PMID- 29304224 OWN - NLM STAT- MEDLINE DCOM- 20190121 LR - 20220321 IS - 1460-2350 (Electronic) IS - 0268-1161 (Linking) VI - 33 IP - 3 DP - 2018 Mar 1 TI - In vitro differentiation of human oocyte-like cells from oogonial stem cells: single-cell isolation and molecular characterization. PG - 464-473 LID - 10.1093/humrep/dex377 [doi] AB - STUDY QUESTION: Are the large cells derived from cultured DEAD box polypeptide 4 (DDX4)-positive oogonial stem cells (OSCs), isolated from the ovarian cortex of non-menopausal and menopausal women, oocyte-like cells? SUMMARY ANSWER: Under appropriate culture conditions, DDX4-positive OSCs from non-menopausal and menopausal women differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. WHAT IS KNOWN ALREADY: The recent reports of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, several questions remain regarding the isolation of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes. STUDY DESIGN, SIZE, DURATION: DDX4-positive OSCs were obtained from 19 menopausal and 13 non-menopausal women (who underwent hysterectomy for uterine fibroma, ovarian cyst, or other benign pathologies) and cultured for up to 3 weeks. Large and small cells were individually isolated and typed for early and late differentiation markers. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortex fragments were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and the positive populations were measured by assessing both FRAGILIS and stage-specific embryonic antigen 4 (SSEA-4) expression. After 3 weeks in culture, large oocyte-like cells were individually isolated by DEPArray based on PKH26 red staining and cell size determination. GDF-9 and SYCP3 as final, and developmental pluripotency-associated protein 3 (DPPA3) as primordial, germline markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content of chromosomes X and 5 was investigated using fluorescence in situ hybridization (FISH). MAIN RESULTS AND THE ROLE OF CHANCE: SSEA-4+ and FRAGILIS+ subsets of DDX4-positive populations were present at lower mean levels in menopausal (SSEA-4+: 46.7%; FRAGILIS+: 47.5%) than in non-menopausal (SSEA-4+: 64.9%; FRAGILIS+: 64.8) women (P < 0.05). A comparison of the women's age with the ratio of DDX4-positive cells/cm3 of ovarian cortex revealed an inverse correlation with OSC number (P < 0.05). Once cultured, cells from both groups differentiated to form large (up to 80 mum) mature oocyte-like cells with typical oocyte morphology. Despite the higher numbers of these cells in cultures from non-menopausal women (37.4 versus 23.7/well; P < 0.001), the intra-culture percentages of large oocyte-like cells did not differ significantly between the two groups. Single large oocyte-like cells isolated from non-menopausal and menopausal women expressed equivalent levels of GDF-9 (e.g. 2.0 and 2.6 copies/mul RNA, respectively) and SYCP3 (e.g. 1.2 and 1.5 copies/mul RNA, respectively) mRNA. The remaining small cells isolated from the cultures expressed large amounts of DPPA3 mRNA (e.g. 5.0 and 5.1 copies/mul RNA, from menopausal and non-menopausal women, respectively), which was undetectable in the large oocyte-like cells. FISH analysis of the large and small cells using probes for chromosomes X and 5 revealed a single signal in the large cells, indicative of chromosome haploidy, whereas in the small cells two distinct signals for each chromosome indicated diploidy. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our study demonstrated the final differentiation of OSCs, collected from the ovarian cortex of adult women, to oocyte-like cells. However, because the rate of differentiation was low, a major role of the stem cell niche housing these OSCs cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: Since the ability of OSCs to generate mature oocytes in vitro is highly variable, the viability of these cells in the ovarian cortex of non-menopausal and menopausal women may well be determined by the stem cell niche and the woman's concurrent reproductive state. Our study showed that the oocyte-like cells obtained by OSC differentiation in vitro, including those from the OSCs of menopausal women, express markers of meiosis. This model of ovarian neo-oogenesis will contribute to the development of approaches to treat female infertility. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Italian Association for Cancer Research (IG grant 17536), and from the Apulia Region ('Oncogenomic Project' and 'Jonico-Salentino Project'). All Authors declare no competing interests. FAU - Silvestris, Erica AU - Silvestris E AD - Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Aldo Moro, P.za G. Cesare, 11-70124 Bari, Italy. AD - Department of Emergency and Organ Transplantation, Section of Obstetrics and Gynecology, University of Bari Aldo Moro, P.za G. Cesare, 11-70124 Bari, Italy. FAU - Cafforio, Paola AU - Cafforio P AD - Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Aldo Moro, P.za G. Cesare, 11-70124 Bari, Italy. FAU - D'Oronzo, Stella AU - D'Oronzo S AD - Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Aldo Moro, P.za G. Cesare, 11-70124 Bari, Italy. FAU - Felici, Claudia AU - Felici C AD - Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Aldo Moro, P.za G. Cesare, 11-70124 Bari, Italy. FAU - Silvestris, Franco AU - Silvestris F AD - Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Aldo Moro, P.za G. Cesare, 11-70124 Bari, Italy. FAU - Loverro, Giuseppe AU - Loverro G AD - Department of Emergency and Organ Transplantation, Section of Obstetrics and Gynecology, University of Bari Aldo Moro, P.za G. Cesare, 11-70124 Bari, Italy. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - England TA - Hum Reprod JT - Human reproduction (Oxford, England) JID - 8701199 RN - 0 (Cell Cycle Proteins) RN - 0 (DNA-Binding Proteins) RN - 0 (Growth Differentiation Factor 9) RN - 0 (Nuclear Proteins) RN - 0 (SYCP3 protein, human) RN - EC 3.6.1.- (DDX4 protein, human) RN - EC 3.6.4.13 (DEAD-box RNA Helicases) SB - IM CIN - Hum Reprod. 2018 May 1;33(5):979. PMID: 29474551 CIN - Hum Reprod. 2018 May 1;33(5):978-979. PMID: 29474556 MH - Cell Cycle Proteins MH - Cell Differentiation/*physiology MH - Cell Separation MH - DEAD-box RNA Helicases/genetics/metabolism MH - DNA-Binding Proteins MH - Female MH - Growth Differentiation Factor 9/genetics/metabolism MH - Humans MH - In Situ Hybridization, Fluorescence MH - In Vitro Techniques MH - Nuclear Proteins/genetics/metabolism MH - Oocytes/*cytology/metabolism MH - Oogonial Stem Cells/*cytology/metabolism EDAT- 2018/01/06 06:00 MHDA- 2019/01/22 06:00 CRDT- 2018/01/06 06:00 PHST- 2017/07/25 00:00 [received] PHST- 2017/12/18 00:00 [accepted] PHST- 2018/01/06 06:00 [pubmed] PHST- 2019/01/22 06:00 [medline] PHST- 2018/01/06 06:00 [entrez] AID - 4785907 [pii] AID - 10.1093/humrep/dex377 [doi] PST - ppublish SO - Hum Reprod. 2018 Mar 1;33(3):464-473. doi: 10.1093/humrep/dex377.