A comparison between the colony formation of adult mouse spermatogonial stem cells in Co cultures with sertoli and STO (mouse embryonic fibroblast cell line)

Objective: The aim of this study was to compare the colony formation of spermatogonial stem cells (SSCs) on Sertoli and STO (Mouse embryonic fibroblast cell line) feeder cell layers during a two-week period. Materials and Methods: Initially, sertoli cells and SSCs were isolated from adult mouse testes using a two-step enzymatic digestion and lectin immobilization. Characteristics of the isolated cells were immunocytochemically confirmed by examining for the presence of Oct-4, CDH1, promyelocytic leukaemia zinc finger factor (PLZF), SSC C-kit, and the distribution of Sertoli cell vimentin. SSCs were then cultured above the Sertoli, STO and the control (without co-culture) separately for two weeks. In all three groups, the number and diameter of colonies were evaluated using an invert microscope on the 3rd, 7th, 10th and 14th day. β1 and α6-integrin m-RNA expressions were assessed using a reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, Oct-4 m RNA expression was assessed using real time PCR. Statistical analysis was performed using ANOVA; and the paired two-sample t test and Tukey's test were used as post-hoc tests for the data analysis of the three sertoli, STO and control cocultures. Results: At the four specified time points, our results showed significant differences (p<0.05) in colony numbers and diameters among the sertoli, STO and control groups. The number and diameter of colonies increased more rapidly in the sertoli coculture than in the other two Our results at all four time points also showed significant differences (p<0.05) in the mean colony numbers and diameters between the three groups, with the Sertoli coculture having the highest mean values for colony numbers and diameters. The RT-PCR results, after two-weeks of culturing, showed that β1-integrin was expressed in all three groups co-cultures, but α6-integrin was not expressed. Additionally, based on real time PCR results, the three genes (β1-integrin, α6-integrin, Oct-4) mentioned were also expressed in all three co cultures groups. Conclusion: Based on the optimal effects of Sertoli feeder cells on spermatogonial stem cells in a co culture system, as also confirmed by several other studies, their use is suggested to achieve better colonization of SSCs

Evaluation of Physical Parameters of Skin by Consecutive Ultrasonic Image Processing During Ultraviolet Radiation in an Animal Model of Wrinkled Skin

BACKGROUND AND OBJECTIVE: Skin aging is divided into two categories of intrinsic and extrinsic aging. Skin aging due to repeated exposure to ultraviolet radiation (extrinsic aging) is different from aging caused by time (intrinsic aging). The appearance of wrinkles caused by sunlight is due to subcutaneous fat atrophy and reduced production of collagen and elastin, thereby altering the biomechanical properties of the skin tissue. This study was conducted to investigate the skin damage caused by ultraviolet B (UVB) radiation by consecutive ultrasonic image processing with high resolution. METHODS: In this experimental study, we evaluated the skin injury process among 25 C57BL/6 mice in healthy group (zero dose), and case group exposed to UVB radiation at 0.03 milliwatts per square centimeter (5 times a week for 5 weeks) due to differences in skin characteristics. Physical parameters of dermal and epidermal layers were measured and evaluated weekly from day 7 to day 35 using ultrasonic image processing. FINDINGS: The thickness of the dermal and epidermal layers obtained by ultrasonic processing during the process of ultraviolet radiation injury in the mouse model significantly increased during the 5 – week study (p < 0.05). In addition, the percentage of changes in the thickness of the epidermis layer (from 0.22±0.01 mm on day zero to 0.37 ± 0.02 mm on the thirty-fifth day) and the dermal layer (from 0.57 ± 0.05 on day zero to 0.90 ± 0.08 mm on the thirty-fifth day) showed 68% and 57% increase, respectively. CONCLUSION: The results showed that UVB radiation increased the thickness of the skin layers

The Epigenetic Assessment of Human Spermatogenic Cells Derived from Obstructive Azoospermic Patients in Different Culture Systems

Purpose: Generating functional gametes for patients with male infertility is of great interest. We investigated different cultural systems for proliferation of SSCs derived from obstructive azoospermic patients. Materials and Methods: Testicular cells were obtained from men with obstructive azoospermia. After enzymatic digestion process, cells were assigned to various groups: culture of SSCs in the dish without cover (control group), co-culture of SSCs with infertile Sertoli cells (I), co-culture of SSCs with fertile Sertoli cells (II), culture of SSCs on nanofiber (covered with laminin) (III), culture of testicular cell suspension (IV). Then cells were cultured and colony formation, gene-specific methylation (by MSP), quantitative genes expression of pluripotency (Nanog, C-Myc, Oct-4) and specific germ cell (Integrin α6, Integrin β1, PLZF) genes were evaluated in five different culture systems. Results: Our findings indicate a significant increase in the number and diameter of colonies in IV group in compare to control group and other groups. Expression of germ specific genes in IV group were significantly increased (P < 0.05) and levels of expression of pluripotency genes were significantly decreased in this group (P < 0.05) compared with other groups. Gene-specific pattern of methylation of examined genes showed no changes in culture systems during the culture era. Conclusion: A microenvironment capable of controlling the proliferation of cell colonies can be restored by testicular cell suspension

Stem cells research and its applications: A review

Research in developmental biology has led to the discovery of different types of stem cells (totipotent, pluripotent and multipotent stem cells) that can give rise to multiple tissue types. This review summarizes a description about the stem cell concept, different type of stem cells and their potential applications. The face of extraordinary advances in the prevention, diagnosis and treatment of human diseases, devastating illnesses such as heart disease, diabetes, cancer and diseases of the nervous system, continue to deprive people of health, independence and well-being has been reviewed in this study. Stem cell research leading to prospective therapies in reparative medicine has the potential to affect the lives of millions of people around the world and there is a good reason to be optimistic. The road towards the development of an effective cell-based therapy for widespread use is long and involves overcoming numerous technical, legislative, ethical and safety issues. © 2011 Asian Network for Scientific Information

Isolation and proliferation of spermatogonial cells from ghezel sheep

Background: Sheep industry has taken steps toward transforming itself into a more efficient and competitive field. There are many varieties of sheep breeds in the world that each of them serves a useful purpose in the economies of different civilizations. Ghezel sheep is one of the Iranian important breeds that are raised for meat, milk and wool. Field of spermatogonial cell technologies provides tools for genetic improvement of sheep herd and multiple opportunities for research. Spermatogonial cells are the only stem cells capable of transmitting genetic information to future generations. Methods: This study was designed to extend the technique of isolation and in vitro proliferation of spermatogonial cells in Ghezel sheep. Results: Isolated cells were characterized further by using specific markers for type A spermatogonia, including PLZF. Also, sertoli cells were characterized by vimentin which is a specific marker for sertoli cells. After 10 days of co-culture, viability rates of the cells was above 94.7, but after the freezing process the viability rates were 74 percent. Conclusion: In this study, a standard method for isolation and in vitro proliferation of spermatogonial stem cells in Ghezel sheep was developed. © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved

Isolation and proliferation of spermatogonial cells from ghezel sheep

Background: Sheep industry has taken steps toward transforming itself into a more efficient and competitive field. There are many varieties of sheep breeds in the world that each of them serves a useful purpose in the economies of different civilizations. Ghezel sheep is one of the Iranian important breeds that are raised for meat, milk and wool. Field of spermatogonial cell technologies provides tools for genetic improvement of sheep herd and multiple opportunities for research. Spermatogonial cells are the only stem cells capable of transmitting genetic information to future generations. Methods: This study was designed to extend the technique of isolation and in vitro proliferation of spermatogonial cells in Ghezel sheep. Results: Isolated cells were characterized further by using specific markers for type A spermatogonia, including PLZF. Also, sertoli cells were characterized by vimentin which is a specific marker for sertoli cells. After 10 days of co-culture, viability rates of the cells was above 94.7, but after the freezing process the viability rates were 74 percent. Conclusion: In this study, a standard method for isolation and in vitro proliferation of spermatogonial stem cells in Ghezel sheep was developed. © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved

Nestin, a neuroectodermal stem cell marker, is expressed by bovine sertoli cells

Nestin, an intermediate filament protein is expressed by neuroectodermal stem cells and tumors originating from cells of neuroectodermal and mesenchymal lineages. Nestin expression is prominent in embryos and remains upregulated until 3-6 weeks after birth but is downregulated afterward. Sertoli cells are nucleated somatic cells that are spanned in the seminiferous epithelium and play a critical role in supporting and controlling germ-cell development. In this context, we employed immunocytochemical, Western blot, and Flow cytometric analyses to demonstrate nestin expression in bovine sertoli cells. Immunostaining clearly showed that setoli cells express high levels of nestin, a result which was confirmed by Western blot analysis of purified cells. Intracellular staining of sertoli cells by flow cytometry revealed that around 74 of the cells express this marker. Given the high expression of vimentin by sertoli cells, it is proposed that the expression of nestin in these cells might be required for the formation of stable vimentin/nestin intermediate filament network. In light of these findings, it seems that sertoli cells of mature bull have potentiality of proliferation. © 2010 Springer-Verlag London Limited

Genetic Changes during Differentiation of Spermatogonial Stem Cells into Oligoprogenitor Cells

BACKGROUND AND OBJECTIVE: The culture of spermatogonial cells and the production of embryonic stem cells (ES-like cells), suggest these cells as a sufficient new source for cell therapy and for the treatment of diseases, including neurodegenerative diseases. The aim of this study is to evaluate the pattern of genetic changes during differentiation of spermatogonial cells into oligodendrocyte precursor cells. METHODS: In this experimental study, spermatogonial cells were isolated from the testicles of 2 – 6 days old newborn mice (6 – 10 mice each time) through two stages of enzymatic digestion. The cells were divided into three groups of quasi-embryonic stem cells, neuro-progenitive and oligodendrocyte precursor. Specific markers stra8, mvh, piwil2, C-myc, Nanog, NF68, Nestin, Olig2, and NG2 were evaluated using Real Time-PCR and immunocytochemistry method at each differentiation step. FINDINGS: Molecular evaluations showed that increase in Nestin gene expression in neuronal precursor cells was 1.3 times more than quasi-embryonic stem cells. In the molecular evaluations at the end of the second stage of differentiation, it was determined that culture at the end of the induction steps resulted in a significant increase in the expression of the genes of Olig2 and NG2 and decrease in the expression of Nestin gene (p<0.05). Molecular evaluations showed that this increase in oligodendrocyte-like cells was respectively 1.4 and 1.6 times more than neuronal precursor cells. CONCLUSION: In this study, it was demonstrated that quasi-embryonic stem cells have the potential to express the NF68 and Nestin neuron markers. The quasi-embryonic stem cells of NG2 and Olig2 genes were expressed in the cells after the induction stage

Xenotransplantation assessment: Morphometric study of human spermatogonial stem cells in recipient mouse testes

The purpose of this study was (i) To establish in vitro propagation of human spermatogonial stem cells (hSSCs) from small testicular biopsies to obtain a high number of cells; (ii) to evaluate the presence of functional hSSCs in culture system by RT-PCR using DAZL, α6-Integrin, β1-Integrin genes; and (iii) to evaluate the effects of cell concentration on successful xenotransplantation of hSSCs in mice testis. Donor hSSCs were obtained from men with maturation arrest of spermatogenesis duration 1 year ago. These cells were propagated in DMEM containing 1 ng ml-1 bFGF (basic fibroblast grow factor) and 1500 U ml LIF (leucaemia inhibitory factor) for 5 weeks. Different concentrations of hSSCs transplanted into seminiferous tubules of busulfan-treated immunodeficient mice and analysed up to 8 weeks after transplantation. The results showed that expression of DAZL and α6-Integrin mRNA was increased as well as the colony formation of SSCs in vtro culture during 5 weeks. Proliferation occurred about 4 weeks after transplantation, but meiotic differentiation was not observed in recipient testis after 8 weeks. The difference in donor cells concentration had effect on homing spermatogenesis in recipient testis. Homologous transplantation of proliferated SSCs to seminiferous tubules of that patient individually may allow successful differentiation of transplanted cells. © 2014 Blackwell Verlag GmbH