Transcriptome profiling reveals signaling conditions dictating human spermatogonia fate in vitro

Abstract

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-β, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition—AKT pathway inhibition—had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.

Transcriptional states of retroelement-inserted regions and KRAB zinc finger protein association regulate DNA methylation of retroelements in human male germ cells

“…5C). Although approximately half of the "High and Low" SVA_B-F copies were located in the non-genic regions, RNA-seq reads from previously reported undifferentiated spermatogonia (Tan et al 2020) were more frequently mapped around the non-genic "High and Low" SVA_B-F copies than "Common Low" B-F copies (Fig. 5D).…”

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

“…We also detected a slightly increased expression of UCHL1 in LT-ITGA6 + PTCs, corresponding to expression of the UCHL1 transcript by some somatic cells originating from the testicular somatic cell population present in our cultured cell fractions. The issue of marker specificity to detect human spermatogonia in vitro has been raised in previous reports and is an ongoing topic of debate [ 27 , 28 , 60 , 61 ].…”

“…Follow up studies using single cell RNA-Seq could provide more clarity into the precise expression patterns of different testicular cell types, both in vivo and in vitro. Recently, substantial progress has been made in unravelling the transcriptomic properties of several testicular cell types through single cell RNA-Seq [ 61 , 62 , 63 ]. Alternatively, Q-PCR and FACS analyses of sorted cell populations within the LT-ITGA6 + PTCs might be another approach to further characterize each of the sorted population for expression of well-known cell type specific markers.…”

The role of the NMD factor UPF3B in olfactory sensory neurons

“…As described previously ( Sohni et al, 2019 ; Tan et al, 2020b ; Tan et al, 2020a ), de-multiplexed raw sequencing reads were processed and mapped to the mouse genome (mm10) using Cell Ranger software (v2.0) with default parameters. We filtered raw count matrices by excluding cells expressing less than 200 detectably expressed genes and genes expressed in less than 3 cells.…”