Transplantation of putative mSSCs in germ cell depleted mice

For functional analysis of cells, co-culture-derived putative mSSCs were transplanted into the seminiferous tubules of germ cell depleted mice (2.2.4.8). While the recipient’s endogenous spermatogenesis was destroyed by busulfan treatment, subsequent transplantation of co-culture-derived putative mSSCs may lead to colonization and repopulation of SSCs within the tubules as well as induction and regeneration of spermatogenesis. In general, four to six weeks after busulfan treatment mice were mated with females for at least four weeks to ensure the infertility of males before their use for transplantations. In the transplantation assay, freshly α6-Integrin MACSorted putative mSSCs were either directly transplanted into one testis of the host or cultured for 24 hours in StemPro+3 medium on MEF, which was removed before transplantation procedure. 30-40 µl of the cell suspension (~1 x 10⁸ cells/ml) were injected. The remaining testis was not transplanted and therefore used as an internal negative control. Tab. 8 summarizes the performed cell transplantations in germ cell

depleted CD-1 and 129SV mice and indicates the analyses, which were performed in order to check for colonization of transplanted cells as well as for repopulation of the seminiferous tubules.

Tab. 8: Overview of performed cell transplantations in germ cell depleted mice.

Analysis after transplantation

number of transplanted mice

CD-1 129SV

A Test mating with wild type females for at least 4 weeks

following: H&E Staining 6 3

B Genotyping-PCR for Stra8-EGFP construct at certain time points

after transplantation 5 10

C Histochemical analysis at certain time points after transplantation 2 3

The first transplantations of putative mSSCs were carried out with germ cell depleted mice of CD-1 genetic background (Tab. 8 A). Because a complete spermatogenesis and its regeneration after depletion take about 35 days, mice were mated with females approximately four weeks after transplantation. Since after several weeks of mating no offspring was born as well as no teratoma formation was observed, transplanted mice were sacrificed and their testes histologically analyzed by H&E staining. The H&E staining of testis sections revealed no seminiferous tubules with regenerated spermatogenesis (Fig. 37).

Fig. 37: H&E staining of testis sections after transplantation of co-culture-derived putative mSSCs in germ cell depleted CD-1 mice. After transplantation of putative mSSCs derived from (A-F) HTF-1-Co and (G-J) HTF-2-Co in germ cell depleted CD-1 mice, their testes were histologically analyzed by H&E staining, which revealed no regeneration of spermatogenesis in seminiferous tubules. Transplanted and non-transplanted testis as an internal negative control were analyzed. H&E staining with CD-1 wild type testis demonstrated normal spermatogenesis. #: mouse number. Representative scale bar in K: 50 µm.

Aiming to check whether putative mSSCs derived from co-culture were able to colonize within the seminiferous tubules after transplantation, germ cell depleted CD-1 mice were transplanted with cell suspension containing the prepared putative mSSCs and sacrificed at certain time points after transplantation. The presence of transplanted cells within the testis was checked by genotyping PCR on genomic DNA derived from transplanted testes (Tab.

8 B) as well as immunohistochemistry on testis cryosections (Tab. 8 C) for the transgene construct Stra8-EGFP included in the starting cell line SSC 12 of co-culture. While

problems to detect EGFP signals on testis cryosections, the presence of Stra8-EGFP-specific PCR products was observed 7 days after transplantation by genotyping PCR, whereas no Stra8-EGFP PCR bands were detected 14 days after transplantation (Fig. 38 A).

The non-transplanted testis was used as an internal negative control and TP2 (Transition Protein 2) -specific PCR was performed as loading control of DNA. Further transplantations were performed to narrow down the time span of settling and loss of transplanted cells within the seminiferous tubules. These transplantations demonstrated the presence of Stra8-EGFP-specific PCR products in transplanted testes 6 and 9 days after transplantation by genotyping PCR (Fig. 38 B). No Stra8-EGFP-specific PCR bands were detected 12 days after transplantation (Fig. 38 B). Consequently, transplanted mSSCs became lost between 10 and 12 days after transplantations in CD-1 mice.

Fig. 38: Proof of Stra8-EGFP-specific PCR products in testes of transplanted germ cell depleted CD-1 mice by genotyping PCR. Stra8-EGFP genotyping PCR results indicated the presence of Stra8-EGFP positive cells (A) 7 days as well as (B) 6 and 9 days after transplantation of co-culture-derived putative mSSCs in transplanted testes of germ cell depleted CD-1 mice. Putative mSSCs derived from (A) HTF-1-Co and (B) HTF-15-Co were used for transplantation. Fragment sizes of genotyping PCRs are indicated within the figure.

TP2: PCR for DNA loading control; d: day after transplantation; +: transplanted testis; -: non-transplanted testis;

PC: positive control with SSC 12 genomic DNA; NC: negative control with H2O.

Because an ES-RI cell line, established from a murine 129SV-background (Nagy et al., 1993), is the origin of the starting cell line SSC 12 of co-culture, further transplantations of putative mSSCs derived from co-culture were performed using germ cell depleted 129SV

testis. Genotyping PCR was performed to check the presence of Stra8-EGFP-specific PCR products in transplanted testes (Tab. 8 B). After transplantation into germ cell depleted 129SV mice, Stra8-EGFP PCR bands were detected 6, 9 and 12 days after transplantation by genotyping PCR (Fig. 39 A), which was confirmed in three biological replicates.

Furthermore the presence of Stra8-EGFP-specific PCR products could be confirmed 15 days after transplantation in 129SV mice (Fig. 39 B). The non-transplanted testis and TP2 PCR were used as an internal negative control and loading control of DNA, respectively.

Fig. 39: Proof of Stra8-EGFP-specific PCR products in testes of transplanted germ cell depleted 129SV mice by genotyping PCR. Stra8-EGFP genotyping PCR results indicated the presence of Stra8-EGFP positive cells (A) 6, 9 and 12 days as well as (B) 15 days after transplantation of HTF-1-Co-derived putative mSSCs in transplanted testes of germ cell depleted 129SV mice. Fragment sizes of genotyping PCRs are indicated within the figure. TP2: PCR for DNA loading control; d: day after transplantation; +: transplanted testis; -: non-transplanted testis; PC: positive control with SSC 12 genomic DNA; NC: negative control with H2O.

In order to check whether the presence of Stra8-EGFP-specific PCR products in transplanted testes revealed by genotyping PCR corresponded to a possible colonization of transplanted cells within the seminiferous tubules, putative mSSCs derived from co-culture were transplanted into germ cell depleted 129SV mice and analyzed at the given time points by immunohistochemical staining (Tab. 8 C). The staining pattern of SALL4, a pluripotency factor, which is expressed in undifferentiated spermatogonia in adult testes (Gassei and Orwig, 2013), should offer a valuable clue to a potential colonization of transplanted cells within the seminiferous tubules. The analyses of SALL4 staining in histological sections of testes prepared 9, 12 and 15 days after transplantation are still in progress.

In addition, germ cell depleted 129SV mice were mated with females approximately four

No offspring was born as well as no teratoma formation was observed after several weeks of breeding. Two mice are still in test matings, while one transplanted mouse was already sacrificed and its testes histological analyzed by H&E staining, which revealed no seminiferous tubules with regenerated spermatogenesis.

3.3.5 In vitro spermatogenesis experiments with putative mSSCs