O-132 A systematic spatial HiPlex proteomics exploration of human testicular tissues from birth to adulthood

Abstract

Abstract Study question Can we map the key maturation stages of human spermatogonial stem cells (SSCs) within their niche during infancy and puberty? Summary answer Using highplex spatial proteomics, we describe the key cellular events taking place during human testis maturation. What is known already The human testis is a complex “ecosystem” that matures in response to endocrine and exocrine signals and interactions between its diverse cellular components. Unlike animal models, in humans, testicular maturation is a slow process that takes place over more than a decade. In contrast to ovaries, the human testis retains its immature state until the onset of puberty. Single-cell RNA sequencing data has revealed significant developmental changes for germ cell and the somatic niche during infancy and puberty. Study design, size, duration Using the hyperplex imaging technique (CellDIVE) and a panel of 30 protein markers - selected to label key components of the developing human testis - we have systematically analyzed a time series of ∼75 testicular biopsies from boys aged 8 months to adulthood. Following sequential staining and capture of high-resolution images, our analysis pipeline involves the conversion of image into single-cell data, which allows a systematic description of the spatial SSC/niche microenvironment interactions across development. Participants/materials, setting, methods The panel of antibody markers was defined using integrated scRNAseq data and represents all key cell types, including germ cell markers (e.g., MAGEA4), Sertoli cell markers (e.g., SOX9), Leydig cell markers (e.g., INSL3), myoid cell markers (e.g., ACTA2) ... Main results and the role of chance To describe the events occurring during human testicular maturation, we build a Tissue Microarray (TMA) containing ∼75 biopsies of boys of different ages (0-16yrs old) and using an in-house panel of 30 markers for high-dimensional multiplex CELL DIVE images. Our analytical strategy uses Uniform Manifold Approximation and Projection (UMAP) maps to quantify and statistically compare single-cell data. Our marker panel allows us to phenotype testicular cells at different stages of development, including SSCs (state 0 to state 4), Sertoli cells, Leydig cells, and so on. Moreover, employing spatial-latent Dirichlet allocation (LDA), we identified recurrent cellular neighbourhoods (RCNs), revealing distinct cell clusters and their corresponding RCNs. These RCNs reflect recurring spatial arrangements of cells within testicular tissues, allowing us to infer the spatial organization and interplay among different cell types. Additionally, we explored spatial distances and neighbourhood frequencies across different tissues and conducted spatial co-occurrence analysis to assess the likelihood of finding adjacent cell types. The identification of RCNs offers valuable insights into the microenvironmental dynamics and cellular relationships taking place within the developing testis. Looking ahead, we aim to delve into the functional significance of RCNs in shaping the developmental trajectories and physiological processes within human testicular tissues. Limitations, reasons for caution Due to limitations in proteomics techniques, we could only include ∼30 protein markers on our panel. Although the biopsy samples used in our study are all from chemo-naïve patients, it possible that the conditions affecting these paediatric patients may impact the architecture or development of their testicular tissues. Wider implications of the findings Our study provides a systematic approach to study the development and organization of human testicular structures, which will allow to enumerate the cellular content of individual testicular biopsies and infer their stage of maturation, therefore providing a personalised baseline assessment for each patient at the time of cryopreservation. Trial registration number not applicable