Maximal Donor Germline Transmission from DAZL-Deficient Rats.

<p>(A) Southern blot analysis of progeny from Wildtype (WT) and DAZL-Deficient (Dazl) recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13 (i.e. 158 days in culture); left testes of each animal were not transplanted. At 75 days post-transplantation recipients (R) were paired with WT females (F) and allowed to produce pups by natural breeding (See R942-R949 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Shown are blots from representative litters probed for <i>EGFP</i> to distinguish progeny produced by donor cells. OMP = loading control. Genomic DNA Controls were from untreated <i>GCS-EGFP</i> and <i>DAZL</i>-Deficient transgenic rats. LTR = PCR primers specific for lentiviral transgene in <i>DAZL</i>-deficient rats. GAPDH = PCR loading control. (B) Bright field and green fluorescence images of testes from Wildtype (<i>Left</i>) and DAZL-deficient (<i>Right</i>) recipient rats at 212 days post-transplantation. Scale bar = 1 cm. (C) Graph of germline transmission rates for the donor, <i>GCS-EGFP</i> transgene from Wildtype and DAZL-deficient recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13; left testes were not transplanted. <i>DAZL</i>-deficient recipients transmitted the <i>GCS-EGFP</i> transgene to 100%+/−0% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 73 of 73 total F1 pups born from donor cells. Wildtype recipients transmitted the <i>GCS-EGFP</i> transgene to 14%+/−5.9% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 16 of 116 total F1 pups born from donor cells. (D) Genealogy tree showing stable transmission of donor haplotypes from <i>DAZL</i>-Deficient recipients (F0) R989 and R990 to F1 and F2 progeny. Recipients were each transplanted with 150,000 rat spermatogonia/testis from line RSGL-GCS9 at passage 17 (See R988-R990 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Spermatogonial line RSGL-GCS9 was derived from a rat homozygous for the <i>GCS-EGFP</i> transgene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone.0006308-Wu1" target="_blank">[36]</a>. Thus, F1 progeny represent half-siblings; some of which were crossed to re-derive transgenic F2 progeny homozygous for the <i>tgGCS-EGFP</i> allele.</p

Analysis of Spermatogenic Failure in DAZL-Deficient Rats.

<p>(A) Relative numbers of spermatogenic cell types/1000 Sertoli cells in testes of 4 month old wildtype and transgenic <i>DAZL</i>-deficient rats (+/−SEM, n = 3 rats/group). Undifferentiated type A spermatogonia (Undif), differentiating type A spermatogonia (Type A), intermediate to type B spermatogonia (Int-Type B), preleptotene spermatocytes (PL), leptotene-zygotene spermatocytes (lept-zygo), pachytene-diplotene spermatocytes (Pachy-Di), secondary spermatocytes-round spermatids (SS-RS), elongating spermatids (ES). Significant differences (i.e. p<0.05) in testis cell types scored in wildtype and DAZL-deficient rats were determined for SS-RS (p = 0.022) and ES (p<0.0001) using unpaired two-tailed students t-test. (B) Histological cross-sections of seminiferous tubules from 4 month old wildtype (<i>Top left</i>) and transgenic <i>DAZL</i>-deficient rats (<i>Top right</i>). <i>Bottom left and right</i> show higher magnification images of boxed regions in the <i>Top</i> panels. Scale bars = 100 µm. (C) Images of spermatogonial types in testis sections from wildtype rats at stages VIII, XI, XIII, XIV, II and V of spermatogenesis. Types of undifferentiated (Undif) and differentiating spermatogonia (A1, A2, A3, A4, Int, B) were determined by analysis of staining patterns in nuclei at specific stages of spermatogenesis. Scale bar = 30 µm. (D) Images of spermatogonial types in testis sections from <i>DAZL</i>-deficient rats. Because stages of the epithelial cycle could not be classified in these rats, types of undifferentiated (Undif) and differentiating spermatogonia (A1-like, A2-like, A3-like, A4-like, Int-like, B-like) were estimated based on staining profiles of spermatogonia in wildtype rats. Scale bar = 30 µm.</p

Long-Term Spermatogenic Potential of Donor Spermatogonia.

<p>(<i>Left</i>) Graph showing relative numbers of Round and Elongating Spermatids in seminiferous tubules of non-transplanted, non-busulfan-treated, Wildtype and <i>DAZL</i>-deficient rat lines at 4 months of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-g001" target="_blank">Fig. 1a</a>), in comparison to Spermatid numbers in busulfan-treated, <i>DAZL</i>-deficient recipient rats at 212 days (i.e. ∼8 months of age) after being transplanted with rat spermatogonial line, RSGL-GCS9, at passage 13 (i.e. culture day 158). Cell counts were normalized/1000 Sertoli cells. +/−SEM, n = 3 rats/group. (<i>Right</i>) Images of histological sections of seminiferous tubules from the <i>DAZL</i>-deficient recipient rats described in the “Left” panel after being transplanted with spermatogonia from RSGL-GCS9. <i>Bottom Right</i> shows a higher magnification image within the boxed region of the <i>Top Right</i> panel. Scale bars = 100 µm.</p

DAZL-Deficient Rats are Efficient Spermatogonial Recipients.

<p>(A) Relative abundance of EGFP in testes of Wildtype, <i>DAZL</i>-deficient and <i>GCS-EGFP</i> rats. <i>Left</i>: Data expressed as the equivalents of recombinant, histidine-tagged EGFP (rEGFP)/testis (+/−SEM, n = 3 testes/rat strain) as determined by fluorometry of testis extracts at 24 days of age. <i>Right</i>: Bright field (top) and green fluorescence (bottom) images of testes dissected from <i>GCS-EGFP</i>, wildtype (WT) and <i>DAZL</i>-deficient (Dazl-Def) rats at 24 days of age. Scale bar = 1 cm. (B) Spermatogenesis colony forming assays using <i>DAZL</i>-deficient rats as recipients. <i>Left</i>: Numbers of spermatogenic colonies formed/testis by donor <i>GCS-EGFP</i> rat spermatogonia in Wildtype (10.25+/−0.68 colonies/testis, +/−SEM, n = 8 testes) and <i>DAZL</i>-Deficient rats (30.69+/−0.62 colonies/testes, +/−SEM, n = 8 testes) at 30 days following transplantation; p<0.0001, unpaired two-tailed students t-test. Donor spermatogonia were transplanted at passages 15 and 17 (i.e. culture days 182 and 204) at 2000 <i>GCS-EGFP</i>⁺ cells/testis. <i>Right</i>: Images of individual colonies of spermatogenesis in Wildtype and <i>DAZL</i>-deficient recipient rats that were generated by the donor <i>GCS-EGFP</i> spermatogonia (green fluorescence is from donor cells). Images are representative of colonies scored and plotted in the <i>Left</i> panel. Scale bar = 100 µm.</p

Progeny from Wildtype and <i>DAZL</i>-Deficient Recipient Rats Transplanted with GCS-EGFP Rat Spermatogonia.

<p>Wildtype or DAZL-deficient (DAZL-def) recipient rats were transplanted with either 0.5 or 1.5×10⁵ EGFP⁺ cells/testis from rat spermatogonial line GCS9 at 12 days after busulfan treatment (i.e. 12 mg/kg i.p.) on postnatal day 24. At ∼75 days post-transplantation recipients were paired with 75–80 day old wildtype female rats. Spermatogonia line GCS9 was harvested from passages number 13 and 17, which corresponded to 158 and 204 days in culture, respectively, prior to their transplantation. Recipients R942-R949 were littermates born from a hemizygous, transgenic DAZL-deficient female and a wildtype Sprague Dawley male. No progeny were born from breeder pairs of un-transplanted, busulfan-treated DAZL-deficient males and wild-type females (n = 3 breeder pairs). Breeder pairs of untreated, wild-type male litter mates of DAZL-deficient rats and wild-type female rats from Harlan, Inc. produced 15.5±4.5 pups/litter (+/−SEM, n = 8 litters from 3 breeder pairs).</p>1<p>p = 0.0209 Average Group 1 versus Average Group 2; p = 0.0251 Average Group 2 versus Average Group 3.</p>2<p>p = 0.0031 Average Group 1 versus Average Group 2.</p>3<p>Percent <i>GCS-EGFP</i>⁺ F1 progeny.</p

Prospective Procedures for Curing Azoospermia with Stem Cells.

<p>In many cases of spermatogenic failure, healthy sources of spermatogonia could be obtained from either a biopsy of an azoospermic patient's own testis, or prospectively, from a biopsy of his own somatic cells following induction into a pluripotent state. Pure spermatogonial lines derived from either source would require that they are maintained in a spermatogenic lineage and prevented from acquiring a pluripotent state when in culture. If such protocols were established, naturally healthy spermatogonial lines could be selected for from a background of potentially unhealthy cell lines, such as from cancer patients, or azoospermic men diagnosed as germline mosaics (later example is shown). Once selected for, the healthy lines could then be induced to develop into sperm by transplanting them back into the patient's own testes. As potential alternatives to sterile-testis complementation that are currently being investigated, spermatogonial stem cells could be induced to develop through meiosis during culture <i>in vitro</i>, or within tissue grafts for production of haploid gametes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone.0006308-Honaramooz1" target="_blank">[50]</a>. The resulting haploid spermatids could then be used for assisted fertilization.</p

Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

<div><p>Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse “GS” (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated <i>in vitro</i>, contribute to the germline <i>in vivo</i> following testicular transplantation or become reprogrammed to pluripotency <i>in vitro</i>.</p></div

Retention of the spermatogonial phenotype following gene correction.

<p>Immunostaining was performed on gene-corrected GT59 (left) and GT65 cells (right): DAZL, a germ cell specific marker; GFRA1, POU5F1, ETV5, CDH1, and SOHLH1, markers of undifferentiated spermatogonia. Additionally, GT59 and GT65 cells were treated with the differentiation factor, retinoic acid (1 µM) or a vehicle control and then immunostained to examine levels of ZBTB16, a marker of undifferentiated spermatogonia. Bar represents 50 microns.</p

Colonization by gene-corrected GS cells following testicular transplantation.

<p>(A) Whole mounted squash preparation of seminiferous tubules depicting a seminiferous tubule (arrows) extensively colonized by gene-corrected GT59 SSCs two months following transplantation into a <i>Kit^W-v</i>/<i>Kit^W</i> sterile pup testis. (B) Non-transplanted control. (C) Whole mounted squash preparation of seminiferous tubules depicting a colony of GT65 SSCs two months following transplantation into a busulfan treated adult testis. Visualization of GT65 cells was facilitated by modification with Histone-GFP lentivirus prior to transplant. (A–C) Large arrowheads indicate GFP+ colonies and small arrows indicate autofluorescence in nearby tubules. Bar  = 100 microns. (C′) Higher magnification image of the boxed area in (C). Bar  = 50 microns. (D–G) Immunostaining with anti-GFP antibody (E, G, E′) or DAPI staining (D, F, D′, F′) of a cryosection of a GT59 colony 6 months following transplantation into <i>Kit^W-v</i>/<i>Kit^W</i> pup testis (D, E, D′, E′) or non-transplanted control testes (F, G, F′). Boxed area in D corresponds to the higher magnification view in D′ and E′. Triangles indicate donor-derived GFP+ cells. Boxed area in F corresponds to the higher magnification view in F′ and depicts the “Sertoli Cell Only” phenotype of non-transplanted <i>Kit^W-v</i>/<i>Kit^W</i> testes. The Sertoli cells are indicated by open triangles. Bar  = 25 microns.</p

ZFN-mediated genome editing in GS cells.

<p>(A) Neon transfection (1200/30/1) was used to transfect 3×10e5 cells with 1.0 µg of em-GFP plasmid DNA (pCDNA6.2/emGFP) or 1.0 µg of capped and poly-adenylated mRNA coding for pmaxGFP and transfection efficiency was quantified by flow cytometry three days after transfection. Lipofectamine-2000 was used to transfect the same ratio of cells:DNA or cells:mRNA as in the Neon experiment. The mean and standard deviation of percentage of GFP+ cells from three experiments are shown. (*p<0.05, **p<0.01,***p<0.001, Student T test). (B) Schematic depicting the two plasmids used in genome editing experiments. The donor DNA (“³⁷GFP”; plasmid BE356) contains a fragment of the GFP coding sequence lacking the first 37 nucleotides and serves as a donor template. “Ubc-ZFN1-T2A-ZFN2” (plasmid M500) contains a bicistronic expression cassette with a human Ubiquitin C promoter driving expression of two ZFNs directed to a recognition site in the GFP gene and separated by a T2A skip sequence. GS cell lines were derived from mice carrying a mutated GFP gene, with a 85 nucleotide stop codon and frame shift insertion (labeled “STOP”), introduced into the <i>ROSA26</i> locus by standard knockin technology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112652#pone.0112652-Connelly1" target="_blank">[25]</a>. (C) 0.8 µg of Ubc-ZFN1-T2A-ZFN2 (M500) plasmid or 0.8 µg each of synthesized mRNA ZFN1 and ZFN2, together with 2.4 µg donor plasmid (BE356), were transfected (1400/20/1) into MPG6 cells on day 1 and genome editing events were quantified on day 5 or 7 (data pooled). Histogram shows mean +/- standard error mean. The dot plot shows sample results of a single transfection of donor DNA and ZFN mRNAs. (D) GFP fluorescence (left) or corresponding transmitted light image in GT59 cells following two sorts to enrich for GFP+ cells. Bar represents 50 microns. (E) Chromatogram showing corrected GFP gene sequence of PCR amplified genomic DNA from GT59 cells. The ZFN recognition sites are indicated by boxes and the site in which the mutation was replaced by donor DNA is indicated by a line.</p