3 Results
3.3 Co-culture of mouse ESCs with human testicular feeder cells… 100
3.3.3 Characterization of putative mSSCs derived from co-culture with
3.3.3.2 Expression patterns of SSC- and PGC marker genes in putative
The characterization of co-culture-derived putative mSSCs was performed by staining with alkaline phosphatase (AP) as well as immunocytochemical and qRT-PCR analyses of phenotypic mSSC markers, which are generally used for the identification of SSCs.
AP-staining as well as immunocytochemical analysis were performed with putative mSSCs co-cultured with HTF cells. The putative mSSCs were positively stained for AP, whereas the HTF cells were negative for AP-staining (Fig. 30 B). Additionally the AP-staining revealed the high number of mSSC-like cells generated in co-culture (Fig. 30 C).
Fig. 30: Alkaline Phosphatase staining of co-culture-derived putative mSSCs. (A) Brightfield microscopy image of putative mSSCs derived from HTF-2-Co before AP-staining showed the typical colonies of round cells with grape-like structures. (B) In contrast to HTF-2 cells (arrow heads), the putative mSSCs were positively stained for AP perceptible from the dark brown staining of the cells (arrows). (C) Overview of culture plate after AP-staining demonstrated the high number of putative mSSCs generated in co-culture. AP: Alkaline Phosphatase. Scale bars: A: 10 µm; B: 20 µm; C: 100 µm.
Within cell clusters both immunopositive and immunonegative cells could be observed for all tested SSC markers OCT4, SALL4 (Sal-Like Protein 4), PLZF and α6-Integrin (Fig. 31).
OCT4 showed a heterogeneous expression pattern within cell clusters, some cells were strongly immunopositive, whereas some remaining cells depicted a weaker staining for OCT4 (Fig. 31 C+F). In comparison to the OCT4 expression the SALL4-, PLZF- and α6-Integrin stainings showed a weaker signal (Fig. 31 I, L, O).
Fig. 31: Immunocytochemical stainings of putative mSSCs derived from co-culture with HTF-2 cells.
Immunocytochemical stainings were performed with HTF-2-Co cells using specific antibodies for SSC markers (A-F) OCT4, (G-I) SALL4, (J-L) PLZF and (M-O) α6-Integrin. Representative overlay images are shown in the last column (C, F, I, L and O). Incubation with IgG instead of first antibody was used as negative control (P-R).
Representative scale bars are indicated in the overlay images. Scale bars: C, F, I, L, O: 10 µm; R: 20 µm.
A similar staining pattern was observed in co-stainings using antibodies against OCT4 and SALL4 as well as OCT4 and DDX4 (). Within cell clusters OCT4 and SALL4 or rather OCT4 and DDX4 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 4; aliases: VASA) were co-localized in some cells (Fig. 32, 4th column), while remaining cells were stained positive for either one or none of the markers (Fig. 32; 2nd and 3rd columns). Mostly the staining intensity of markers differed in cells showing co-localization. No staining was detected in negative controls using IgG instead of first antibody (Fig. 32 M-P).
Fig. 32: Co-Immunocytochemical stainings of putative mSSCs derived from co-culture with HTF-2.
Co-Immunocytochemical stainings were performed with HTF-2-Co using specific antibodies for SSC markers (A-H) OCT4 / SALL4 and (I-L) OCT4 and DDX4. Representative overlay images are shown in the last column (D, H and L). Incubation with IgG instead of first antibody was used as negative control (M-P). Red and green arrows indicate cells, which are positive for the respective marker, whereas white arrows indicate those cells that stained positive for both markers. Representative scale bars are indicated in the overlay images. Scale bars: D, H, L:
10 µm; P: 20 µm.
Therefore, the evaluation of the expression pattern of mSSC phenotypic markers by immunocytochemistry indicated that the putative mSSC clusters do not represent a homogeneous mSSC population. An enrichment of those cells in culture could be achieved by MACSorting of the putative mSSCs derived from the co-cultures HTF-1-Co, HTF-2-Co and HTF-3-Co using the mSSC surface marker antibody α6-Integrin, followed by an immunostaining with α6-Integrin in order to quantify the α6-Integrin expression in putative mSSCs after MACSorting. After examination of a total of 500 cells for each cell line regarding their α6-Integrin expression (Fig. 33 II), the putative mSSCs derived from the co-cultures HTF-1-Co, HTF-2-Co and HTF-3-Co revealed 64-70% α6-Integrin positive cells after MACSorting (Fig. 33 I). This comprised a two times higher percentage of α6-Integrin positive compared to the starting cell line SSC 12 of co-culture (33%) and even a five times higher percentage than the original ES cell line ES-RI (13%).
Fig. 33: Enrichment of α6-Integrin positive cells by MACSorting of co-culture-derived putative mSSCs.
After MACSorting and immunostaining with α6-Integrin a total of 500 cells for HTF-1-Co, HTF-2-Co and HTF-3-Co were examined regarding their α6-Integrin expression, respectively. (I) Counting of positive cells revealed a two times- and five times increase of α6-Integrin positive cells in the co-culture-derived mSSCs compared to SSC 12 and ES-RI, respectively. (II) Representative images of α6-Integrin immunostaining with SSC 12, ES-RI, HTF-1-Co, HTF-2-Co and HTF-3-Co used for evaluation of α6-Integrin positive cells. Overlay images are shown in C, F, I, L and O. Representative overlay images of incubations with IgG instead of first antibody used as negative controls are shown in inlays in C, F and I. Representative scale bars are indicated in the overlay images.
Scale bars: 20 µm.
In addition, qRT-PCR experiments were performed to analyze the expression of a subset of mPGC- and mSSC markers in putative mSSCs derived from co-culture (Fig. 34). These analyses were carried out with putative mSSCs co-cultured with HTF cells as well as with freshly α6-Integrin MACSorted putative mSSCs derived from co-cultures HTF-1-Co, HTF-2-Co and HTF-3-Co in comparison to ES-RI cells as well as the starting cell line 2011). For this reason the expression of these genes indicates the commitment of cells to the germ line lineage and provides the opportunity to discriminate between PGCs and ESCs in vivo and in vitro. Independent of preparation of co-culture samples, the results of PGC marker gene expression demonstrated a significantly increased expression level of Fkbp6 and Mov10l1 in cells derived from co-cultures in comparison to the ESC line ES-RI as well as to SSC 12 cells (Fig. 34 A). The calculated significances normalized to ES-RI are indicated within Fig. 34 A. The expression analysis of Gpr125 (G-Protein-coupled Receptor 125), which is a well established germ cell marker and expressed in undifferentiated spermatogonia within the testis (Seandel at al., 2007), revealed a moderate expression compared to ES-RI (Fig. 34 B).
Fig. 34: qRT-PCR expression analyses of PGC marker genes Fkbp6 and Mov10l1 and the SSC marker gene Gpr125 in co-cultured cell lines. Mean values with standard deviations for (A) PGC marker genes Fkbp6 and Mov10l1 and (B) SSC marker gene Gpr125 are shown. Data comprised at least two biological replicates before and after MACSorting of putative mSSCs derived from HTF-1-Co, HTF-2-Co and HTF-3-Co. Significant increase of marker gene expression in co-culture samples was calculated in comparison to ES-RI and indicated with *: p ≤ 0.05, **: p ≤ 0.01; ***: p ≤ 0.001. SSC 12: starting cell line of co-culture. mTestis: positive control;
mBrain, mSpleen: negative controls.