The ability to culture human spermatogonial stem cells (SSCs) in vitro is critical to develop SSC therapeutic approaches to treat male infertility. To achieve this goal, it is important to define molecular pathways in human SSCs. Toward this end, researchers at University of California San Diego School of Medicine developed an approach to purify human primitive undifferentiated spermatogonia (uSPG) highly enriched in SSCs. Comparative RNA-sequencing analysis of these primitive uSPG versus differentiating SPG identified differentially expressed genes encoding key components in several signaling pathways. Leveraging this information, coupled with single-cell RNA-sequencing analysis, the researchers studied the effects of modulating signaling pathways on human SPG fate in vitro. Inhibition of AKT signaling favored primitive uSPG fate, which can be applied to culture SSCs for therapeutic applications.
scRNA-seq analysis elucidates the constituents in human ITGA6+ cell cultures
(A) t-Distributed stochastic neighbor-embedding (tSNE) plot showing the cell clusters present in testicular ITGA6+ cells cultured for 2 wk with the agents shown. (B) Reclustering of the SPG cell cluster defined in A reveals five subclusters. (Lower) The percentage of cells in each subcluster. (C) Monocle trajectory analysis of subclusters defined in B. Arrow shows the pseudotime direction. (D) The expression pattern of genes preferentially expressed in the C1 subcluster, as shown on the pseudotime axis defined in C. (E) The expression pattern of differentiation-associated genes plotted along the pseudotime axis defined in C. (F, Upper) tSNE plot inferring the cell-cycle phase of the cells in the five subclusters defined in B, based on expression of a large set of G1-, G2/M-, and S-phase genes. (Lower) Percentage of cells in the cell-cycle phases indicated. (G) Enriched signaling pathways in each cell cluster defined in B.