scholarly journals Successful Transplantation of Spermatogonial Stem Cells Into the Seminiferous Tubules of Busulfan-treated Mice

2020 ◽  
Author(s):  
Hossein Azizi ◽  
Amirreza Niazi Tabar ◽  
Thomas Skutella
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Spermatogonial Stem Cells ◽  
Pcr Analysis ◽  
Seminiferous Tubules ◽  
Flow Cytometry Analysis ◽  
Rt Pcr ◽  
Expression Levels ◽  
Successful Transplantation

Abstract Background: Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods: In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology - immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer.Results: ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P< 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions: In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

scholarly journals Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Hossein Azizi ◽  
Amirreza Niazi Tabar ◽  
Thomas Skutella
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Spermatogonial Stem Cells ◽  
Pcr Analysis ◽  
Seminiferous Tubules ◽  
Flow Cytometry Analysis ◽  
Rt Pcr ◽  
Expression Levels ◽  
Successful Transplantation

Abstract Background Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology—immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer. Results ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells

Zygote ◽  
2019 ◽  
Vol 27 (02) ◽  
pp. 82-88 ◽  
Author(s):  
Vivek Pandey ◽  
Anima Tripathi ◽  
Pawan K. Dubey
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Germ Cells ◽  
Expression Patterns ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Single Type ◽  
Rt Pcr

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

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

2013 ◽  
Vol 25 (1) ◽  
pp. 290 ◽  
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.

scholarly journals Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Sujeong Jang ◽  
Jong-Seong Park ◽  
Han-Seong Jeong
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Neural Differentiation ◽  
Neural Induction ◽  
Stem Cell Differentiation ◽  
Human Adipose Tissue ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Jnk Pathway ◽  
Expression Levels

Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs.

scholarly journals Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice

2020 ◽  
Vol 117 (14) ◽  
pp. 7837-7844
Author(s):  
Mito Kanatsu-Shinohara ◽  
Narumi Ogonuki ◽  
Shogo Matoba ◽  
Atsuo Ogura ◽  
Takashi Shinohara
Keyword(s):  
Stem Cells ◽  
Sertoli Cells ◽  
Expression Patterns ◽  
Autologous Transplantation ◽  
Spermatogonial Stem Cells ◽  
Congenital Defects ◽  
Seminiferous Tubules ◽  
Special Environment ◽  
Deficient Mice

The blood–testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role ofCldn11, a major component of the BTB, in spermatogenesis using spermatogonial transplantation.Cldn11-deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage.Cldn11-deficient SSCs failed to colonize wild-type testes efficiently, andCldn11-deficient SSCs that underwent double depletion ofCldn3andCldn5showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However,Cldn11-deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intactCldn11-deficient testes, those from WT orCldn11-deficient testes regenerated sperm inCldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion ofCldn3/5regenerated endogenous spermatogenesis inCldn11-deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue.

Human CB-Derived Cells Give Rise to Insulin-Producing-Cells in Xenogeneic Hosts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3603-3603
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Noriaki Kawano ◽  
Hua Zhang ◽  
Yuan Kong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Chromosome ◽  
Human Insulin ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Fish Analysis ◽  
Rt Pcr ◽  
Post Transplantation ◽  
Insulin Producing Cells

Abstract (Purpose) We examine the capacity of human cord blood (CB)-derived cells to generate insulin-producing cells and other lineages of cells in pancreatic tissue in vivo. (Method) Ten millions of human CB-derived T-cell-depleted mononuclear cells were intravenously transplanted into conditioned newborn NOD/SCID/b2-microglobulinnull mice with or without chemical injury by an intraperitoneal injection of streptozotocin (STZ) at dose of 100 mg/g body weight. At 1–3 months post-transplantation, pancreatic tissues of the recipient mice were analyzed for the presence of human CB-derived cells by performing immunofluorescence study (insulin, amylase, or CD45) and FISH analysis for human chromosomes on the same specimens. RNA was isolated from pancreatic tissues of recipient mice, and RT-PCR analysis using human insulin specific primer was performed to examine human insulin at RNA level. Finally, double FISH analysis for human- and murine chromosomes was performed to get an insight into the mechanism for the generation of human CB-derived insulin-producing cells in vivo. (Results) At 1–3 months post-transplantation, human CB-derived T-cell-depleted mononuclear cells gave rise to both myeloid and lymphoid progeny (CD33+, CD19+, and CD3+ cells) in bone marrow and peripheral blood of the recipient mice. In recipient pancreatic tissues, human CB-derived cells were identified inside and outside islets. Outside pancreatic islets, the vast majority of human chromosome+ cells were CD45+ hematopoietic cells, while human chromosome+ amylase+ acinar cells were also identified. Inside islets, human chromosome+ cells accounted for 1.01 +/− 0.73 % (n=6) without STZ treatment. Among them, human CB-derived insulin-producing cells were identified at a frequency of 0.65 +/− 0.64 % (n=6) of total insulin+ cells in xenogeneic hosts. RT-PCR analysis demonstrated the presence of human insulin, whose sequence was fully identical to that of already-known human insulin cDNA. Chemical injury with STZ treatment led to the significant destruction of islet tissue and reduction of cell numbers in islets. In STZ-treated recipient mice, however, human insulin-producing cells were identified at a frequency of 0.23 +/− 0.27 % (n=4) in islets, which was lower than the mice without STZ treatment. Finally, double FISH analyses using species-specific probes demonstrated the presence of human chromosome+ murine chromosome+ insulin-producing cells and human chromosome+ murine chromosome- insulin-producing cells in recipient islets. (Conclusion) It is concluded that human CB cells contain the progenitor cells to generate the insulin-producing cells in vivo. The mechanism of CB-derived insulin-producing cells includes both fusion-dependent and independent mechanisms. Although the capacity of CB-derived cells needs to be compared with other stem cell sources such as tissue stem cells or embryonic stem cells, the present study suggests the possibility of CB cells as new source for future regenerative medicine for diabetes mellitus.

scholarly journals Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2265
Author(s):  
Whitney F. Alpaugh ◽  
Anna L. Voigt ◽  
Rkia Dardari ◽  
Lin Su ◽  
Iman Al Khatib ◽  
...  
Keyword(s):  
Stem Cells ◽  
Metabolic Regulation ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Testis Weight ◽  
Stem And Progenitor Cells ◽  
Cell Transcriptome ◽  
Carboxy Terminal

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1−/− mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1−/− mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured Uch-l1−/− SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

scholarly journals Human levator veli palatini muscle: A novel source of mesenchymal stem cells for use in the rehabilitation of patients with congenital craniofacial malformations

2020 ◽  
Author(s):  
Daniela Franco Bueno ◽  
Gerson Shigueru Kabayashi ◽  
Carla Cristina Gomes Pinheiro ◽  
Daniela Y S Tanikawa ◽  
Cassio Eduardo Raposo-Amaral ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Potential ◽  
Flow Cytometry Analysis ◽  
Non Invasive ◽  
Levator Veli Palatini

Abstract Background. Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stem cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent stem cells that can be isolated via non-invasive procedures. Here we analysed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during surgical rehabilitation of cleft p­­atients during palatoplasty, represent a novel source of MSCs with osteogenic potential. Methods. We obtained levator veli palatini muscle fragments, in non-invasive procedure during surgical rehabilitation of 5 unrelated cleft palate patients (palatoplasty surgery). The levator veli palatini muscle fragments was used to obtain the mesenchymal cells using pre-plating technique in a clean rooms infrastructure and all procedures were performed at good practices of manipulation conditions. To prove that levator veli palatini muscle are mesenchymal stem cells they were induced to flow cytometry analysis and to differentiation into bone, cartilage, fat and muscle. To demonstrate the osteogenic potential of these cells in vivo a bilateral full thickness calvarial defect model was made in immunocompentent rats.Results. Flow cytometry analysis showed that the cells were positive for mesenchymal stem cell antigens (CD29, CD73, CD90), while negative for hematopoietic (CD45) or endothelial cell markers (CD31). Moreover, these cells were capable of undergoing chondrogenic, adipogenic, osteogenic and skeletal muscle cell differentiation under appropriate cell culture conditions characterizing them as mesenchymal stem cell. Defects treated with CellCeramTM scaffolds seeded with levator veli palatini muscle cells showed significantly greater bone healing compared to defects treated with acellular scaffolds. Conclusion. We have demonstrated that cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stem cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

scholarly journals Propagation of bovine spermatogonial stem cells in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (5) ◽  
pp. 543-557 ◽  
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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