Here, we aimed to answer important and fundamental questions in germ cell biology with special focus on the age of the male donor cells and the possibility to generate embryonic stem cell- (ESC-) like cells. While it is believed that spermatogonial stem cells (SSCs) and truly pluripotent ESC-like cells can be isolated from adult mice, it remained unknown if the spontaneous conversion of SSCs to ESC-like cells fails at some age. Similarly, there have been differences in the literature about the duration of cultures during which ESC-like cells may appear. We demonstrate the possibility to derive ESC-like cells from SSC cultures until they reach adolescence or up to 7 weeks of age, but we point out the impossibility to derive these cells from older, mature adult mice. The inability of real adult SSCs to shift to a pluripotent state coincides with a decline in expression of the core pluripotency genes Oct4, Nanog, and Sox2 in SSCs with age. At the same time genes of the spermatogonial differentiation pathway increase. The generated ESC-like cells were similar to ESCs and express pluripotency markers.
Pluripotent stem cells (PSCs) are undifferentiated cells which have the potential for proliferation, self-renewal, and differentiation into ectodermal, mesodermal, and endodermal cells of all three embryonic germ layers
The generation of PSCs of mouse testis cells dates back to 2004 by Kanatsu-Shinohara et al. [
Also Seandel et al. generated adult spermatogonial-derived stem cells from GPR125-positive cells in 3-week- to 8-month-old mice, but these cells were only multipotent [
In our experiments, we identified the spontaneous conversion of SSCs in ESC-like cells from neonate and nearly adult testis up to 7-week-old mice. On the contrary, it was impossible to generate ESC-like cells from mice older than 7 weeks. According to the NIH criteria (
The potential generation of pluripotent cells from SSCs can apparently only be realized up to the age of 7 weeks. Therefore, it is a debatable point whether generation of pluripotent SSCs depends on their development status in correlation with the completion of puberty. The possibility of generating ESC-like cells from this cell type seems to stall before donor mice are fully matured adults.
All animal experiments were confirmed to the local and international guidelines for the use of experimental animals and were approved by the Royan Institutional Review Board and Institutional Ethical Committee (Tehran, Iran) and by the regional authorities in Germany (Regierungspräsidium Karlsruhe). Testis cells were isolated from C57BL/6, 129/Sv mouse strains of 6-day- to 6-month-old transgenic Oct4-GFP-reporter mice. After removing the tunica albuginea, the seminiferous tubules were separated and placed in a digestion solution which contained collagenase IV (0.5 mg/mL, Sigma), DNAse I (0.5 mg/mL, Sigma), and Dispase I (0.5 mg/mL, Roche) in HBSS buffer with Mg++ and Ca++ (PAA) at 37°C for 8 minutes. Digestion enzymes were stopped with 10% ESC-qualified FBS (Invitrogen) and additionally the cell suspension was triturated by pipetting to obtain a single cell suspension. After centrifugation, the cell pellet was washed with DMEM/F12 (PAA), filtered through a 70
These generated ESC-like cells were manually selected and subcultured on a MEF feeder layer in mouse ESC (mESC) medium with KO-DMEM, (Invitrogen) 15% ESC-qualified FBS (Invitrogen), 1% NEAA (PAA), 1% L-glutamine (PAA), 1% Pen-Strep (PAA), 0.1%
In the supplementary method section (in Supplementary Material available online at
After mild digestion with collagenase, the seminiferous testicular tubules from neonatal, 7-week-old, and 12-week-old mice were separated and could be microscopically investigated under UV-light. The Oct4-GFP signal was clearly observable in the freshly isolated seminiferous tubules of neonate mouse testis (Figure
Number and intensity of GFP signals in the neonate and adult mouse testicular tubules (a, b) and SSC cultures (c, d) from transgenic Oct4-GFP reporter mice. (A1–A3) In the freshly dissected testicular tubules, the number of Oct4-GFP positive cells and the intensity of the Oct-GFP signal were higher and stronger in neonate mice than in adult mice >7 weeks (B1–B3). (C1–C3) Oct4-GFP positive SSCs were clearly present during initial cultures from neonate mice, while in adult mice >7 weeks SCCs Oct4-GFP signals were much weaker from the beginning (D1–D3). SSC colonies were grown on MEF feeder layers. (A1–D1) bright field; (A2–D2) green fluorescence for Oct4-GFP; (A3–D3) merged images. Scale bars: (a)–(c) 50
Up to 14–21 days after initiation of the primary testis cultures with SSC medium, SSCs with a positive Oct4-GFP signal were observed during the culture of neonate but were observed very rarely during the culture of adult mice (Figures
We quantified and analyzed the expression of important germ cell-enriched genes (
Hierarchical clustering (dendrogram) and principal component analysis (PCA), as in Figure
Different gene expression profiles of neonatal and adult SSCs with germ cell-enriched and pluripotency associated genes. Adult SSCs were obtained from 7- and 12-week-old mice. (a) Dendrogram and (b) PCA demonstrate that neonate and adult SSCs are distinct and localize in separated trees in the dendrogram or areas in the PCA. (c) Heat map shows array of pluripotency and germ cell associated genes with a cluster of different populations of neonatal SSCs (coloured dark blue), while adult SSCs cluster from 7- and 12-week-old mice in a separate tree (coloured light blue and green).
The heat map analysis with an array of pluripotency and germ cell associated genes revealed that a cluster of various populations of neonatal SSCs was significantly different from the other groups, while adult SSCs from 7- and 12-week-old mice clustered in a separate tree.
The neonatal SSCs expressed a significantly higher level of the pluripotency
Bar plot showing expression of pluripotency and germ cell associated genes between neonatal SSCs (coloured dark blue), 7-week-old adult SSCs (coloured light blue), and 12-week-old adult SSCs (coloured green blue). Red arrows mark significantly downregulated genes and purple arrows mark upregulated genes in adult SSCs (more than 2-fold and
In contrast, several germ cell associated genes in the adult SSCs were expressed in descending order
Not significantly regulated between neonate and adult SSCs were
In a comparison between neonatal SSCs and SSCs obtained from 12-week-old mice, these differences became even more apparent (see Supplementary Tables). Moreover, comparing SSCs from 7-week-old and 12-week-old mice, the pluripotency genes are significantly higher expressed in SSCs obtained from 7-week-old mice.
As apparent in the bar plot (Figure
(a) Heat map and (b–d) correlation analyses reveal expression levels of core pluripotency genes Oct4, Nanog, and Sox2 decrease in SSCs with age of the animal. A decline in Oct4, Nanog, and Sox2 expression is clearly observable after 7 weeks of age and becomes even more evident after 12 weeks of age. Arrows in (b–d) mark the localization of Oct4, Nanog, and Sox2 in the correlations.
As documented in Figures
(a) Table and (b) graph demonstrate that ESC-like cells can only be obtained with SSCs obtained from mice until the age of 7 weeks. SSCs obtained from older adult animals are unable to show a shift to pluripotency (red arrows).
Schematic illustration pinpoints that occurrence of pluripotent ESC like cells from Oct4 GFP positive cells with the production of chimera and formation of teratoma is restricted to neonatal up to 7-week-old mice.
We observed no development of ESC-like colonies from the SSC cultures at all before 46 days and after 143 days.
ESC-like colonies had a packed spindle- to round-shaped morphology with smooth borders (Supplementary Figure 2A1). Moreover, they displayed a high intensity of the Oct4-GFP signal (Supplementary Figures 2A2, 2A3). The ESC-like cell lines were passaged 1 : 5–1 : 8 for more than 15 times following trypsin digestion, with an estimated doubling time of 48–72 h. They still expressed Oct4-GFP after long-term cultivation. The cells preserved their undifferentiated state in multiple passages. The established ESC-like cell lines were successfully expanded, cryopreserved, and thawed with no loss in proliferation or differentiation capacities. Figures
Similar gene expression profiles of ESC-like cells and mESCs with germ cell-enriched and pluripotency associated genes. (a) Dendrogram and (b) PCA clearly demonstrate that ESC-like cells and mESCs are similar to each other but distinct to fibroblasts which localize in separated trees or areas. (c) Heat map shows array of pluripotency and germ cell associated genes with a cluster of ESC-like cells and mESCs (both underlined with orange bar), while fibroblasts cluster in a separate tree (underlined with black bar).
Bar plot showing expression of pluripotency and germ cell associated genes between ESC-like cells (coloured pink or dark red) and mESCs (coloured light red). Note that the core pluripotency genes Oct4, Nanog, and Sox2 are not differentially regulated between ESC-like cells and mESCs.
The ESC-like cell lines showed the ability to differentiate spontaneously
Thus, the expression of lineage specific marker genes,
We tested the capacity of the ESC-like cells to form a teratoma and to generate chimeric mice to further confirm their pluripotency. We subcutaneously transplanted 2 × 106 ESC-like cells into SCID mice. At four weeks after injection, ESC-like cells resulted in teratomas that contained all three germ layers (Figures
Pluripotency characterization of ESC-like cells with teratoma formation including tissue structures of all germ layers (a–d) and chimera formation (e–h). (a) Skin formation with keratinizing squamous epithelium, (b) respiratory epithelium with goblet cells, (c) neuronal rosette, and (d) primitive cartilage. (e, f) Blastocysts injected with GFP-marked ESC-like cells and chimera formation. Scale bars: (a), (e), and (f) 50
SSCs are the only source of naturally occurring truly pluripotent stem cells in the organism after birth, which do not have to be artificially reprogrammed such as iPSCs. The molecular mechanisms underlying the natural shift from a unipotent to a completely pluripotent cell during the establishment of mouse ESC-like cells from SSCs are not completely understood until now. However, it appears that the age of animals, the mouse strain used, the culture conditions with growth factors involved, the cell density of SSCs during culture, the time period after initiation of culture, and the length of culture might all be key players in the transition process [
In our work, we demonstrated the scarcity of the conversion of SSCs to ESC-like cells. Conversion occurred spontaneously from SSCs of neonate and up to 7-week-old mouse testis but not from older mice considered adult or even mature adult [
We observed that the amount of positive cells and signal density of Oct4-GFP in the seminiferous tubules of the neonate mouse testis was higher than in old mouse testis (after 7 weeks of age). Reduction of
We also observed another limitation for appearance of ESC-like cells after initiation of culture that only occurred during a special time window (46 until 143 days) after initiation of SSCs cultures. Several reports concerning long-term cultivation for SSCs failed to describe this spontaneous shift of SSCs to pluripotent ESC-like cells [
These results give the impression of a critical time window for the generation of pluripotent cells from SSCs and the impracticality of ESC-like cells being generated from continuous Oct4-GFP SSC culture. Kanatsu-Shinohara et al. also generated ESC-like cells during a time window about 4–7 weeks after initiation of culture in the neonate mouse SSCs [
Ko et al. [
We demonstrated that the ESC-like cells are fully pluripotent, express pluripotency markers, have the potential for complex teratoma formation, and produce chimera in the recipient mouse similar to mouse ESCs. Moreover, they are highly capable of differentiating into neuronal and cardiomyocyte phenotypes after
It would be of major interest to study factors, including small molecules, that could increase the probability and also the restricted time window of SSC to PSC conversion. Recently it has been shown that the addition of glycogen synthase kinase-3 inhibitors to the testis-derived SC cultures increases the likelihood for the occurrence of ESC-like cells from SSCs [
In the primary culture of isolated cells from Oct4 transgenic reporter mice, we observed that the Oct4-GFP signal was expressed at a moderate level in neonate up to a low level in older or adult SSCs. This expression was completely downregulated during short- and long-term SSC culture [
mRNA expression profiling confirmed that the expression of germ cell specific genes increased with age and was therefore significantly higher in SSCs from 7- to 12-week-old mice compared with neonatal SSCs. In parallel, we observed that the expression of
In PSCs, core transcriptional genes control the expression of different lineage specific genes and prevent pluripotent cells from differentiation [
This study has come to the conclusion that the natural reprogramming of unipotent SSCs into pluripotent cells cannot occur during adulthood and implies that this conversion is only observable until adolescence and during a special time window after initiation of culture.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to convey thanks to all the colleagues who kindly helped them in this research: Steffen Albrecht, Gerald Bendner (Institute of Anatomy and Cell Biology, University of Heidelberg), and all other people and institutions supporting this research.