Induction of human foreskin fibroblasts (hFSKs) and human mesenchymal stem cells (hMSCs) using germ line factors triggers the formation of germ cell-like cells

Initially, we identified a pool of 12 candidate genes (i12F), with unequivocal contribution in the mammalian germ line determination, migration and meiotic progression in the mouse model: PRDM12,12, PRDM1413, LIN28A2, NANOG14, NANOS315, DAZ2, BOLL, DAZL16, VASA (also known as DDX4)17,18,19, STRA820, DMC121 and SYCP322. Next, we cloned their Consensus Coding Sequences (CCDSs) into a lentiviral expression backbone with a CMV promoter and a green fluorescent protein (GFP) reporter (pLenti7.3/V5-DEST, Life Technologies) and used lentiviral particles to transduce them into human foreskin fibroblasts (hFSK) (46, XY) (Supplementary Figure S1C).

Initially, fibroblasts transduced with i12F were cultured in standard medium comprising DMEM/F12 with 10% FBS. Starting between days 3–7 post-transduction, we observed morphological changes in the fibroblast-like cultures with the appearance of round single cells that formed clumps of compacted small cells. However, after a few days, these clumps disappeared, suggesting that the culture conditions were not sufficient for their survival (Supplementary Figure S3A). Therefore, we based in previous reports on in vitro derivation of Spermatogonial Stem Cells (SSCs)23,24,25 to design a Germ Cell Medium (GC-M) enriched with several growth factors to promote the survival of the putative germ cells resulting from genetic induction (see Methods section for further details). Replacing stardard medium by GC-M at 24h post-transduction resulted in an increase of cell clumps formation (Supplementary Figure S3A). Thus, GC-M was employed for culturing both MOCK and induced cells in vitro in following experiments.

Transduced fibroblasts showed a clear up-regulation of all 12 induced factors during the first week post-transduction, with a marked decrease during the second and third week for most of the transgenes, probably due to the silencing of the CMV promoter driving its expression (Supplementary Figure S1A). However, further expression analysis at day 14 post-transduction indicated that transgenes continued their expression still at moderate levels (Supplementary Figure S1B). This observation was corroborated by a detectable GFP signal that did not disappear along time (Supplementary Figure S2A).

Initial characterization of i12F transduced hFSK cells indicated a significant up-regulation of the epithelial marker E-Cadherin (CDH1) and the PGC germ cell marker STELLA specifically in the clumps two weeks post-transduction. Although not significant, FRAGILIS, another known PGC marker, also showed a relative up-regulation in the clumps, suggesting their possible germ cell-like identity (Supplementary Figure S3B). Next, we sought to find the minimal combination of factors necessary for the phenotypic switch achieved with the i12F cocktail. For this, we screened among the different combinations of factors within i12F employing as a read out the efficiency of clump formation from hFSK cells. We individually transduced all twelve factors and selected those factors that induced the appearance of clumps. Afterwards, we designed factorial combinations of factors to achieve the maximum efficiency of clump formation by microscopic observation (Supplementary Figure S2). As a result of this screening, the most effective combination was the combined ectopic expression of: PRDM1, PRDM14, LIN28, DAZL and VASA. Additionally to these five factors, ectopic expression of SYCP3 resulted essential for achieving the meiotic-like phenotype described below (see Discussion). Thus, next experiments employed a cocktail of 6 factors comprising PRDM1, PRDM14, LIN28, DAZL, VASA and SYCP3 (i6F) (Fig. 1A).

Figure 1 Characterization of induced fibroblasts (hFSKs). (A) Schematic diagram of the experimental setup of the study. (B) Principal Component Analyses and Venn diagrams of up- and down-regulated genes when compared MOCK, i12F and i6F- induced hFSKs all together (n = 5). (C) RT-qPCR expression analysis of human PGC markers over i6F induced hFSK cells. (D) RT-qPCR expression analysis of the germ line markers GFRA1, PIWIL2, TNP2, PRM1 and ACR over i6F induced hFSK cells at 7 (D7), 14 (14D) and 21 (21D) days post-transduction (n = 8). Human testis cDNA physiological expression fold change relative to MOCK samples is also shown as a control. (E) Illustrative pictures of immunofluorescencent stainings for VIM, PLZF, UTF1, VASA, DAZL and HIWI over MOCK and i6F clumps from hFSK cells. Data is presented as normalized fold change mean +/− SEM. (*) represent significant differences (p < 0.05) with MOCK controls; (+) represents significant differences (p < 0.05) between i12F/i6F conditions and their respective clumps; (∧) represent significant differences (p < 0.05) with day 7 expression within sample groups; (∨) represent significant differences (p < 0.05) with day 14 expression within sample groups. Scale bar represents a distance of 50 μm. Full size image

Principal components analysis (PCA) of gene expression profile 14 days after transduction clustered i12F/i6F and i12F clumps/i6F clumps in two defined groups different that MOCK controls (Fig. 1B and Supplementary Figure S3D). Moreover, i6F cells showed a switch in their genetic expression program, with the significant up-regulation of 293 genes and the down-regulation of 322 genes compared to MOCK controls. Manually isolated i6F clumps showed significant up-regulation of 442 genes (226 of them shared with i12F) and down-regulation of 402 genes (254 of them shared with i12F treatment) compared to MOCK controls. Further comparisons between i12F and i6F identified 140 significant up-regulated genes and 167 down-regulated genes shared between induced cells, compared to MOCK controls (Fig. 1B). Functional enrichment analysis of the list of differentially regulated genes in i6F indicated their implication in germ cell-related processes such as “Integrin cell surface interactions” (REACT_13552), “Cell cycle” (REACT_152), “DNA Replication” (REACT_383), “Telomere maintenance (REACT_7970), as well as several Gene Ontology biological processes related to “Positive regulation of MAP kinase activity” (GO:0043406), “ovarian follicle development” (GO:0001541), “positive regulation of tyrosine phosphorylation of STAT protein” (GO:0042531), “Retinoid acid metabolic process” (GO:0042573) and “transforming growth factor beta receptor signaling pathway” (GO:0007179), among others. Interestingly, we observed the significant down-regulation of several genes related to the mitotic cell cycle regulation and the significant up-regulation of genes related to the TGFβ and LIF/STAT3 pathways. On the other hand, clumps showed a significant down-regulation of several genes related to chromatin stability and several somatic lineage determinant factors (Supplementary Table S1).

In a similar way that happened with i12F, i6F induced cell clumps showed significant up-regulation for the human PGC markers CDH1 and STELLA (Fig. 1C), but also for the recently described human PGC markers T (Brachyury), SOX17 and the naïve pluripotency marker TFCP2L1, indicating the presence of PGC-like cells within clumps (Fig. 1C). However, further RT-qPCR time course analysis showed a significantly increased expression of the pre-meiotic spermatogonial marker GDNF receptor type 1α (GFRA1) during the first week post-transduction that remained over the second week and declined at the third week post-transduction. Concomitant with this, we observed a trend of the data to show up-regulation of the meiotic (PIWIL2) and post-meiotic markers Transition Protein 2 (TNP2), Protamin 1 (PRM1) and Acrosin (ACR), that resulted statistically significant from the second week post-transduction onwards in the case of TNP2 and from the third week post-transduction in the case of PRM1 and ACR (Fig. 1D). More significant differences with MOCK controls were found along time in manually isolated clumps for PIWIL2, TNP2, PRM1 and ACR, but not for GFRA1, suggesting that a more differentiated germ cell-like population might have arised and co-exist with pre-meiotic germ cell-like cells within the clumps. However, only PIWIL2 and TNP2 showed significant differences 14 days post-transduction in clumps compared with the whole i6F culture (Fig. 1D). Similar results were observed in i12F induced cultures, highlighting the similarities between 12F and i6F phenotypes (Supplementary Figure S3C). Immunocytological analysis demonstrated the expression of the germ line-related markers VASA and DAZL (both exogenously induced), HIWI, PLZF and UTF1 in all the analyzed clumps, whereas the mesenchymal filament VIMENTIN (VIM) was found to be expressed only in fibroblastic cells surrounding the clumps but not within them (Fig. 1E).

In order to test if this phenotype may also appear by using another cell source, we also transduced human mesenchymal cells derived from bone marrow (hMSCs, 46, XY) with the i12F and i6F cocktails. In contrast with our observations in hFSK induced cells, where most of the cells kept their fibroblastic morphology and some clumps raised, in hMSCs most of the cells detached from the culture plates during the first week after the lentiviral transduction and those that remained attached switched their morphology to form clumps (Supplementary Figure S5A). Interestingly, hMSCs responded with the maximal expression of post-meiotic markers during the first week post-transduction, whereas in hFSK cells it occurred at the third week, indicating a different timing in the reprogramming process between both cell types (Supplementary Figure S5D).

Induced germ cell-like cells progress through meiosis in vitro

The expression of post-meiotic markers in induced cells prompted us to analyze their meiotic status. We assessed the localization and distribution of the axial element of the synaptonemal complex protein 3 (SYCP3) to determine the prophase I meiotic status on day 14 post-transduction. Although slightly different from the nuclear meiotic patterns previously reported in mammalian spermatocytes26, SYCP3 positive nuclei were considered meiotic when showed a punctate staining pattern corresponding to leptotene, or an elongated pattern corresponding to late zygotene and forward stages of the prophase I (Fig. 2A), as previously reported7,8,9,11,27.

Figure 2 Meiotic progression analysis 14 days post-transduction of fibroblasts. (A) Illustrative pictures of the SYCP3 staining pattern over i6F transduced hFSK cells. (B) SYCP3 and SYCP1 co-localization over transduced cells indicates effective chromosomal synapsis. (C) SYCP3 and ɣH2A.X co-localization over transduced cells indicates putative DSB loci. (D) Representative FISH results for probes against chromosomes 18 (aqua), X (green) and Y (red) over 1N sorted cells. (E) Molecular assessment of the ploidy in single cells. PCR products of the Amelogenin gene results in a peak of 118pb for the copy in X and a 124pb peak for the copy in Y. (F) Illustrative aCGH results of a diploid (46, XY) cell from MOCK and a haploid (23, Y) cell from i6F, co-hybridized with male (upper panels) and female (lower panels) diploid references. (G) Combined SYCP3 staining and FISH analysis reveals that meiotic-like cells recapitulate all the stages of the meiosis. The upper section shows the expected pattern of centromeric probes for the chromosomes 18, X and Y over meiotic cells. The bottom section shows representative pictures of the combined SYCP3 stainning (dark red) and FISH analysis corresponding to each of the meiotic sub-stages. In diploid cells, the expected FISH pattern in (46, XY) cells is 2 aqua signals: 1 green signal: 1 red signal (2:1:1) and any SYCP3 staining. During leptotene, cells show a FISH pattern 2:1:1, with a punctate SYCP3 staining. Since zygotene is a transitional sub-stage until homologue chromosomes are totally paired, both 1:1:1 and 2:1:1 FISH patterns are possible, co-localizing with an elongated SYCP3 staining. In pachytene, totally paired homologue chromosomes show a FISH pattern 1:1:1 with overlap of X and Y signals in the bivalent structure and an elongated SYCP3 staining. After the first reductional meiotic division, the synaptonemal complex is undetectable and nuclei show a 1:1:1 FISH pattern with overlapped X and Y signals. Finally, after the second equational meiotic division, haploid cells can either show a 1:0:1 or a 1:1:0 FISH pattern. Data is presented as mean +/− SEM. (**) represent significant differences (p < 0.01) with MOCK controls. Scale bar represents a distance of 10 μm. Full size image

Although several nuclei resulted positive for SYCP3 staining due to the exogenous expression of SYCP3, most of them showed an aberrant pattern resembling protein-like aggregations (Supplementary Figure S4C). Thus, only 1.88% of hFSK i6F cells showed a proper punctuate/elongated SYCP3 staining pattern corresponding to the prophase I of the meiosis, indicating a low efficiency of spontaneous meiotic entry of induced cells. Approximately 30.85% of meiotic-like nuclei (0.65% of the total population) had an elongated SYCP3 pattern, whereas 69.15% (1.23% of the total population) showed a punctate pattern (Supplementary Figure S4B). Similar results were obtained when cells were induced with the complete i12F cocktail (Supplementary Figure S4A,B). When hMSCs were subjected to the same genetic induction with both i12F and i6F cocktails, approximately 2.02% showed a correct positive SYCP3 staining pattern. The percentage of meiotic-like cells with elongated SYCP3 pattern in the i6F induced hMSCs was 56.93% (1.15% of the total population), whereas 43.07% (0.87% of the total population) showed a punctate staining pattern (Supplementary Figure S5C).

Chromosomal synapsis was detected by co-localization of SYCP1 and SYCP3 (Fig. 2B). Additionally, ɣH2A.X co-localization with SYCP3 indicated the appearance of SPO11-independent double strand breaks (DSBs) (Fig. 2C). However, even that we observed chromosomal synapsis between homologs and putative DSBs, we did not detect recombination events by MLH1 staining (data not shown).

In order to confirm if meiotic-like cells were able to fully complete meiosis, we next stained cells with propidium iodide (PI) and isolated the putative haploid (1N) population by fluorescence activated cell sorting (FACS) (Supplementary Figure S4D) as previously reported7,8,9. We were able to isolate 1–2% putative haploid cells from i6F induced cultures, a figure which correlates with the percentage of meiotic-like cells found by SYCP3 staining (Supplementary Figure S4B). When fluorescence in situ hybridization (FISH) with centromeric probes for the chromosomes 18, X and Y was performed (Fig. 2D), we found that 12.15% of hFSK cells within the putative 1N i6F sorted populations resulted haploid, compared to 1.07% of haploid cells within the unsorted populations (Supplementary Figure S4F). Since i6F clumps showed a significant enrichment of the expression of post-meiotic markers, we also performed FISH analysis over them after their manual isolation, resulting in an enrichment of 5.86% of haploid cells (Supplementary Figure S4F). In the case of hMSCs, we found that 4.11% of the i6F induced hMSCs resulted haploid, whereas we observed an enrichment of 20% haploid cells within the putative 1N sorted cell population (Supplementary Figure S5C). Complete FISH-based counts can be found in Supplementary Tables S2 and S3.

Haploidy of cells was validated by re-hybridization of the same nuclei with centromeric probes for the chromosomes 10, 12 and 3 (Supplementary Figure S4E). These results were also supported by an alternative molecular approach based in the detection of the AMELOGENIN gene, which has been widely used in sex determination of unknown human samples by PCR28. Here, we employed the 6bp length difference between both X and Y chromosome gene copies of AMELOGENIN to determine the presence/absence of sexual chromosomes in induced single cells individually isolated and used it to confirm their ploidy (Fig. 2E). Further array Comparative Genomic Hybridization (aCGH) analysis with single cells from clumps confirmed the haploidy of the cells previously analyzed by AMELOGENIN PCR (Fig. 2F).

Finally, combination of both meiotic analysis by SYCP3 staining and ploidy analysis by FISH over the same nuclei led us to identify all the intermediate meiotic stages of the prophase I from pre-leptotene to pachytene (Fig. 2G).

i6F induction leads to the activation of the TET-mediated methylation erasure pathway

We observed an up-regulation of the Ten-eleven translocation methylcytosine dioxygenases TET1, TET2 and TET3 in i6F induced fibroblasts that was significantly higher in the clumps compared to the rest of the culture (Fig. 3A), suggesting the activation of this de-methylation pathway. This finding was supported by the detection of the marker 5-hydroxi-methyl-Cytosine (5hmC), which was previously described as a mark for the CpGs to be de-methylated by the TET pathway29. Interestingly, we also observed a relative enrichment of 5hmC within the clumps during the second week of culture after transduction (Fig. 3B). Slight differences in the DNA methyltransferases DNMT1, DNMT3A and DNMT3B in induced fibroblasts versus controls were observed, except a decrease in the expression of the de novo DNMT3B in i6F clumps (Fig. 3A).

Figure 3 Epigenetic characterization of the in vitro induced fibroblasts. (A) RT-qPCR expression analysis of the DNA methyl-transferases DNMT1, DNMT3A and DNMT3B and the TET-mediated de-methylases TET1, TET2 and TET3 over i6F induced fibroblasts 14 days post-transduction (n = 3). Human testis cDNA physiological expression fold change relative to MOCK samples is also shown as a control. Data is presented as normalized fold change mean +/− SEM. (*) represent significant differences (p < 0.05) with controls; (+) represents significant differences (p < 0.05) between i6F whole culture condition and i6F clumps. (B) Representative pictures of the co-localization of 5-methyl-Cytosine (5mC) and 5-hydroxi-methyl-Cytosine (5hmC) over MOCK cells and i6F clumps at 14 days post-transduction. Dashed lines indicate cell nuclei enriched for 5hmC (red) in MOCK control (0/15 nuclei in the picture) and in two illustrative pictures of i6F clumps (7/17 nuclei (41.1%) and 5/18 nuclei (27.7%), respectively) compared with the 5mC signal (green). Scale bar represents a distance of 10 μm. (C) Circular heat map presentation of the methylation for 37 annotated human imprinted loci in induced fibroblasts 14 days post-transduction (n = 3). (D) Bisulphite sequencing results at 14 days post-transduction at the DMR of the H19, SNRPN, PEG3 and KvDMR1 loci in putative 1N sorted i6F cells (n = 3). Diagrams represent methylation status of each CpG dinucleotide on individual DNA clones. Lines represent different clones and columns are different CpG dinucleotides. Methylated CpGs are represented as filled circles and unmethylated CpGs are represented as open circles. Empty CpG sites represent the CpGs that could not be determined. In the graph, blue line indicates the methylation change in the analyzed maternally imprinted loci, whereas pink line indicates the methylation change observed for the paternally imprinted loci H19. Full size image

Methylation arrays indicated very slight differences between MOCK controls and induced cells (Fig. 3C). Only haploid enriched 1N i6F sorted cells showed a specific decrease of methylation in the paternally imprinted H19 DMR, whereas the maternally imprinted DMRs in SNRPN, PEG3 and KvDMR1 loci increased their methylation (Fig. 3D), suggesting that this population of cells enriched for putative haploid cells may have acquired a female gamete-like epigenetic profile.

Germ cell xenotransplantation assay demonstrates that i6F induced cells are able to colonize seminiferous tubules

To test the functionality of i6F induced hFSK cells, we employed busulfan treated NUDE male mice as recipients to test the ability of MOCK and i6F transduced cells to colonize their germ cell-depleted seminiferous epithelium as previously described30,31.

Small green fluorescence protein (GFP) dots appeared in some seminiferous tubules of i6F-transplanted testis, suggesting the presence of colonies of induced cells within them (Supplementary Figure S6A). Additionally and even that we observed focal spontaneous recovery of mouse spermatogenesis in both transplanted and not transplanted busulfan treated mice, we observed that i6F transplanted testis showed a slight increase in the normalized testis weight when compared to other conditions (Supplementary Figure S6B).

Immunostainings for co-localization of VASA and human NuMA32 were performed in frozen sections to detect human germ cells within the mouse testes as previously described27,33,34 (Fig. 4A and Supplementary Figure S6C). In 5 out of 8 transplanted testis, we detected double positive VASA+/NuMA+ cells. On average, 3.5% of tubule cross-sections showed colonizing donor cells located in the basal layer of both germ cell-depleted tubules and in tubules showing spontaneous recovery of the mouse spermatogenesis, with an efficiency of 0.76 colonizing cells per 105 injected cells (Fig. 4B–D). Also, we observed that the engrafted cells stained for DAZL (Supplementary Figure S6D), UTF1 (Supplementary Figure S6E), 5mC (Fig. 4E) and 5hmC (Fig. 4F).