros TT, we used the horse as a model for rhinoceros (both species are in the same order: Perissodactyla). In this study, we (1) identified and compared the expression of markers specific for SSC (GFRa1, GPR125, PLZF) and pluripotent markers (SSEA-1, SSEA-4, OCT-4) in rhinoceros and horse mixed germ cells; (2) evaluated whether rhinoceros and horse SSC could be purified and enriched by sequential culture with collagen (2 days) and laminin (3 weeks); and (3) determined whether age may affect the percentages of SSC by comparing the expression of SSC and pluripotent markers in horse mixed germ cells at different reproductive stages: pubertal (PU=1-1.5 years) v. post-pubertal (PP=2-3 years) v. adult (AD=>5 years). Mixed germ cells were isolated from testes of one male of each of 3 rhinoceros species: NWR, southern white rhinoceros (SWR; Ceratotherium simum simum, 49 years), and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis, 5 years), and horses at different reproductive stages (PU=2, PP=2, AD=7). The SWR and GOHR testes were refrigerated overnight, whereas NWR mixed germ cells were isolated from frozen-thawed TT. Flow cytometry analysis showed expression of SSC and pluripotent markers but not for transcription factor PLZF. The marker expression was similarly distributed between the 3 species of rhinoceros, with a larger portion of cells positive for SSEA-4 (mean%±s.e.m.: 6.3±1.1%) and smaller proportion for GDNFa1 (0.4±0.2%), GRP125 (0.1±0.1%), OCT-4 (0.4±0.2%), and SSEA-1 (0.4±0.2%; P<0.05). Similarly, horse expressed the markers GDNFa1 (0.9±0.3%), GRP125 (0.9±0.4%), OCT-4 (1.7±0.5%), and SSEA-1 (0.12±0.1%), but the abundance of SSEA-4 (1.8±0.8%) was less than that of rhinoceros (P<0.05). In rhinoceros, sequential culture enhanced the numbers of cells expressing all markers compared with that before culture, whereas in horses, the increase was observed only for cells expressing GDNFa1, GPR125, and SSEA-1. Reproductive stage did not affect the percentages of horse cells expressing germ cell and pluripotent markers. Overall, these results showed that rhinoceros SSC can be isolated from TT and expressed the same SSC markers as horses, and that differential culture enriched a population of SSC.

scholarly journals Human Pluripotent Stem Cells in Reproductive Science—A Comparison of Protocols Used to Generate and Define Male Germ Cells from Pluripotent Stem Cells

2020 ◽  
Vol 21 (3) ◽  
pp. 1028
Author(s):  
Magdalena Kurek ◽  
Halima Albalushi ◽  
Outi Hovatta ◽  
Jan-Bernd Stukenborg
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Male Germ Cells ◽  
Germ Cell Differentiation

Globally, fertility-related issues affect around 15% of couples. In 20%–30% of cases men are solely responsible, and they contribute in around 50% of all cases. Hence, understanding of in vivo germ-cell specification and exploring different angles of fertility preservation and infertility intervention are considered hot topics nowadays, with special focus on the use of human pluripotent stem cells (hPSCs) as a source of in vitro germ-cell generation. However, the generation of male germ cells from hPSCs can currently be considered challenging, making a judgment on the real perspective of these innovative approaches difficult. Ever since the first spontaneous germ-cell differentiation studies, using human embryonic stem cells, various strategies, including specific co-cultures, gene over-expression, and addition of growth factors, have been applied for human germ-cell derivation. In line with the variety of differentiation methods, the outcomes have ranged from early and migratory primordial germ cells up to post-meiotic spermatids. This variety of culture approaches and cell lines makes comparisons between protocols difficult. Considering the diverse strategies and outcomes, we aim in this mini-review to summarize the literature regarding in vitro derivation of human male germ cells from hPSCs, while keeping a particular focus on the culture methods, growth factors, and cell lines used.

229 DERIVATION OF PRESUMPTIVE GONOCYTES IN VITRO FROM PRIMATE EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 231
Author(s):  
T. Teramura ◽  
N. Kawata ◽  
T. Takehara ◽  
N. Fujinami ◽  
M. Takenoshita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Germ Cells ◽  
Nonhuman Primates ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Embryoid Bodies

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis, because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES (cyES) cell lines and attempted to induce their differentiation into germ cells in order to obtain further information on the development of primate germ cells by observing the transcripts of some markers reported as specific for germ cells. CyES cell lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing superovulation, females were treated with 25 IU kg-1 pregnant mare serum gonadotropin once a day for 9 days, followed by 400 IU kg-1 hCG. Oocytes were collected at 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For cyES cell establishment, inner cell masses (ICMs) were isolated by immunosurgery. The ESC colonies developed at about 10 days after ICM plating, and 3 cell lines were successfully established (3/11; 27.3%). All cell lines expressed Oct3/4, SSEA-4, and ALP activity. These ESCs formed teratomas containing 3 different embryonic layers when injected into SCID mice. And the cells could be passaged over 50 times without losing their original properties. To observe in vitro gametogenesis, we attempted to induce differentiation by non-adherent conditions. When cyES cells differentiated spontaneously, the aggregated structures (i.e. embryoid bodies; EBs) accumulated vasa, the expression of which is restricted to germ cells, and some meiotic markers such as dmc1 and sycp1 that exist only in synaptonemal complexes in meiosis. The existence of these markers was also confirmed by immunocytochemistry on cryosections. Interestingly, these products expressed oct4 and nanog again at Day 16, though the expression of both genes diminished at once with onset of differentiation. In vivo, it is reported that vasa, oct4, and nanog are expressed in migrating PGCs, posibly throughout the development of germ cells into spermatocytes/oocytes. Given the results obtained with the meiotic markers, it is possible that developing germ cells such as PGCs or gonocytes could be formed in cynomolgus EBs as in previous cases with mouse or human EBs. These results demonstrate that cyES cells might contribute to putative germ cells in vitro by differentiating into EBs and could be used as a model for studying mechanisms of germ cell development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

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