ITGA6+ Human Testicular Cell Populations Acquire a Mesenchymal Rather than Germ Cell Transcriptional Signature during Long-Term Culture.

Robert B Struijk,Aldo Jongejan,Ans M M Van Pelt,Cindy M De Winter-Korver,Saskia K M Van Daalen,Sjoerd Repping,Callista L Mulder

doi:10.3390/ijms21218269

Abstract

Autologous spermatogonial stem cell transplantation is an experimental technique aimed at restoring fertility in infertile men. Although effective in animal models, in vitro propagation of human spermatogonia prior to transplantation has proven to be difficult. A major limiting factor is endogenous somatic testicular cell overgrowth during long-term culture. This makes the culture both inefficient and necessitates highly specific cell sorting strategies in order to enrich cultured germ cell fractions prior to transplantation. Here, we employed RNA-Seq to determine cell type composition in sorted integrin alpha-6 (ITGA6+) primary human testicular cells (n = 4 donors) cultured for up to two months, using differential gene expression and cell deconvolution analyses. Our data and analyses reveal that long-term cultured ITGA6+ testicular cells are composed mainly of cells expressing markers of peritubular myoid cells, (progenitor) Leydig cells, fibroblasts and mesenchymal stromal cells and only a limited percentage of spermatogonial cells as compared to their uncultured counterparts. These findings provide valuable insights into the cell type composition of cultured human ITGA6+ testicular cells during in vitro propagation and may serve as a basis for optimizing future cell sorting strategies as well as optimizing the current human testicular cell culture system for clinical use.

Highlights

Spermatogonial stem cells (SSC) are the progenitor cells of male gametes, developing to spermatozoa in the seminiferous epithelium of the testis through a specialized process called spermatogenesis
Based on cell type decomposition analyses over time during culture, ITGA6 + Primary testicular cells (PTC) appear to have a distinct cellular composition that is characterized by cell populations with a decreasing spermatogonial-related gene expression profile and an increasing gene expression profile related to cells of mesenchymal, fibroblast and peritubular myoid origins
In the primary testicular cell cultures examined here, a notable macroscopic shift from mostly non-adherent round cells to a pronounced monolayer of cells morphologically resembling spindle shaped fibroblast-like cells was observed, as well as a reduction in the percentage of ITGA6 + PTCs in long-term PTC cultures

Summary

Introduction

Spermatogonial stem cells (SSC) are the progenitor cells of male gametes, developing to spermatozoa in the seminiferous epithelium of the testis through a specialized process called spermatogenesis. Since SSCs are sensitive to damage originating from common gonadotoxic therapies, including chemo- and radiotherapy [1,2,3], survivors of cancer or hematological diseases are predisposed to develop infertility after they have been cured [4]. This is especially devastating for prepubertal patients that cannot benefit from sperm cryopreservation, in case they received gonadotoxic treatment before spermatogenesis has been initiated. Upon recolonization in the testicular niche, the transplanted in vitro propagated SSCs are capable of starting the process of life long spermatogenesis, allowing for natural conception to fulfill their child wish

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