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

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.

Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis

“…Some of our findings include culture conditions that have been implemented for years, such as the beneficial effect of differential plating of cells after isolation to reduce the amount of somatic cells at the start of culture, which are readily present within testicular biopsies and represent a heterogeneous population ( Struijk et al, 2020b ). However, the presence of feeder cells in culture may also contribute to establishment of the microenvironment to support propagation and differentiation of spermatogonia ( van der Sanden et al, 2010 ).…”

“…The manner of verifying the presence of these SSCs in propagation cultures by use of protein or gene markers is hindered by the current lack of an unambiguously established SSC marker for human culture ( Di Persio and Neuhaus, 2023 ). Therefore, a wide array of spermatogonial markers for PCR or immunofluorescence is used by researchers in an attempt to characterize the cultured cells, though these markers are often not specific for just one cell type and some markers have since been disputed due to their observed presence in somatic cells as well ( Kossack et al, 2013 ; Struijk et al, 2020b ). Conversely, instead of looking for specific markers researchers can also study specific behaviors of stem cells.…”

Spermatogonial Stem Cell-Based Therapies: Taking Preclinical Research to the Next Level

“…At this moment there is no agreement on a specific, unambiguous marker for human and/or non-human primate SSCs in vivo and in vitro . For many currently used markers, including ITGA6, KIT, GPR125 and DAZL, expression is not strictly limited to spermatogonia and can also be found in testicular somatic cells ( 61 , 62 ). For other markers, spermatogonial expression of the marker is controversial, as not all study results are in agreement.…”

“…Furthermore, it is uncertain whether various testicular cell types retain their transcriptomic and metabolic signatures when isolated from their natural niche ( 62 ). Due to these dynamic processes, the heterogeneity of the testicular cell populations and the many potential markers to choose from, it is difficult to establish inter-study comparisons of culture outcomes and efficiency with regard to SSC propagation.…”

“…developed a successful culture protocol, using medium based on StemPro-34 Serum Free medium, for long-term in vitro propagation of murine SSCs ( 58 ). Although a comparable medium can also support long-term propagation of human SSCs ( 59 , 60 ), overgrowth of testicular somatic cells within the culture system remains problematic, as the SSC signature is diluted over time ( 62 ). The presence of feeder cells is beneficial to the survival of SSCs as they provide mechanical and metabolic support and paracrine signals ( 85 , 86 ).…”