Prior exposure to alkylating agents negatively impacts testicular organoid formation in cells obtained from childhood cancer patients.

Study Question: Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)?

Summary Answer: Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy.

What Is Known Already: Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples.

Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids.

Human primordial germ cells (hPGCs), the precursors of eggs and sperm, start their complex development shortly after specification and during their migration to the primitive gonads. Here, we describe protocols for specifying hPGC-like cells (hPGCLCs) from resetting precursors and progressing them with the support of human hindgut organoids. Resetting hPGCLCs (rhPGCLCs) are specified from human embryonic stem cells (hESCs) transitioning from the primed into the naive state of pluripotency.

Epigenetic resetting in the human germ line entails histone modification remodeling.

Epigenetic resetting in the mammalian germ line entails acute DNA demethylation, which lays the foundation for gametogenesis, totipotency, and embryonic development. We characterize the epigenome of hypomethylated human primordial germ cells (hPGCs) to reveal mechanisms preventing the widespread derepression of genes and transposable elements (TEs). Along with the loss of DNA methylation, we show that hPGCs exhibit a profound reduction of repressive histone modifications resulting in diminished heterochromatic signatures at most genes and TEs and the acquisition of a neutral or paused epigenetic state without transcriptional activation.

Specification of human germ cell fate with enhanced progression capability supported by hindgut organoids.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, are specified during weeks 2-3 after fertilization. Few studies on ex vivo and in vitro cultured human embryos reported plausible hPGCs on embryonic day (E) 12-13 and in an E16-17 gastrulating embryo. In vitro, hPGC-like cells (hPGCLCs) can be specified from the intermediary pluripotent stage or peri-gastrulation precursors.

Self-organising human gonads generated by a Matrigel-based gradient system.

Background: Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro.

Spermatogonia Loss Correlates with LAMA 1 Expression in Human Prepubertal Testes Stored for Fertility Preservation.

Fertility preservation for male childhood cancer survivors not yet capable of producing mature spermatozoa, relies on experimental approaches such as testicular explant culture. Although the first steps in somatic maturation can be observed in human testicular explant cultures, germ cell depletion is a common obstacle. Hence, understanding the spermatogonial stem cell (SSC) niche environment and in particular, specific components such as the seminiferous basement membrane (BM) will allow progression of testicular explant cultures.

Use of a three-layer gradient system of cells for rat testicular organoid generation.

We have recently developed a 3D culture system that allows the reorganization of rat primary testicular cells into organoids with a functional blood-testis barrier, as well as the establishment and maintenance of germ cells. The innovative aspect of our model, the three-layer gradient system (3-LGS), comprises cells combined with Matrigel placed between two layers of Matrigel without cells, which creates a gradient of cells and allows the reorganization of testicular cells into organized structures after 5-7 d in culture. This reorganization is not observed when testicular cells are suspended in only one layer of Matrigel, the methodology used in the majority of the protocols for generating organoids.

Testicular organoids: a new model to study the testicular microenvironment in vitro?

Background: In recent decades, a broad range of strategies have been applied to model the testicular microenvironment in vitro. These models have been utilized to study testicular physiology and development. However, a system that allows investigations into testicular organogenesis and its impact in the spermatogonial stem-cell (SSC) niche in vitro has not been developed yet.

Testicular organoid generation by a novel in vitro three-layer gradient system.

A system that models the testicular microenvironment and spermatogonial stem-cell (SSC) niche in vitro has not been produced yet. Here, we developed and characterized a novel three-dimensional multilayer model, the Three-Layer Gradient System (3-LGS), which permits the generation of rat testicular organoids with a functional blood-testis barrier (BTB) and germ cell establishment and proliferation. The model is unique as regards the formation of cellular organizations that more closely represent the in vivo germ-to-somatic cell associations in vitro.

Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties.

So far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e.