Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture

Abstract

Study question: Can human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia?

Summary answer: Germ cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1-2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures.

What is known already: The development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures.

Study design, size, duration: Testicular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures.

Participants/materials, setting, methods: Immunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1-2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4).

Main results and the role of chance: UTF1, FGFR3, DAZL and ZBTB16 qRT-PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1-2 weeks), their expression steadily declined over time in vitro. After 6 weeks in culture only somatic cells were detected.

Limitations, reasons for caution: Different groups attempting SSC culture have utilized different sources of human testes and minor differences in the preparation and maintenance of the testicular cell cultures. Differences in outcome may be explained by genetic background of the source tissue or technical differences.

Wider implications of the findings: The ability to propagate human SSCs in vitro is a prerequisite for proposed autologous transplantation therapy aimed at restoring fertility to men who have been treated for childhood cancer. By applying the assays validated here it will be possible to quantitatively compare human SSC culture conditions. The eventual development of conditions for long-term propagation of human SSCs in vitro will greatly facilitate learning about the basic biology of these cells and in turn the ability to use human SSCs in therapy.

Study funding/competing interests: The experiments presented in this manuscript were funded by a Project Development Team within the ICTSI NIH/NCRR Grant Number TR000006. The authors declare no competing interests.

Trial registration number: Not applicable.

Keywords: cell culture; fertility; germ cells; stage-specific embryonic antigens; testis.

Figure 1

Summary of methods used to establish primary testicular cell cultures. See Materials and Methods for full description.

Figure 2

Immunofluorescence analysis of germ and somatic cell markers in seminiferous tubules. Immunostaining on paraffin sections (A, B and C) or whole tubule pieces (D). Co-immunostaining was performed to detect (A) DAZL (green) and VIM (red), (B) SALL4 (green) and UTF1 (red) or (C) SALL4 (green) and GATA4 (red). DAPI was used to stain the DNA (blue). Merges of green and red show that the germ cell and somatic cell staining were mutually exclusive. SALL4 immunostaining was unexpectedly observed in post meiotic cells, although it may be background staining associated with this lot of polyclonal antibody (Eildermann et al., 2012). Bars represent 50 μm.

Figure 3

Immunofluorescence analysis of germ and somatic cell markers in early primary testicular cell cultures. Primary testicular cells were cultured in germ cell maintenance medium prior to fixation and co-immunostaining to detect (A) DAZL (green) and VIM (red) or (B) SALL4 (green) and UTF1 (red) or (C) SALL4 (green) and GATA4 (red). Merges of green and red are shown. DAPI was used to stain the DNA (blue). Bars represent 50 μm.

Figure 4

Immunofluorescence analysis of somatic cell markers in cultured somatic cells. The bound fraction of dissociated testicular cells were cultured in somatic cell maintenance medium (F12/FBS) prior to fixation and immunostaining to detect (A) GATA4, (B) ACTA2 or (C) VIM. DAPI was used to stain the DNA (blue). Bars represent 50 μm. (D and E) Oil Red O staining of 293 human embryonic kidney fibroblasts (D) or SOM cells (E). (F) Agarose gel analysis of the indicated qRT–PCR products using Hu2 SOM RNA (top image) or Hu4 SOM RNA. ‘+’ and ‘−’ refer to the presence or absence of reverse-transcriptase in cDNA reactions. ‘Ladder’ refers to the DNA size markers of 100–400 base pairs.

Figure 5

Quantitative RT–PCR analysis of early primary testicular cell cultures. (A) Representative images of PTC on Day 7 and SOM on Day 20 are shown. (B–H) RNA was isolated from cultures of primary testicular cells on Day 7 (n = 2, PTC from Hu2 and Hu4) and somatic cells on Day 20 (n = 3, SOM from Hu2 twice and Hu4 once). Related data from additional culture experiments are presented in Supplementary data, Fig. S2. Graphs depict the mean ± SD of the relative mRNA levels of the indicated mRNAs normalized to GAPDH mRNA levels for each sample. The y-axis represents arbitrary units. The P-values and means are shown. Bar represents 100 μm.

Figure 6

Flow cytometry analysis of SSEA4 and CD9 in primary testicular cell cultures. PTC and SOM cells from Day 8 of culture were immunostained with antibodies to CD9 (A) or SSEA4 (B and C) or the corresponding IgG isotype control for each. Representative dot plots depicting the forward/side scatter for SOM (A, left) and PTC (B and C, left) are shown. Dot plots from the same samples are shown in (B) and (C) except a more restrictive forward/side scatter gate is applied in (C). The plots shown in this figure are from an experiment using frozen Hu5 testes. The same results were obtained using cultures derived from frozen Hu6 testes.

Figure 7

Immunofluorescence analysis of SSEA4 in primary testicular cultures. Primary testicular cells (A and B) or SOM cells (C and D) were cultured for 6 days prior to fixation and immunostaining to detect SSEA4. DAPI was used to stain the DNA (blue). The images shown are from cultures derived from Hu2 and the same results were found with cultures from Hu4 and other donors. Bar represents 50 μm.

Figure 8

Quantitative RT–PCR analysis of SSEA4 sorted cells. Dot blots show SSEA4 fluorescence on the x-axis in (A) primary testicular cells or (B) somatic cells on Day 11 of a culture derived from frozen Hu2 testes. (C) Isotype control. RNA was isolated from SSEA4^bright, SSEA4^dim and SSEA4^negative cells. Graphs depict the mean relative mRNA levels of (D) FGFR3, (E) UTF1 or (F) VIM normalized to GAPDH mRNA levels for that sample. The y-axis represents arbitrary units.

Figure 9

Quantitative analysis of validated germ cell markers in primary testicular cells over time in culture. (A and B) Representative images of PTC on Day 7 and Day 42 derived from either fresh (A) or frozen/thawed (B) testes. (C) The graph depicts the mean ± SD of the percentage SSEA4^bright cells on Day 1 (n = 2: Hu8 and Hu9 fresh; n = 2: Hu2 and Hu4 frozen), Day 7 (n = 2: Hu8 and Hu9 fresh; n = 2: Hu2 and Hu4 frozen) and Day 42 (n = 1: Hu9 fresh; n = 2 Hu2 and Hu4 frozen) of culture. (D–K) Graphs depict the mean of the relative mRNA levels of the indicated mRNAs normalized to GAPDH mRNA levels for each timepoint. Each graph depicts data from a PTC of a single donor with the mean from technical duplicates displayed at each timepoint for either fresh (D–G) or frozen (H–K). The y-axis represents arbitrary units. Similar qRT–PCR results from different donors are shown in Supplementary data, Fig. S3.

Figure 10

Immunofluorescence analysis of somatic cell markers in Day 48 primary testicular cultures. Primary testicular cells derived from fresh Hu9 testes were cultured for 48 days prior to fixation and immunostaining to detect GATA4 (A), ACTA2 (B) or VIM (C). DAPI was used to stain the DNA (blue). These same results were obtained using cultures from frozen Hu4 testes. Bar represents 100 μm.