In vitro transplantation of spermatogonial stem cells isolated from human frozen–thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions

Abstract

Background Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. Results Testicular tissue samples from azoospermic patients were obtained and then these were freeze–thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P < 0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF , SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF , Tekt1 and TP1 , the results indicated that the genes were positive in the test group, while not in the control group. Conclusion These results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-dimensional testicular tissue culture.

Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys

“…The authors presented a single donor cell in mouse seminiferous tubules, which was not further characterized. Mohaqiq et al [56] observed spermatogenesis from transplanted human SSCs in mouse seminiferous tubules after in vitro xenotransplantation which has different conditions compared to nude mice in vivo xenotransplantation. In the present study, the donor-derived colonies with more than four cells were identified in the recipient mouse testis three and six weeks after transplantation.…”

Cellular Therapy via Spermatogonial Stem Cells for Treating Impaired Spermatogenesis, Non-Obstructive Azoospermia

“…The cell content is diluted to 5 × 10 6 cells/mL. Then the process of cell sorting is performed via magnetic-activated Cell Sorting (MACS) to select Thymus cell antigen 1 (Thy1+) or octamer-binding transcription factor 4 (Oct4+) or the promyelocytic leukaemia zinc finger (PLZF+) cells which are specific for SSCs [ 125 , 130 , 131 ]. Thy1 antibody-conjugated microbeads are added to the cell suspension (cell pellet with PBS) containing the SSCs with specific dilutions, and then incubated at a specific temperature for a certain time.…”

“…For instance, matrigel-based and hydrogel-based culture systems were successfully reported for mice SCC culture [ 135 , 136 ]. While in human, three-dimensional culture system using agarose gel was used to improve the differentiation of SCC to spermatocytes and spermatids [ 131 ]. Hydrogel-coated or gelatin-coated dishes together with growth factor (GDNG, bFGF, GFRA1-Fc fusion protein, NUDT6, TGFB, EGF, and LIF) were used for propagation of human GPR125 + spermatogonia [ 137 , 138 , 139 , 140 ].…”

Advanced bioengineering of male germ stem cells to preserve fertility

“… 33 By culturing tissue fragments or entire organ in vitro, the tissue structure can be preserved to support the natural developmental processes. 34 Organ cultures provide an opportunity to manipulate the paracrine environment and also to examine the role of each growth factor individually on the spermatogenesis process. 35…”

“…54 – 57 It is worth saying that this agarose-based system can be used as a support layer for testis organ culture in either fragmented or whole testis and for the culture of host testis fragments. 34 , 58 Some researchers have reported successful spermatogenesis induction by use of agar gel supports in 3D organ culture ( Table 1 ). In 2011, Sato et al 58 designed a new in vitro organ culture system onto which mouse SSCs lines are transplanted and can form colonies and differentiate up into fertile sperm.…”