Male gametes originate from a small population of spermatogonial stem cells (SSCs). These cells are believed to divide infinitely and to support spermatogenesis throughout life in the male. Here, we developed a strategy for the establishment of SSC lines from embryonic stem (ES) cells. These cells are able to undergo meiosis, are able to generate haploid male gametes in vitro, and are functional, as shown by fertilization after intracytoplasmic injection into mouse oocytes. Resulting two-cell embryos were transferred into oviducts, and live mice were born. Six of seven animals developed to adult mice. This is a clear indication that male gametes derived in vitro from ES cells by this strategy are able to induce normal fertilization and development. Our approach provides an accessible in vitro model system for studies of mammalian gametogenesis, as well as for the development of new strategies for the generation of transgenic mice and treatment of infertility.
Recent studies have demonstrated that somatic stem cells have a more flexible potential than expected, whether put into tissue or cultured under different conditions. Bone marrow (BM)-derived stem cells can transdifferentiate into multilineage cells, such as muscle of mesoderm, lung and liver of endoderm, and brain and skin of ectoderm origin. Here we show that BM stem cells are able to transdifferentiate into male germ cells. For derivation of male germ cells from adult BM stem (BMS) cells, we used the Stra8-enhanced green fluoresence protein (EGFP) transgenic mouse line expressing EGFP specifically in male germ cells. BMS cellderived germ cells expressed the known molecular markers of primordial germ cells, such as fragilis, stella, Rnf17, Mvh and Oct4; as well as molecular markers of spermatogonial stem cells and spermatogonia including Rbm, c-Kit, Tex18, Stra8, Piwil2, Dazl, Hsp90a, b1-and a6-integrins. Our ability to derive male germ cells from BMS cells reveals novel aspects of germ cell development and opens the possibilities for use of these cells in reproductive medicine.
Spermatogonial stem cells (SSCs) isolated from the adult mouse testis and cultured have been shown to respond to culture conditions and become pluripotent, so called multipotent adult germline stem cells (maGSCs). microRNAs (miRNAs) belonging to the 290 and 302 miRNA clusters have been previously classified as embryonic stem cell (ESC) specific. Here, we show that these miRNAs generally characterize pluripotent cells. They are expressed not only in ESCs but also in maGSCs as well as in the F9 embryonic carcinoma cell (ECC) line. In addition, we tested the time-dependent influence of different factors that promote loss of pluripotency on levels of these miRNAs in all three pluripotent cell types. Despite the differences regarding time and extent of differentiation observed between ESCs and maGSCs, expression profiles of both miRNA families showed similarities between these two cell types, suggesting similar underlying mechanisms in maintenance of pluripotency and differentiation. Our results indicate that the 290-miRNA family is connected with Oct-4 and maintenance of the pluripotent state. In contrast, members of the 302-miRNA family are induced during first stages of in vitro differentiation in all cell types tested. Therefore, detection of miRNAs of miR-302 family in pluripotent cells can be attributed to the proportion of spontaneously differentiating cells in cultures of pluripotent cells. These results are consistent with ESC-like nature of maGSCs and their potential as an alternative source of pluripotent cells from non-embryonic tissues.
CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.
Previous studies showed that in the mouse mutant Lis1GT/GT gene trap integration in intron 2 of Lis1 gene leads to male infertility in homozygous Lis1GT/GT mice. We further analyzed this line and could confirm the suggested downregulation of a testis-specific Lis1 transcript in mutant animals in a quantitative manner. Moreover, we analyzed the gene trap mutation on different genetic backgrounds in incipient congenic animals and could exclude a genetic background effect. To gain further insights into the role and requirement of LIS1 in spermatogenesis, 3 transgenic lines were generated, that overexpress Lis1 under control of the testis-specific promoters hEF-1α, which is exclusively active in spermatogonial cells, PGK2, which is active in pachytene spermatocytes and following stages of spermatogenesis, and Tnp2 which is active in round spermatids and following stages of spermatogenesis, respectively. All 3 transgenic lines remained fertile and testis sections displayed no abnormalities. To overcome the infertility of Lis1GT/GT males, these transgenic Lis1-overexpressing animals were mated with Lis1GT/GT mice to generate ‘rescued’ Lis1GT/GT/Lis1Tpos males. ‘Rescued’ animals from all transgenic lines remained infertile, thus overexpression of Lis1 in different stages of spermatogenesis could not rescue the infertility phenotype of homozygous gene trap males.
Azacitidine combined with donor lymphocyte infusions (DLI) is an established treatment for relapse of myeloid malignancies after allogeneic transplantation. Based on its immunomodulatory and anti-leukemic properties we considered Lenalidomide to act synergistically with Azacitidine/DLI to improve outcome. We therefore prospectively investigated tolerability and efficacy of this combination as first salvage therapy for adults with post-transplant relapse of AML, MDS and CMML. Patients were scheduled for 8 cycles Azacitidine (75 mg/m2 day 1-7), Lenalidomide (2.5 or 5mg, day 1-21) and up to 3 DLI with increasing T cell dosages (0.5×106-1.5×107 cells/kg). Primary endpoint was safety, while secondary endpoints included response, graft-versus-host disease (GvHD) and overall survival (OS). Fifty patients with molecular (52%) or hematological (48%) relapse of MDS (n=24), AML (n=23) or CMML (n=3) received a median of 7 (range, 1 to 8) cycles including 14 patients with 2.5mg and 36 with 5mg Lenalidomide daily dosage. Concomitantly, 34 patients (68%) received at least one DLI. Overall response rate was 56% and 25 patients (50%) achieved complete remission being durable in 80%. Median OS was 21 months and 1-year OS rate 65% with no impact of type of or time to relapse and Lenalidomide dosages. Treatment was well tolerated indicated by febrile neutropenia being the only grade ≥3 non-hematologic adverse event in >10% of patients and modest acute (grade II-IV 24%) and chronic (moderate/severe 28%) GvHD incidences. In summary, Lenalidomide can be safely added to Azacitidine/DLI without excess of GvHD and toxicity. Its significant anti-leukemic activity suggests that this combination is a novel salvage option for post-transplant relapse. (NCT02472691)
Background Azacitidine (Aza) in combination with donor lymphocyte infusions (DLI) is an established treatment option for pts with relapse of myeloid malignancies after allo-SCT. Accounting for its immunomodulatory and anti-leukemic properties, we considered Lenalidomide (Len) to be a synergistic partner for Aza and DLI that may further improve response rate and outcome. To investigate the tolerability and efficacy of the combination of Aza, Len and DLI as first salvage therapy for relapsed MDS, AML and CMML after allo-SCT we performed a prospective, multicenter, single-arm phase-II trial. Results from two safety interim analyses have previously been reported. Here, we report the final results from this investigator-initiated trial. Design/Methods: Patients with relapse of MDS, AML and CMML after first allo-SCT were eligible. Envisaged treatment according to the protocol consisted of up to 8 cycles Aza (75 mg/m 2/d d1-7, every 28 days) and up to 3 DLI with increasing T cell dosages (0.5×10 6 - 1.5×10 7 cells/kg). Len was administered concomitantly for 21 days of a 28-day cycle. Following a positive first interim safety analysis in 10 patients the daily dose of Len was increased from 2.5 to 5mg. The primary endpoint of the study was safety, while secondary efficacy endpoints included response type and rates, time to and duration of response and overall survival. Results: Overall, 50 pts with molecular (n=29, 58%) or hematological (n=21, 42%) relapse of MDS (n=24, 48%), AML (n=23, 46%) or CMML (n=3, 6%) detected after median of 233 days (range, 98 to 2659) after allo-SCT were included. Fourteen patients (28%) received Len at a daily dosage of 2.5 mg and 36 patients (72%) at a daily dosage of 5 mg with no DLTs observed in the interim analyses. Median number of Len cycles per patient was 7 (range, 1 to 8) with no differences between the two dose levels. Concomitantly, 34 pts (68%) received at least one DLI (median: 3, range: 1-11). Overall response rate (ORR) during treatment was 56% (CR n=25, 50%, PR n=3, 6%). ORR and CR rates did not differ between Len dose levels. Of interest, CR rate did not differ between pts treated at the stage of molecular relapse and those initiated at hematological relapse (52% vs. 48%). Median time to CR was 112 days (range 1-286) corresponding to 4 cycles (range 1 to 8). At the time of data lock, 20 patients (80%) were still in CR without additional therapy for a median of 15 months, while 5 patients (20%) had relapsed again after a median of 8 months. With a median follow-up of 20 months median OS was 21 months and 1-year OS rate 65%. While therapy-related CTC grade III/IV neutropenia (92%), thrombopenia (80%) or anemia (36%) occurred frequently, drug-related non-hematological adverse events (AE) >grade II were rare and mainly consisted of gastrointestinal toxicity (6%), laboratory findings (28%) and infections (22%). Twenty-three pts (46%) developed acute GvHD including 5 patients (10%) with grade III/IV aGvHD, and 26 pts (52%) chronic GvHD (mild n=10; moderate n=11; severe n=5). During the study period, 3 secondary malignancies (squamous cell, basal cell and vulvar carcinoma) occurred. There were no therapy related deaths. Conclusion: Len up to a dosage to 5 mg/day can be safely added to the combination of AZA and DLI without excess of GvHD and toxicity. Furthermore, these data suggest that the combination of Aza, Len and DLI has promising clinical activity for relapse of myeloid malignancies after allo-SCT and is able to induce durable responses and survival in a substantial proportion of pts. Disclosures Schroeder: Celgene: Honoraria, Other: Travel support, Research Funding. Stelljes: Kite/Gilead: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Medac: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau. Holtick: Celgene: Honoraria; Sanofi: Honoraria. Germing: Janssen: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis: Honoraria, Research Funding. Kröger: AOP Pharma: Honoraria; Celgene: Honoraria, Research Funding; Gilead/Kite: Honoraria; Jazz: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Novartis: Honoraria; Riemser: Honoraria, Research Funding; Sanofi: Honoraria. Kobbe: Celgene: Research Funding. OffLabel Disclosure: Lenalidomide is not licensed for AML, CMML and advanced MDS except for MDS with isolated del5q
Determination of integration sites in transgenic hEF-1α-Lis1-c-myc Tag lines -------3.4.6 Analysis of "rescued" L39 GT/GT / hEF-1α-Lis1-c-myc Tag Tpos males
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