In Vitro Generation of Haploid Germ Cells from Human XY and XXY Immature Testes in a 3D Organoid System

Abstract

Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

Keywords: Klinefelter syndrome; cancer survivors; fertility preservation; in vitro; male infertility; organoid; spermatogenesis 3D culture; spermatogonia; spermatogonia stem cells.

Figure 1

Hematoxylin and eosin (HE) staining on histology testicular tissue slides from (a) prepubertal XY donor, (b) Klinefelter syndrome peripubertal patient, and (c) XY mature control.

Figure 2

Immunostaining of testicular tissue. (a,b) Immunofluorescence for the protamine-1 (PRM1) early postmeiotic marker shows late-stage round spermatid (red) and nucleus (DAPI, blue) on (a) prepubertal donor tissue samples and (b) adult testis control. The differences in seminiferous tubules structure and the absence of PRM-1 positive cells in prepubertal patient sample confirmed the immaturity of the tissue. (c,d) Optical immunostaining for the undifferentiated spermatogonia marker PGP 9.5 (UCHL1) was demonstrated by the DAB chromogen (brown) and hematoxylin counterstain (blue) on (c) Klinefelter peripubertal tissue samples and (d) age-matched XY.

Figure 3

Characterization of 2D cultured prepubertal testicular cells with qRT-PCR for different cell-type markers. (I) Spermatogonial cell markers: ZTBT16, UCHL1, and THY1. (II) Sertoli cell markers: GATA4, CLU (Clusterin), and SOX9. (III) Leydig cell markers: STAR, TSPO, and CYP11A1. (IV) Peritubular cell markers: CD34 and ACTA2 on whole testis and isolated cells from the study patient. POLR2A (DNA-directed RNA polymerase II subunit RPB1) was utilized as a reference marker in all cell types. All the primers were tested to be exon spanning, both genomic DNA and water were used as negative controls with no band shown (no amplification).

Figure 4

Flow cytometry using enriching markers for spermatogonia stem cells (SSC) in 2D cultures of human prepubertal testicular cells. (A) Histogram of HLA-ABC FITC-conjugated positive cells (green) compared to isotype control (black). (B) Table of percentages of positive cells for different SSC enrichment markers combined: CD90, FGF-R3, SSEA-4, and CD9 without HLA-ABC.

Figure 5

Schematic picture illustrating the creation of three-dimensional (3D) testicular organoids from cultured testicular cells. (A) Two-dimensional cultured cells were trypsinized and made single cells in a suspension of formation media. (B) Single cells were plated in 10,000 cells/100 µL/well. (C) Plates were centrifuged at 150× g for 30 s to make cell aggregates. (D) After 48 h, single spheroids were formed.

Figure 6

Prepubertal (top two rows) and Klinefelter (bottom two rows) HTO morphology and viability. For each condition, the upper row represents live/dead staining, and the lower row is HE staining at each study time point. Scale bars 100 μm.

Figure 7

Evolution of the HTOs in culture. As no difference was observed between 46XY and 47XXY organoid size and ATP, results show data averages. (A) Average size at every time point of the study. (B) Average ATP production compared to initial determination. Data presented as mean ± SD. Significance: **** p < 0.000. Data showed the overall average of 46XY and KS HTOs as the two groups had no significant differences. Negative control: HTO medium without ATP assay regents was used to subtract the background signal.

Figure 8

Testosterone production of the HTOs in culture with or without previous hCG pulse stimulation. Data presented as mean ± SD. Significance: * p < 0.05.

Figure 9

Gene expression analysis of the HTO in differentiation culture media over time using qPCR-RT and dPCR. The data show no significant differences were found. (a) Gene expression comparison between HTOs on day 2 and day 23 in culture expressed as a fold increase in expression level. (b) Heat map of gene expression for germ cells at sequential differentiation stages: undifferentiated spermatogonia (ZBTB16), differentiating spermatogonia (DAZL), differentiating spermatocyte (SYCP3), and differentiated spermatid (PRM1). (c) Digital PCR analysis for postmeiotic markers PRM1 and acrosin combined with housekeeping gene POLR2A to assess the proportion of cells expressing these genes by the end of the culture period. Data presented as mean ± SD. Significance: ** p < 0.01; *** p < 0.001.

Figure 10

Fluorescence in situ hybridization (FISH) for Cr X (green), Cr Y (orange), and Cr 18 (yellow) was performed on fixed HTOs at every time point. Haploid cells with only one chromosome 18 and one sex chromosome, either X or Y, are identified with red arrows. Diploid cells are identified with white arrows for comparison. (A) FISH staining of prepubertal XY HTOs after 23 days in culture. (B) Pie chart showing the percentage of the haploid cells amongst the total population of the cells in the prepubertal XY HTOs. (C) FISH staining of peripubertal Klinefelter HTOs after 23 days in culture. (D) Pie chart showing the percentage of the haploid cells amongst the total population of the cells in the peripubertal Klinefelter HTOs. Scale bar 100 μm.