Isolation of hMGSCs from Human Testis Biopsies
In initial attempts to isolate hMGSCs, we obtained testis biopsies and generated cell suspensions by enzymatic digestion. We then sought to enrich for the spermatogonial stem cell population by MACS with the cell surface marker GFR-α (the receptor for GDNF). GFR-α had previously been reported to localize to a subset of type A spermatogonia in mice [36
]. Isolated cells were cultured on gelatin-coated dishes in MEM-α. However, although the resulting cells were capable of being propagated in vitro, they had an elongated spindle-shaped appearance (similar to fibroblasts), distinctly different from that of hESCs, and lacked characteristic expression of cell surface markers of pluripotent cells.
Thus, we explored alternative methods to induce the propagation of hESC-like cells from testis biopsies: (a) culture of testicular cells in hESC medium post-biopsy digestion; (b) culture of testicular cells in hESC medium for 8 days postdigestion, with subsequent transfer onto MEFs; and (c) transfer directly onto MEFs in hESC media. We noted that all three of these approaches, in contrast to MACS separation, resulted in the formation of colonies. However, these colonies could not be successfully propagated in vitro; with passaging via trypsin digestion, the cultures would progressively become devoid of stem cell-like cell colonies. Thus, in 17 of 17 biopsies subjected to these protocols, no hMGSC line was derived. In contrast, as described below, by manual passaging we succeeded in the derivation of two hMGSC lines (although one patient withdrew from the study, and materials were discarded in that case).
As an alternative, manual passaging of colonies was explored. Following enzymatic dissociation of the testis biopsy, after approximately 7–10 days of culture, very small colonies started to grow on top of the monolayer of testicular cells; these colonies were manually transferred onto MEFs and cultured under hESC conditions (, ). These cells, which we have termed hMGSCs, have been propagated for approximately 20 passages in vitro; the current line is designated NK7. The putative NK7 hMGSCs were passaged once every week and have maintained the ability to form colonies with characteristic hESC morphology. However, although the cells in the middle of the colonies have a distinctive hESC-like appearance, some of the cells at the periphery appear to differentiate and acquire a spindle-shaped morphology, suggesting the need to optimize medium and/or culture and derivation conditions (). In suspension, NK7 cells continued to divide and formed EB-like structures ().
Figure 1 Morphological analysis and analysis for the expression of both pluripotency- and germ cell-specific markers. (A): Light microscopy of spermatogonial stem cell colonies growing on top of a monolayer of testicular cells approximately 2 weeks after plating (more ...)
Gene Expression Analysis
RT-PCR was performed to analyze the expression of a subset of pluripotency markers, as well as germ cell-specific genes, in the isolated hMGSCs at passages 2 and 7 relative to a normal human testis sample (). Results demonstrated that the hMGSCs at passages 2 and 7, grown on MEFs, express a subset of those genes expressed in the testis, as shown (compare a, b with d), which includes the pluripotency markers OCT4 (octamer-binding transcription factor-4) and SOX2 (SRY-box 2). NANOG expression, however, could not be detected in either the isolated hMGSCs or the testis sample. Apart from that, expression of the hESC- and germ cell-enriched genes STELLAR (STELLA-related), GDF3 (growth and differentiation factor 3), PUM1 (PUMILIO 1), and PUM2 (PUMILIO 2) was observed. In addition, the hMGSCs expressed the germ cell-specific gene DAZL (Deleted in AZoospermia-Like), as well as SCP3 (Syntaptonemal Complex Protein 3) and MLH1 (Mut-L Homolog 1). In contrast, expression of the markers VASA and SCP1 was not detected, nor was the expression of the two developmentally late germ cell markers BOULE and TEKT1 (). Notably, however, when we cultured NK7 cells on human testicular stromal cells, we observed the induction of expression of later germ cell markers, including BOULE and TEKT1, and loss of SOX2 expression (c). We therefore concluded that NK7 cells lose the expression of later germ cell markers if cultured under human ESC conditions and regain the expression of pluripotency genes, such as SOX2, if cultured on MEFs in human ESC conditions.
Our next aim was to examine the expression of pluripotency markers in hMGSCs by immunofluorescence (). Putative hMGSCs were shown to express the human pluripotency markers SSEA4 (), TRA1–81 (keratin sulfate-related antigens; ), OCT4 (), and SOX2 (). In addition, the hMGSCs also stained positive for the early germ cell and hESC marker TNAP (), as well as the germ cell lineage marker DAZL (). Negative controls for all experiments demonstrated that antibodies were specific, as expected.
To determine the karyotype of the derived NK7 hMGSC line, SKY analysis was performed. Results demonstrated that the NK7 hMGSC line has a normal karyotype (46, XY) and no Y chromosome microdeletions. No indications of other cytogenetic abnormalities were detected (). This indicated that the derived cell line was karyotypically identical to the patient's somatic cells, at this level of analysis.
Figure 2 Human multipotent germline stem cells (hMGSCs) have a normal karyotype and express telomerase. (A): The DNA spectral karyotyping experiment of undifferentiated hMGSCs at passage 8 demonstrates a normal (46,XY) karyotype. The spectral image, the 4,6-diamidino-2-phenylindole (more ...)
Telomerase Activity and Methylation of the H19 Differentially Methylated Region and the OCT4 Promoter Region
Telomerase activity is indicative of pluripotent stem cells. We examined telomerase activity of the hMGSCs at passages 6 and 8 relative to the human XY-bearing ESC line HSF8, as a positive control. As expected, hESCs exhibited a very high telomerase activity with little or no residual activity in the heat-inactivated control. Telomerase activity was also detected in the two hMGSC extracts, with the level of telomerase activity slightly reduced in cells that had been cultured for eight passages relative to those cultured for six passages ().
Short Tandem Repeat/Variable Number of Tandem Repeat Analysis
Short tandem repeat (STR)/variable number of tandem repeat (VNTR) analysis was performed to determine the origin of the NK7 hMGSCs. Samples analyzed were genomic DNA isolated from NK7 hMGSCs, genomic DNA from the tissue donor's blood sample, and genomic DNA from H9 hESCs. The results () demonstrate that the number of short tandem repeats on both alleles of the 15 loci that were analyzed is identical in NK7 hMGSCs and the tissue donor's blood sample. The probability that two randomly selected individuals would have an identical genotype at these 15 loci is minuscule (5.01 × 10−18
]). Although H9 cells have the same number of short tandem repeats as the NK7 hMGSCs on both alleles of the HUMTHO1
locus and on one allele of the D16S539
, and D5S818
loci, the number of short tandem repeats at all other examined loci differed between H9 hESCs and the NK7 hMGSC line.160:
Variable number of tandem repeat/STR analysis of 15 STR loci
Bisulfite sequencing was performed to investigate the methylation status of 18 CpG (cytosine guanine) dinucleotides in the differentially methylated region upstream of the H19
promoter (). Although the maternal H19
allele is active and therefore unmethylated, the paternal H19
allele is methylated in all somatic cells [38
]. Human ESCs, as well as human somatic cells, carry one paternal and one maternal allele and showed a ratio of 70%:30% and 50%:50% unmethylated to methylated sequences, respectively, as shown (). In contrast, in mature sperm, the paternal allele of the H19
gene was completely methylated (100% of clones), indicative of the establishment of the unique male-specific methylation pattern at this locus during this stage of development, (). In contrast, when we examined the methylation status of H19
in NK7 hMGSCs at passage 8, we observed that this locus was hypomethylated, with 87% of clones unmethylated and only 13% methylated (, ).
In addition, the DNA methylation profile of the 5′-flanking region of the human OCT4 gene was analyzed. The region investigated contains the PE, the DE, and the PP, as indicated in Figure . In undifferentiated cells the majority of CpG repeats in this region are unmethylated and the gene is therefore expressed. Analysis showed that 94% of CpG sites in the OCT4 promoter region of human ESCs are unmethylated, whereas only 19% of CpG repeats in blood cells and 2% of CpG repeats in sperm cells were unmethylated. Analysis of the methylation status of the OCT4 promoter region of NK7 cells at passages 2 and 8 showed that 36% and 32% of CpG repeats were unmethylated, respectively (, ). This partial demethylation is in accordance with the finding that the OCT4 gene is activated in NK7 cells, as demonstrated by RT-PCR and immunofluorescence staining.
Pluripotent stem cells can self-renew or differentiate to the three primary germ layers: endoderm, mesoderm, and ectoderm. To assess whether hMGSCs are able to spontaneously differentiate into derivatives of the three germ layers in vitro, expression of ectoderm-, endoderm-, and mesoderm-specific genes and proteins was analyzed at different time points during differentiation. H9 hESCs were used as a positive control (). As shown, expression of the pluripotency marker OCT4 decreased with differentiation, with a concomitant increase in the expression of the somatic markers MSI1 (ectoderm marker), GATA4 (endoderm marker), and KDR (mesoderm marker) in both hMGSCs and hESCs. Notably, we also found that although NCAM is commonly used as an ectoderm marker in hESC research and would thus be expected to increase with differentiation, its expression decreased with differentiation of hMGSCs. This is contrast to hESCs, which exhibited an increase in the expression levels of NCAM ().
Figure 4 In vitro differentiation of human multipotent germline stem cells (hMGSCs). hMGSCs and H9 human embryonic stem cells were spontaneously differentiated in vitro over a period of 21 days. RNA was isolated at six time points, and TaqMan reverse transcriptase-polymerase (more ...)
Once we examined the expression of ectoderm-, endoderm-, and mesoderm-specific genes at the mRNA level, our next aim was to evaluate germ layer marker expression at the protein level by immunofluorescence. After 7 days of in vitro differentiation, differentiated hMGSCs were positive for the endoderm-specific VWF (, ); ASMA, which specifically recognizes α-smooth muscle actin (, ; mesoderm); and NES, an intermediate filament that is expressed in early embryonic neuroepithelial stem cells (, ; ectoderm).
Figure 5 Immunofluorescence staining of day 7 differentiated human multipotent germline stem cells and teratoma analysis. Attached EBs were assessed for protein expression as shown in (A-F). (A): von Willebrand factor (VWF). (B): VWF with 4,6-diamidino-2-phenylindole (more ...)
Finally, we tested the ability of hMGSCs to form teratomas under the kidney capsule of a female immunodeficient (SCID) mouse to investigate their differentiation capacity in vivo. The grafts were recovered 2 months post-transplantation and weighed 1.2 and 0.5 mg; histological evaluation showed that extensive teratoma formation was not detected (). However, as discussed further below, human cells were present in the graft after 2 months, as demonstrated by molecular analysis (). PCR analysis of the human SRY gene product indicated that the two positive control samples, NK7 genomic DNA and sperm genomic DNA, contained a specific 350-bp band, as did the NK7 hMGSC graft DNA. In contrast, no specific band was amplified using female mouse genomic DNA as a template. To investigate whether teratoma formation is supported by an increased cell number and/or support cells, grafts were prepared using approximately 10,000 hMGSCs accompanied by 1 million irradiated MEFs as carrier cells. Again, the grafts were recovered 2 months post-transplantation; histological analysis again revealed a variety of cell types present but no wide-scale expansion to large teratomas as is frequently seen with hESCs.
Immunofluorescence Staining Following Induced Neural Differentiation
NK7 hMGSCs formed colonies when they were cultured on MEFs (), whereas they grew as a monolayer when they were cultured on gelatin (). Prior to differentiation, the hMGSCs were plated onto gelatin and were cultured until 80% confluence was achieved. Subsequently, with 6 weeks of induced differentiation to the neural cell lineage, immunofluorescence staining of neural makers was performed on the NK7 line. Cells positive for NES could be detected after the induced differentiation () but not in the untreated cell population (). In addition, cells stained positive for MAP2 () and TUB III (), demonstrating that NK7 hMGSCs have the potential to differentiate toward the ectodermal (neural) lineage.
Figure 6 Immunofluorescence staining after 6 weeks of induced neural differentiation. Human multipotent germline stem cells were cultured on mouse embryonic fibroblasts in human embryonic stem cell media (A) and were plated onto gelatin (B) prior to the differentiation (more ...)