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| Status |
Public on Jun 29, 2020 |
| Title |
Transcriptome analysis reveals conditions for culturing human primitive undifferentiated spermatogonia |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for male infertility, which afflicts >100 million men world-wide. To devise SSC therapy approaches, it is critical to first develop methods to culture human SSCs in vitro. Here, we report on a transcriptome approach to address this question. Using single-cell RNA-seq (scRNAseq), immunofluorescence, and germ-cell xenograft transplantation analyses, we identified a cell-surface protein, PLPPR3, that purifies human primitive undifferentiated spermatogonia (uSPG) enriched for SSCs. Comparative RNAseq analysis of PLPPR3+ cells (primitive uSPG) with KIT+ cells (enriched for differentiating [d] SPG) revealed that these two stages differentially express a remarkably large number of genes, including genes encoding key components in the TGF, GDNF, AKT, and JAK-STAT signaling pathways. Using scRNAseq analysis and conventional approaches, we tested the effect of manipulating these signaling pathways on cultured human SPG. This revealed that GDNF and BMP8B broadly support the culture of SPG, Activin A supports more advanced SPG, and one condition—AKT pathway inhibition—had the unique ability to selectively support primitive uSPG. These findings have implications for methods to culture and expand human SSCs for therapeutic uses in the future.
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| Overall design |
Bulk RNAseq was performed on PLPPR3+ and KIT+ cells FACS purified from 4 independent human testicular biopsy samples (aged from 30 to 41). For single cell RNA-Seq, testicular cells from biopsies obtained from two fertile men aged 32- and 37-years, respectively, were cultured under different conditions (FGF2 alone vs AKT-I+FGF2) for two weeks and then collected for sequencing.
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| Contributor(s) |
Tan K, Wilkinson MF |
| Citation(s) |
32661178 |
| Submission date |
Jan 22, 2020 |
| Last update date |
Sep 28, 2020 |
| Contact name |
Kun Tan |
| E-mail(s) |
kutan@health.ucsd.edu
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| Organization name |
UCSD
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| Department |
Department of Obstetrics, Gynecology, and Reproductive Sciences
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| Lab |
Miles F. Wilkinson
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| Street address |
2880 Torrey Pines Scenic Dr. Room 4812
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platforms (2) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA602712 |
| SRA |
SRP243934 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE144085_AKT-I+FGF2_Rep1_barcodes.tsv.gz |
15.2 Kb |
(ftp)(http) |
TSV |
| GSE144085_AKT-I+FGF2_Rep1_features.tsv.gz |
264.3 Kb |
(ftp)(http) |
TSV |
| GSE144085_AKT-I+FGF2_Rep1_matrix.mtx.gz |
31.1 Mb |
(ftp)(http) |
MTX |
| GSE144085_AKT-I+FGF2_Rep2_barcodes.tsv.gz |
14.6 Kb |
(ftp)(http) |
TSV |
| GSE144085_AKT-I+FGF2_Rep2_features.tsv.gz |
264.3 Kb |
(ftp)(http) |
TSV |
| GSE144085_AKT-I+FGF2_Rep2_matrix.mtx.gz |
30.9 Mb |
(ftp)(http) |
MTX |
| GSE144085_FGF2_Rep1_barcodes.tsv.gz |
11.9 Kb |
(ftp)(http) |
TSV |
| GSE144085_FGF2_Rep1_features.tsv.gz |
264.3 Kb |
(ftp)(http) |
TSV |
| GSE144085_FGF2_Rep1_matrix.mtx.gz |
23.1 Mb |
(ftp)(http) |
MTX |
| GSE144085_FGF2_Rep2_barcodes.tsv.gz |
10.1 Kb |
(ftp)(http) |
TSV |
| GSE144085_FGF2_Rep2_features.tsv.gz |
264.3 Kb |
(ftp)(http) |
TSV |
| GSE144085_FGF2_Rep2_matrix.mtx.gz |
19.4 Mb |
(ftp)(http) |
MTX |
| GSE144085_fcounts.txt.gz |
5.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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