Improvement On Culture System Of Mouse Spermatogonial Stem Cell And Its Gene Transfer In Vitro

1 IntroductionSpermatogonial stem cells (SSCs), the postnatal male germ-line stem cells,self-renew throughout life and, after puberty, provide daughter cells thatdifferentiate into spermatozoa. Recently, remarkable progress has been made inthis field with the aid of SSCs transplantation technique and other stem cellrelated techniques. The ability to isolate/enrich, characterize, culture,cryopreserve, modify genetically, and transplant these unique cells provides apowerful tool for the studies of stem cell biology, therapy of male infertility,preservation of endangered animals, generation of transgenic animals, andbreeding of farm animals with superior characters. However, many problemssuch as the molecular regulation mechanisms of SSCs self-renewal, in vitropropagation of these cells in large scale, and the methods, efficiency, and safetyof in vitro gene transfer into SSCs are all still needed to be addressed. Based onour previous work, firstly, the present study was carried out to improve theculture system of mouse SSCs so that SSCs can well survive and proliferate invitro. Subsequently, in vitro gene delivery into these cells was investigated basedon our improved culture system.2 MethodsMouse seminiferous epithelium was dissociated to prepare a suspension ofsingle cells by collagenase-trypsin digestion. The germ cell suspension was thensubjected to centrifugation of Percoll discontinue density gradients or panningfollowed with purification by differential plate. The purity of enriched cells wasassessed according to morphology, cytochemistry, and immunocytochemistry.The enriched cells were maintained under different conditions to determine theeffects of testicular abstract (TA), epidermal growth factor (EGF) andestradiol-17β(E2) on the survival and proliferation of SSCs. Routine molecular methods were used to construct expression vectorscarrying the target gene fragment. Transfection mediated by lipofectamine wasemployed for the delivery of foreign DNA into the target cells. The transfectedcell clones were then subjected to PCR, RT-PCR and immunocytochemicalanalysis for the successful integration, transcription, and expression of thetransgene. Recombined retroviral vectors were also constructed and analyzed bystandard molecular methods. Then the regular procedures of packaging, titrationwere used to generate stable virus-producing package cells. The foreign DNAwas introduced into enriched SSCs by lipofectamine-mediated transfected orvirus-containing supernatant infection. Subsequently, the transfected cells weremicroinjected into the seminiferous tubules of recipients using SSCstransplantation technique.3 Results (1)The percentage of viable cells was 90.08% and 4.1×105 cells could becollected by using collagenase-trypsin digestion to dissociation mouseseminiferous epithelium. ( 2 ) After Percoll discontinue density gradients centrifugation, theseminiferous epithelium cells gathered between adjacent Percoll gradients. Thecellular debris and a few dead cells were mainly distributed in Percoll gradientbetween 11%~19%. Sertoli cells and peritubular myoid cells were mainlydistributed in Percoll gradient between 19%~27% and 35%~43% according to themorphological observation after seeding in vitro. Alkaline phosphatase (AP)staining of the cells distributed in Percoll gradient between 27%~35% showedthat most of them were AP+, and having homogeneous morphology and size.Spermatogonia cells were further enriched by differential plate from these cellsand the purity was 68.76% as determined from morphology andimmunocytochemical stain. (3)SSCs were also enriched using a specific antibody anti-β1 integrin bypanning method and further purified by differential plate. The enrichedspermatogonia were homogeneous in morphology. Most of them displayed theactivity of AP and they were assessed at 63.54% purity by the methods mentionedabove. (4)Mouse SSCs could adhere on the STO (a fibroblast cell line derivedfrom mouse) feeder layer after seeding 8-12 h in DEME supplemented with 7.5%newborn calf serum, 7.5% fetal calf serum, 1% glutamine, 1% non-essentialamino acids, 1% Pen/Strep, and 1% sodium pyruvate. Under phase contrastmicroscope, they retained the typical morphological characteristics such ashaving a spherical or oval outline, homogeneous morphology and size, existingas scattered single cell or as aligned/clumped population. AP staining showedthat the cells were AP+ or AP++ and the cytoplasm contained few lipid dropletswhen stained with Oil red O. Immunocytochemical stain using β1 integrin andc-kit receptor antibodies showed that most of the spermatogonia enriched byPercoll method were c-kit+, while most of the spermatogonia enriched by panningwere β1 integrin+. After adding TA, EGF, and E2 into the media, the averagesurvival time of SSCs was elongated in all the groups, no statistical differenceshad been found compared with the control group(P>0.05). A few proliferatingcells were observed only in the groups containing 10% and 20% TA at 3~4 d ofculture. More proliferating SSCs presented in all the groups at 3~6 d after addingwith EGF, especially in group containing EGF at the dose of 40 ng/ml. SSCsproliferated obviously in groups containing 10~100 pg/ml E2 from 3 d to 9 d afterseeding. (5)A mammalian cell expression vector pcDNA3-mGDNF carrying mouseglia cell line-derived neurotrophic factor (mGDNF) cDNA was constructed andanalyzed by restriction enzyme digestion and sequencing. The recombined vectorwas then transduced into normal STO cell line by liposome transfection and theG418-resistant clone was screened with media containing G418. The integrationof mGDNF cDNA into STO genome was demonstrated by PCR amplification,successful transcription of the transgene in STO cells by RT-PCR, and stableexpressing GDNF protein in transgenic cells by immunocytochemical stain with arabbit-anti mouse GDNF antibody subsequently. All of the results suggested thatwe had established a transgenic STO cell line, STOGDNF, which expressed mouseGDNF protein stably at high level. (6)In DMEM media enriched with 7.5% NBS, 7.5% FBS, 1% glutamine,1% non-essential amino acids, 1% Pen/Strep, 1% sodium pyruvate, 200ng/ml VE,80μg/ml VC, 10mM β-mercaptoethanol, 40ng/ml EGF, and 100pg/ml E2, thesurvival and proliferation ability of mouse SSCs cultured on STOGDNF feederlayer (group I), STO feeder layer with 20~100ng/ml GDNF (group IV~VI) wassignificantly improved than that of the cells maintained on STO feeder layer(group III) and in a system of feeder-free but adding with 40ng/ml GDNF (groupII). Proliferation of SSCs also could be found after cultured 20 d in groups of I,IV, V, VI. These cells still retained their typical morphology and expressedspermatogonia marker c-kit, β1 integrin and AP activity. However, the numberof SSCs in group II and III decreased dramatically and few proliferating cellswere observed in these groups. (7)Based on pcDNA3-tPA, a mammalian cellular expression vectorcarrying tissue-type plasminogen activitor gene t-PA, two retroviral vectorsPLNC-tPA and PL-tPA-SN were constructed and examined by restriction enzymedigestion and sequencing. Then, they were introduced into a packaging cell linePA317 by lipofectamine-mediated transfection. Five stable virus-producing cellclones, PA3171-5, were screened with the media containing G418. Among them,PA3171-2 produced PLNC-tPA and PA3173-5 produced PL-tPA-SN virus particle.All the supernatants collected from these clones could infect standard titteringcell line NIH-3T3 and generate G418-resistant cell clone. The virus titerdetermination showed that PA3172 gave the highest pre-concentrated viral titer (7×102CFU/ml) of PLNC-tPA, PA3175 gave the highest pre-concentrated viral titer(1.6×103CFU/ml ) of PL-tPA-SN。 (8)The average testes weight of mice in group IV (40mg/kg) and V(60mg/kg) was significantly lower (P<0.01) than that of mice in group I(physiological saline), II (50%DMSO), III (20mg/kg) at 30 d after intraperitonealinjection of chemotherapeutic agent busulfan. The testes in group II maintainednormal volume, while the testes volume in group III decreased 10%, 30% ingroup IV and V, when compared with that in group I. Histological observationshowed that the seminiferous epithelium of animals in group I and II was normal,while it degenerated in group III and degenerated severely in group IV and V. (9)After transfected with lipofectamine-pcDNA3-tPA or supernatantscollected from virus-producing PA317 cells, SSCs could still survive and nomorphological change was found. The recipient testes were found that about 30%seminiferous tubules in surface could be fulfilled by injection containingtransfected SSCs and trypan blue. The average testes weight of recipients ingroup I (transplanted with the cells not transfected) was significantly (p<0.05)higher than that in groups V (transplanted with the cells transfected repeatedly bysupernatant from PA3172) and in group VI (transplanted with the cells transfectedrepeatedly by supernatant from PA3175) at 30 d after cell transplantation.Histological observation found that the seminiferous epithelium of recipienttestes in group I, II, III, IV recovered well than that in group V and VI. GenomicDNA PCR amplification of recipient testes showed that there was one recipient ingroup II and IV respectively carrying the target gene.4 Conclusions (1)Combined with differential plate, panning is more suitable than Percolldiscontinue density gradients centrifugation for SSCs enrichment. Adding EGFand E2 into media is beneficial to the survival and proliferation of mouse SSCs invitro. ( 2 ) Mouse GDNF cDNA can be integrated into STO genome bylipofectamine-mediated transfection followed screen with media containingG418. Moreover, the transgene can be transcripted successfully and expressedstably at high level. (3)Appropriate feeder layer cells and GDNF are essential factors for SSCsmaintenance in vitro. The survival and self-renewal of SSCs have a dose-effectrelationship with the concentration of GDNF. (4)The transgenic feeder layer STOGDNF can enhance the maintenance ofmouse SSCs in vitro significantly. (5)Transgenic feeder layer which expresses the essential factors for stemcell self-renewal will be a new and effective way for in vitro propagation of stemcells.