Derivation and propagation of spermatogonial stem cells from human pluripotent cells

Abstract 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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Propagation of bovine spermatogonial stem cells in vitro

Reproduction ◽

10.1530/rep-07-0419 ◽

2008 ◽

Vol 136 (5) ◽

pp. 543-557 ◽

Cited By ~ 90

Author(s):

Pedro M Aponte ◽

Takeshi Soda ◽

Katja J Teerds ◽

S Canan Mizrak ◽

Henk J G van de Kant ◽

...

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Cultured Cells ◽

Somatic Cells ◽

Type A ◽

Spermatogonial Stem Cells ◽

Seminiferous Tubules ◽

Type A Spermatogonia ◽

Stimulation Of

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study thein vitrobehavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

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Impact of Graphene Derivatives as Artificial Extracellular Matrices on Mesenchymal Stem Cells

Molecules ◽

10.3390/molecules27020379 ◽

2022 ◽

Vol 27 (2) ◽

pp. 379

Author(s):

Rabia Ikram ◽

Shamsul Azlin Ahmad Shamsuddin ◽

Badrul Mohamed Jan ◽

Muhammad Abdul Qadir ◽

George Kenanakis ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Regenerative Medicine ◽

Cell Types ◽

Biological Properties ◽

Research Progress ◽

Differentiation Potential ◽

Graphene Derivatives ◽

Potential Applicability

Thanks to stem cells’ capability to differentiate into multiple cell types, damaged human tissues and organs can be rapidly well-repaired. Therefore, their applicability in the emerging field of regenerative medicine can be further expanded, serving as a promising multifunctional tool for tissue engineering, treatments for various diseases, and other biomedical applications as well. However, the differentiation and survival of the stem cells into specific lineages is crucial to be exclusively controlled. In this frame, growth factors and chemical agents are utilized to stimulate and adjust proliferation and differentiation of the stem cells, although challenges related with degradation, side effects, and high cost should be overcome. Owing to their unique physicochemical and biological properties, graphene-based nanomaterials have been widely used as scaffolds to manipulate stem cell growth and differentiation potential. Herein, we provide the most recent research progress in mesenchymal stem cells (MSCs) growth, differentiation and function utilizing graphene derivatives as extracellular scaffolds. The interaction of graphene derivatives in human and rat MSCs has been also evaluated. Graphene-based nanomaterials are biocompatible, exhibiting a great potential applicability in stem-cell-mediated regenerative medicine as they may promote the behaviour control of the stem cells. Finally, the challenges, prospects and future trends in the field are discussed.

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Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem cells

International Journal of Molecular Sciences ◽

10.3390/ijms222212503 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12503

Author(s):

Martina Culenova ◽

Andreas Nicodemou ◽

Zuzana Varchulova Novakova ◽

Michaela Debreova ◽

Veronika Smolinská ◽

...

Keyword(s):

Stem Cells ◽

Low Cost ◽

Biological Properties ◽

Cytokine Profile ◽

Rice Grain ◽

Differentiation Potential ◽

Secretory Rate ◽

Gm Csf ◽

Non Invasive ◽

Cell Doubling Time

Mesenchymal stem cells (MSCs) represent an attractive source within the field of tissue engineering. However, their harvesting often requires invasive medical procedures. Urine-derived stem cells (UDSCs) display similar properties to MSCs, and their obtention and further processing is non-invasive for the donors as well as low cost. Here, we offer a comprehensive analysis of their biological properties. The goal of this study was to analyze their morphology, stemness, differentiation potential and cytokine profile. We have successfully isolated UDSCs from 25 urine samples. First colonies emerged up to 9 days after the initial seeding. Cell doubling time was 45 ± 0.24 SD, and when seeded at the density of 100 cells/cm2, they formed 42 ± 6.5 SD colonies within 10 days. Morphological analyzes revealed that two different types of the cell populations have been present. The first type had a rice-grain shape and the second one was characterized by a polyhedral shape. In several cell cultures, dome-shaped cells were observed as well. All examined UDSCs expressed typical MSC-like surface markers, CD73, CD90 and CD105. Moreover, conditioned media from UDSCs were harvested, and cytokine profile has been evaluated showing a significantly higher secretory rate of IL-8, IL-6 and chemokines MCP-1 and GM-CSF. We have also successfully induced human UDSCs into chondrogenic, osteogenic and myogenic cell lineages. Our findings indicate that UDSCs might have immense potential in the regeneration of the damaged tissues.

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286 EXPRESSION OF PLURIPOTENT STEM CELL MARKERS IN DOMESTIC CAT SPERMATOGONIAL CELLS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab286 ◽

2013 ◽

Vol 25 (1) ◽

pp. 290 ◽

Cited By ~ 4

Author(s):

R. H. Powell ◽

M. N. Biancardi ◽

J. Galiguis ◽

Q. Qin ◽

C. E. Pope ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Basement Membrane ◽

Germ Cell ◽

Germ Cells ◽

Spermatogonial Stem Cells ◽

Seminiferous Tubules ◽

Cell Populations ◽

Surface Markers ◽

Cell Markers

Spermatogonial stem cells (SSC), progenitor cells capable of both self-renewal and producing daughter cells that will differentiate into sperm, can be manipulated for transplantation to propagate genetically important males. This application was demonstrated in felids by the successful xeno-transplantation of ocelot mixed germ cells into the testes of domestic cats, which resulted in the production of ocelot sperm (Silva et al. 2012 J. Androl. 33, 264–276). Spermatogonial stem cells are in low numbers in the testis, but have been identified and isolated in different mammalian species using SSC surface markers; however, their expression varies among species. Until recently, little was known about the expression of SSC surface markers in feline species. We previously demonstrated that many mixed germ cells collected from adult cat testes express the germ cell markers GFRα1, GPR125, and C-Kit, and a smaller population of cells expresses the pluripotent SSC-specific markers SSEA-1 and SSEA-4 (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222). In the present study, our goal was to identify germ cell and SSC-specific markers in SSC from cat testes. Immunohistochemical (IHC) localization of germ cell markers GFRα1, GPR125, and C-Kit and pluripotent SSC-specific markers SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 was detected in testis tissue from both sexually mature and prepubertal males. Testes were fixed with modified Davidson’s fixative for 24 h before processing, embedding, and sectioning. The EXPOSE Mouse and Rabbit Specific HRP/DAB detection IHC kit (Abcam®, Cambridge, MA, USA) was used for antibody detection. Staining for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 markers was expressed specifically at the basement membrane of the seminiferous tubules in both adult and prepubertal testes. The GFRα1 and GPR125 markers were detected at the basement membrane of the seminiferous tubules and across the seminiferous tubule section. However, C-Kit was not detected in any cell. Using flow cytometry from a pool of cells from seven adult testes, we detected 45% GFRα1, 50% GPR125, 59% C-Kit, 18% TRA-1-60, 16% TRA-1-81 positive cells, and a very small portion of SSEA-1 (7%) and SSEA-4 (3%) positive cells. Dual staining of germ cells pooled from 3 testes revealed 3 distinct cell populations that were positive for GFRα1 only (23%), positive for both GFRα1 and SSEA-4 (6%), and positive for SSEA-4 only (1%). Our IHC staining of cat testes indicated that cells along the basement membrane of seminiferous tubules were positive for SSC-specific markers, and flow cytometry analysis revealed that there were different cell populations expressing both germ cell and SSC-specific markers. Flow cytometry results show overlapping germ cell populations expressing SSEA-4 and GFRα1, and IHC results reveal that SSEA-4 positive cells are spermatogonia, whereas GFRα1 positive cells include other stages of germ cells, indicating that the small population of cells positive only for SSEA-4 is undifferentiated cat SSC.

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Shengjing Capsule Improves Spermatogenesis through Upregulating Integrin α6/β1 in the NOA Rats

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/8494567 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Jiamin Wang ◽

Shankun Zhao ◽

Lianmin Luo ◽

Yangzhou Liu ◽

Ermao Li ◽

...

Keyword(s):

Stem Cells ◽

Sperm Quality ◽

Sperm Count ◽

Spermatogonial Stem Cells ◽

Normal Group ◽

Seminiferous Tubules ◽

High Dose ◽

Sprague Dawley ◽

Underlying Mechanisms ◽

Almost All

Objective. To evaluate the therapeutic effect of Shengjing capsules on nonobstructive azoospermia (NOA) in the rat model. Methods. Twenty-five male Sprague–Dawley rats were randomly divided into five groups as follows (n=5 per group): normal group, NOA group, and three Shengjing capsule treatment groups (low-dose, medium-dose, and high-dose groups, respectively). HE staining and semen smear were performed to assess sperm quality. The expression levels of PI3K/AKT and integrin α6/β1 were measured by qRT-PCR and western blot analyses. Results. In the NOA group, almost all of the seminiferous tubules were vacuolated with a thin layer of basal compartment containing some spermatogonial stem cells. The counts of sperms in the NOA group were strongly lower than those of the normal group (P=0.0001). The expression of PI3K/AKT and integrin α6/β1 was scarcely expressed in the NOA group. All indexes mentioned above were significantly different from those of the medium- and high-dose groups (P=0.001, all). The sperm count of rats treated with Shengjing capsules was significantly higher than that of the NOA group (P=0.0001). The rats of Shengjing capsule groups had more layers of spermatogonial stem cells and spermatocytes, and some had intracavitary sperms. Conclusions. Shengjing capsules may be a promising therapeutic medicine for NOA. The underlying mechanisms might involve activating SSCs by upregulating the integrin α6/β1 expression via the PI3K/AKT pathway.

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Xenogeneic and endogenous spermatogenesis following transplantation of rat germ cells into testes of immunocompetent mice

Reproduction Fertility and Development ◽

10.1071/rd10349 ◽

2012 ◽

Vol 24 (2) ◽

pp. 337 ◽

Cited By ~ 10

Author(s):

Ning Qu ◽

Munekazu Naito ◽

Jun Li ◽

Hayato Terayama ◽

Shuichi Hirai ◽

...

Keyword(s):

Stem Cells ◽

Immune Response ◽

Germ Cells ◽

Spermatogonial Stem Cells ◽

Seminiferous Tubules ◽

Recipient Mouse ◽

Immunocompetent Mice ◽

Rat Spermatogenesis ◽

Privileged Site

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and are characterised by their ability to self-renew and to produce differentiated progeny that form spermatozoa. It has been demonstrated that rat spermatogenesis can occur in the seminiferous tubules of congenitally immunodeficient recipient mice after transplantation of rat SSCs. However, the testis is often viewed as an immune-privileged site in that autoimmunogenic antigens on germ cells do not normally elicit an immune response in situ. In the present study, we tried to transplant rat SSCs into immunocompetent mice after depletion of their own germ cells by means of busulfan. The results showed that some transplanted SSCs could undergo complete spermatogenesis in recipient mouse testes, the rat spermatozoa being detected in 7 of 28 recipient epididymides. A significant increase in mouse spermatozoa was also noted in all 28 epididymides of recipient mice regardless of whether rat spermatozoa were concurrently present or not. These results suggest that transplanted rat SSCs can be tolerated in the testes of immunocompetent mice and that the transplantation of rat SSCs stimulates endogenous spermatogenesis in the recipient mice.

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Expression of Promyelocytic Leukemia (PML) Protein in the Mouse Developing Spermatogonia

10.21203/rs.3.rs-125056/v1 ◽

2020 ◽

Author(s):

Amin Tavassoli ◽

Hesam DEHGHANI

Keyword(s):

Stem Cells ◽

Immunohistochemical Analysis ◽

Cell Types ◽

Spermatogonial Stem Cells ◽

Promyelocytic Leukemia ◽

Nuclear Bodies ◽

Seminiferous Tubules ◽

Open Chromatin ◽

Pml Nuclear Bodies ◽

Oct4 Protein

Abstract Background: Promyelocytic leukemia (PML) as the main protein of PML nuclear bodies regulates various physiological processes such as transcription, DNA repair, apoptosis, senescence, and several signaling pathways in different cell types. It is well known that the PML protein is involved in the regulation of stem cell properties by maintaining an open chromatin conformation for the regulatory regions of the Oct4 gene. However, there is no experimental evidence for the presence and function of PML protein in the testis tissue. Results: In this study, we show the presence of PML protein in the developing mouse testis and its co-expression with the OCT4 protein. Immunohistochemical analysis of testis mirror sections shows that PML is co-expressed with the OCT4 protein in the outermost cellular layer of seminiferous tubules, where the spermatogonial stem cells are located. Conclusions: Our findings suggest that the PML protein might be involved in the stemness of spermatogonial stem cells at different stages of its development, even before earning the ability to produce mature sperm.

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A Mini Review Focused on the Recent Applications of Graphene Oxide in Stem Cell Growth and Differentiation

10.20944/preprints201809.0047.v1 ◽

2018 ◽

Author(s):

Alexander Halim ◽

Qing Luo ◽

Yang Ju ◽

Guanbin Song

Keyword(s):

Stem Cells ◽

Graphene Oxide ◽

Stem Cell ◽

Cell Growth ◽

Disease Modeling ◽

Biological Effects ◽

Stem Cell Differentiation ◽

Differentiation Potential ◽

The World ◽

Cell Growth And Differentiation

Stem cells are undifferentiated cells which can give rise to any types of cells in our body. Hence, they have been utilized for various applications such as drug testing and disease modeling. However, for the successful of those applications, the survival and differentiation of stem cells into specialized lineages should be well controlled. Growth factors and chemical agents are the most common signals to promote the proliferation and differentiation of stem cells. However, those approaches holds several drawbacks such as the negative side effects, degradation or denaturation, and expensive. To address such limitations, nanomaterials have been recently used as a better approach for controlling stem cells behaviors. Graphene oxide is the derivative of graphene, the first 2D materials in the world. Recently, due to its extraordinary properties and great biological effects on stem cells, many scientists around the world have utilized graphene oxide to enhance the differentiation potential of stem cells. In this mini review, we highlight the key advances about the effects of graphene oxide on controlling stem cell growth and various types of stem cell differentiation. We also discuss the possible molecular mechanisms of graphene oxide in controlling stem cell growth and differentiation.

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A Mini Review Focused on the Recent Applications of Graphene Oxide in Stem Cell Growth and Differentiation

Nanomaterials ◽

10.3390/nano8090736 ◽

2018 ◽

Vol 8 (9) ◽

pp. 736 ◽

Cited By ~ 20

Author(s):

Alexander Halim ◽

Qing Luo ◽

Yang Ju ◽

Guanbin Song

Keyword(s):

Stem Cells ◽

Graphene Oxide ◽

Stem Cell ◽

Cell Growth ◽

Disease Modeling ◽

Biological Effects ◽

Stem Cell Differentiation ◽

Differentiation Potential ◽

The World ◽

Cell Growth And Differentiation

Stem cells are undifferentiated cells that can give rise to any types of cells in our body. Hence, they have been utilized for various applications, such as drug testing and disease modeling. However, for the successful of those applications, the survival and differentiation of stem cells into specialized lineages should be well controlled. Growth factors and chemical agents are the most common signals to promote the proliferation and differentiation of stem cells. However, those approaches holds several drawbacks such as the negative side effects, degradation or denaturation, and expensive. To address such limitations, nanomaterials have been recently used as a better approach for controlling stem cells behaviors. Graphene oxide is the derivative of graphene, the first two-dimensional (2D) materials in the world. Recently, due to its extraordinary properties and great biological effects on stem cells, many scientists around the world have utilized graphene oxide to enhance the differentiation potential of stem cells. In this mini review, we highlight the key advances about the effects of graphene oxide on controlling stem cell growth and various types of stem cell differentiation. We also discuss the possible molecular mechanisms of graphene oxide in controlling stem cell growth and differentiation.

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Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

Nature Communications ◽

10.1038/s41467-019-13193-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Zhengpin Wang ◽

Xiaojiang Xu ◽

Jian-Liang Li ◽

Cameron Palmer ◽

Dragan Maric ◽

...

Keyword(s):

Stem Cells ◽

Sertoli Cell ◽

Reproductive Medicine ◽

Spermatogonial Stem Cells ◽

Transcriptional Analysis ◽

Seminiferous Tubules ◽

Transgenic Expression ◽

Genetic Ablation ◽

Male Germline ◽

Lower Expression

AbstractSpermatogonial stem cells (SSCs) have the dual capacity to self-renew and differentiate into progenitor spermatogonia that develop into mature spermatozoa. Here, we document that preferentially expressed antigen of melanoma family member 12 (PRAMEF12) plays a key role in maintenance of the spermatogenic lineage. In male mice, genetic ablation of Pramef12 arrests spermatogenesis and results in sterility which can be rescued by transgenic expression of Pramef12. Pramef12 deficiency globally decreases expression of spermatogenic-related genes, and single-cell transcriptional analysis of post-natal male germline cells identifies four spermatogonial states. In the absence of Pramef12 expression, there are fewer spermatogonial stem cells which exhibit lower expression of SSC maintenance-related genes and are defective in their ability to differentiate. The disruption of the first wave of spermatogenesis in juvenile mice results in agametic seminiferous tubules. These observations mimic a Sertoli cell-only syndrome in humans and may have translational implications for reproductive medicine.

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