Characterization and Survival of Human Infant Testicular Cells After Direct Xenotransplantation

Abstract

BackgroundCryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications.MethodsTestis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1).ResultsFollowing xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes.ConclusionXenotransplantation, without in vitro propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time.

Germline stem cells in human

“…389,390 Germ cell transplantation in interspecies and intraspecies has been applied to zebrafish, rats, dogs, farm animals (goats, sheep, pigs, and cattle), nonhuman primates, and humans, in addition to mice. [391][392][393][394][395][396][397][398][399][400][401][402][403][404][405][406][407][408] In primates, postpubertal SSC transplantation in infertile rhesus monkeys restored functional sperm production after puberty. 409 In humans, the first clinical trial of testis-cell transplantation using cryopreserved single-cell suspension from patients' testis with lymphoma before chemotherapy was reported in 1999.…”

Puberty Blocker and Aging Impact on Testicular Cell States and Function

“…This is because the generation of gametes from human SSC that have not naturally transitioned during puberty to a meiotogenetic state have been unsuccessful ( 45, 46 ). Prepubertal human testicular tissue/SSC has also failed to generate gametes in vitro or when xenografted in animals ( 47, 48 ). Additionally, the actions of GnRH and GnRHa may be influenced by genetics and lifestyle, making it challenging to predict individual responses to their SSC pubertal transition.…”

A 20-year overview of fertility preservation in boys: new insights gained through a comprehensive international survey

“…The majority of centres nowadays perform histological or immunohistochemical analyses of testicular tissues prior to cryopreservation in order to assess the presence and quantity of germ cells, with a particular focus on spermatogonia. Moreover, individual centres have established these analyses also for frozen/thawed testicular tissues, for quality assurance purposes, and have published the results of these assessments ( Keros et al , 2005 , 2007 ; Kvist et al , 2006 ; Gille et al , 2021 ; Kabiri et al , 2022 ; Wang et al , 2022 ). Semi-quantitative analytical techniques have been proposed as a means of standardized quality assessment of freezing techniques and integrity of frozen tissue ( Rives-Feraille et al , 2022 ).…”