Embryonic stem cell lines derived from somatic cell nuclear transferred oocytes fertilizing with sperm

13. Salimi A, Nadri S, Ghollasi M, Khajeh K, Soleimani M. Comparison of different protocols for neural differentiation of human induced pluripotent stem cells. Mol Biol Rep. 2014;41(3):1713-1721. 14. Cho MS, Hwang DY, Kim DW. Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale. Nat Protoc. 2008;3(12):1888-1894. 15. Salvagiotto G, Burton S, Daigh CA, Rajesh D, Slukvin, II, Seay NJ. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs. PLoS One. 2011;6(3):e17829. 16. Dorn I, Klich K, Arauzo-Bravo MJ, et al. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin. Haematologica. 2015;100(1):32-41. 17. Kim K, Zhao R, Doi A, et al. Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat Biotechnol. 2011;29(12):1117-1119. 18. Dias J, Gumenyuk M, Kang H, et al. Generation of red blood cells from human induced pluripotent stem cells. Stem Cells Dev. 2011;20(9):1639-1647. Supported by: This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education(2013R1A1A1076178). P-225 Tuesday, October 31, 2017 HIGHER ABUNDANCE OF STEM CELLS IN HUMAN MYOMETRIAL TISSUE FROM AFRICAN AMERICANS MAY CONTRIBUTE TO THE ETHNIC DISPARITY OF UTERINE FIBROIDS. L. Prusinski,a A. Mas,b A. Al-Hendy.a aAugusta University, Augusta, GA; bGrupo de Investigaciðn de Medicina Reproductiva, Instituto de Investigaciðn Sanitario La Fe, Paterna (Valencia), Spain. OBJECTIVE: Though benign, uterine fibroids (UF) diminish the quality of life of affected women, often causing heavy uterine bleeding and infertility. Currently, the unknown etiology of UF prevents significant improvement in care for affected women. Additionally, UF prevalence in AfricanAmerican (AA) women is four times greater than in Caucasian (CA) women; AA women generally have earlier disease onset and greater UF tumor burden, but the mechanism behind this ethnic disparity remains unknown. It has been previously shown with immunohistochemistry and paraffin-fixed tissues that myometrium of AA women show increased numbers of Stro-1+/CD44+ myometrial stem cells vs. CA women, both in normal and fibroid-affected uteri1. Recent findings link cancer (i.e. tumor) risk to the number of tissue-specific stem cell divisions; increased numbers of stem cells and, thus, DNA replication events, in proliferative tissue like the myometrium, which continually remodels during the female lifespan3, increase the risk of mutation and tumorigenesis. In this work, we investigated this further using flow cytometry, a more specific technique, and fresh human myometrial tissues to quantify the myometrial stem cells in AA vs. CA women. DESIGN: Laboratory research studies using freshly-isolated human myometrial tissues from post-hysterectomy normal and fibroid-containing uteri. MATERIALS AND METHODS: Human myometrial samples were obtained from 20 AA and 11 CA pre-menopausal women undergoing hysterectomy for either UF or other medical indications. In fibroid-uteri, samples were collected at >2 cm away from closest UF lesion. Subjects were between 30 and 55 years of age and BMI>25. No subjects used any hormonal treatment for at least 3 months prior to surgery. This study was approved by Augusta University IRB, and informed consent was collected from all participants. Fresh tissue was digested, and Stro-1+/CD44+ myometrial stem cells (Myo+/+) were isolated from single-cell suspensions as reported previously4. Percentage of Myo+/+ was determined for each sample using flow cytometry. Percentages of Myo+/+ in AA vs. CA women were compared using two-sample, two-tailed Student’s t-test. RESULTS: By flow cytometry, the mean percentage of Stro-1+/CD44+ myometrial stem cells from fresh myometrium obtained from AA uteri (0.41%, 95% CI [0.32%, 0.49%]) was significantly greater than that of CA uteri (0.26%, 95% CI [0.17%, 0.36%]), (p<0.05). CONCLUSIONS: Increasing evidence suggests that somatic mutations in myometrial stem cells convert them into tumor-initiating cells, leading to UF development. Our data support the hypothesis that an increased percentage of myometrial stem cells, i.e. an expanded myometrial stem cell population in African American women, suggests increased risk of mutation and formation of tumor-initiating cells, which may ultimately contribute to increased UF tumor prevalence and burden in African American women.

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ASRM Abstracts

References: 1. Mas A, Stone L, O’Connor P, Yang Q, Kleven D, Simon C, Walker C, Al-Hendy A. Developmental exposure to endocrine disruptors expands the murine myometrial stem cell compartment as a prerequisite to leiomyoma tumorigenesis. Stem Cells, 2016; 35(3):666678. 2. Tomasetti C, Vogelstein B. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science, 2015; 347(6217):78-81. http://dx.doi.org/10.1126/science.1260825. 3. Odunsi K, Pejovic T, Thomas Jr. CR. Gynecologic Cancers: A Multidisciplinary Approach to Diagnosis and Management. Current Multidisciplinary Oncology. 1st ed. Demos Medical, New York: 2013. 4. Mas A, Nair S, Laknaur A, Simon C, Diamond MP, Al-Hendy A. Stro1/CD44 as putative human myometrial and fibroid stem cell markers. Fertil Steril, 2015; 104(1):225-34.e3. Supported by: Augusta University Start-up Package; National Institutes of Health: R01 HD046228-12 P-226 Tuesday, October 31, 2017 EMBRYONIC STEM CELL LINES DERIVED FROM SOMATIC CELL NUCLEAR TRANSFERRED OOCYTES FERTILIZING WITH SPERM. D. Wen,a S. Chow,b G. D. Palermo,c Z. Rosenwaks.d aThe Ronald O. Perelman and Claudia Cohen CRM, Weill Cornell Medical College, New York, NY; b The Ronald O. Perelman and Claudia Cohen CRM, Weill Cornell Medicine, New York, NY; cThe Ronald O. Perelman and Claudia Cohen CRM, Weill Medical College of Cornell University, New York, NY; dThe Ronald O. Perelman and Claudia Cohen CRM, Weill Cornell Medical College, NYC, NY. OBJECTIVE: Metaphase II (MII) oocyte can halve its genome by extrusion of the second polar body at fertilization. We aim to confirm the ability of MII ooplasm to reprogram and haploidyze somatic cell nucleus. DESIGN: We tested, by somatic cell nuclear transfer, the ability to haploidyze mouse cumulus cell nuclei. Generated constructs were then subjected to intracytoplasmic sperm injection (ICSI) to generate embryos and derive embryonic stem cells (ESCs). MATERIALS AND METHODS: MII oocytes were recovered from female mice 16-18h after hCG injection. Superovulation was achieved by injecting PMSG (10IU/mouse) followed by injection of hCG (10IU/mouse) 48h later. H3.3B-HA-iEYFP or tdTomato cumulus cells were used for nuclear transfer, and sperm carrying a different fluorescence reporter was used for ICSI to activate the constructed oocytes. After extrusion of pseudo-polar bodies (psPBs) and fertilization assessment, eventual morulae and blastocysts were used to derive ESCs. RESULTS: psPBs extrusions were observed in 51% (80/154) of the reconstructed oocytes 2-3h after fertilization. Immunofluorescence (IF) staining revealed that bipolar spindle around the cumulus nucleus was visible 1-3h following fertilization and psPBs extrusion. Using H3.3BHA-iEYFP (chromosome 17) heterozygous cumulus cells, we found that half of the embryos were EYFP positive, indicating proper somatic nuclear haploidization in these embryos. In a total of 118 tdTomato nuclear-transferred oocytes, 55 (47%) of them extruded psPBs at fertilization. In these 55 psPB embryos, 48 (87%) of them reached the 2-cell stage, 42 (76%) the 4-cell stage, 23 (42%) the morula stage, and 6 (11%) the early blastocyst stage. The 6 early blastocysts were used for ESC derivation and one line was established (OIH-td-cum-1). Of the 63 oocytes that did not extrude psPBs, 41(65%), 30(48%), 17(27%) and 10(16%) embryos developed to 2-cell, 4-cell, morula and early blastocyst stage, respectively. Two ESC lines were derived from the blastocysts (OIH-td-cum-2, OIH-td-cum-3). All 3 of the ESC lines were tdTomato positive, maintained typical ESC morphology, and expressed pluripotency markers. Karyotyping analysis showed that ESC line OIH-td-cum-1 was a diploid karyotype with a gain of chromosomes 5, 8, 9, 18, and 19. OIH-td-cum-2 and OIH-td-cum-3 were triploid karyotypes (60 chromosomes). CONCLUSIONS: Somatic genome DNA can be reduced in the MII oocyte cytoplasm through the extrusion of pseudo-polar bodies after fertilization. Embryos containing haploidyzed somatic genome and sperm DNA can develop into blastocysts and derive ESC lines. Haploidization of diploid nuclei in MII oocytes would be an alternative approach to producing artificial gametes. References: 1. Palermo GD, Takeuchi T, Rosenwaks Z. Technical approaches to correction of oocyte aneuploidy. Hum Reprod 2002;17(8):2165-73.

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2. Palermo GD, Takeuchi T, Rosenwaks Z. Oocyte-induced haploidization. Reprod Biomed Online 2002; 4(3):237-42. 3. Ma H, O’Neil RC, Marti Gutierrez N, et al. Functional Human Oocytes Generated by Transfer of Polar Body Genomes. Cell Stem Cell 2017;20(1):112-119. Supported by: Starr Foundation Tri-Institutional Stem Cell Initiative P-227 Tuesday, October 31, 2017 DIFFERENT ANGIOGENIC POTENTIALS OF MESENCHYMAL STEM CELLS DERIVED FROM UMBILICAL ARTERY, UMBILICAL VEIN AND WHARTON’S JELLY. L. Xu,a H. Sun,b L. Ding.a aAffiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China; bNanjing Drum Tower Hospital, Nanjing, China. OBJECTIVE: Human mesenchymal stem cells derived from umbilical cord (UC) are a favorable source for allogeneic cell therapy. Here, we successfully isolated the stem cells derived from three different compartments of the human UC, including perivascular stem cells derived from umbilical arteries (UCA-PSCs), perivascular stem cells derived from umbilical vein (UCV-PSCs) and mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs). This assay aims to compare the biological characteristics and angiogenic potential of these three cell populations. DESIGN: Human UC tissue samples (n¼10) were collected from the Affiliated Drum Tower Hospital of Nanjing University Medical School and processed within 12h of natural delivery. The experimental procedure was approved by the Clinical Research Ethics committee at Affiliated Drum Tower Hospital of Nanjing University Medical School.UCA-PSCs, UCVPSCs and WJ-MSCs derived from the same umbilical cord were used for research. MATERIALS AND METHODS: The biological characteristics of UCAPSCs, UCV-PSCs and WJ-MSCs were evaluated with immunofluorescence assay, proliferation assay, flow cytometry analysis and muiltilineage differentiation assay. The angiogenic popential of these cells was evaluated with tube formation assay in vitro and matrigel plug assay in vivo. The possible mechanism was confirmed with gene microarray quantitative real time PCR, western blot and small interfering RNA transfection. RESULTS: These cells had the similar phenotype and differentiation potential toward adipocytes, osteoblasts and neuron-like cells. However, UCA-PSCs and UCV-PSCs had more CD146+ cells than WJ-MSCs (P < 0.05). Tube formation assay in vitro showed a largest number of tube-like structures and branch points in UCA-PSCs among the three stem cells. Additionally, the total tube length in UCA-PSCs and UCV-PSCs was significantly longer than in WJ-MSCs (P < 0.01). Matrigel plug assay in vivo showed that matrigel plugs in UCA-PSCs group expressed higher mCD31 level than PBS, UCV-PSCs and WJ-MSCs group (P < 0.01). Microarray, qRT-PCR and Western blot analysis showed that UCA-PSCs had the highest expression of the Notch ligand Jagged1 (JAG1), which is crucial for blood vessel maturation. Knockdown of Jagged1 significantly impaired the angiogenesis in UCA-PSCs. CONCLUSIONS: UCA-PSCs are promising cell populations for clinical use in ischemic diseases and Jagged1 played an important role in UCMSCs induced angiogenesis. References: 1. Bronckaers A, Hilkens P, Martens W, et al. Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis. PharmacolTher. 2014. 143(2): 181-96. 2. Teixeira FG, Carvalho MM, Sousa N, Salgado AJ. Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration. Cell Mol Life Sci. 2013. 70(20): 3871-82. 3. Carvalho MM, Teixeira FG, Reis RL, Sousa N, Salgado AJ. Mesenchymal stem cells in the umbilical cord: phenotypic characterization, secretome and applications in central nervous system regenerative medicine. Curr Stem Cell Res Ther. 2011. 6(3): 221-8. 4. Kim DW, Staples M, Shinozuka K, Pantcheva P, Kang SD, Borlongan CV. Wharton’s jelly-derived mesenchymal stem cells: phenotypic characterization and optimizing their therapeutic potential for clinical applications. Int J Mol Sci. 2013. 14(6): 11692-712. Supported by: This work was supported by grants Nature Science Foundation of China (81571391, 30900847,31571189), Nanjing Medical Science Development Project (JQ2014004, ZKX16042) and Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01030501).

FERTILITY & STERILITYÒ

P-228 Tuesday, October 31, 2017 HIGH THROUGHPUT SCREENS USING VIABLE FLUORESCENT ESC REPORTERS OF STRESS FORCED DECREASED POTENCY AND INCREASED DIFFERENTIATION TO THE FIRST LINEAGE IDENTIFY EMBRYOTOXIC AND NONEMBRYOTOXIC DRUGS. E. Puscheck,a E. Louden,a D. Rappolee.b aOB/GYN, Wayne State University School of Medicine, Detroit, MI; bOB/GYN, Wayne State University School of Medicine, Grosse Pointe Farms, MI. OBJECTIVE: To test whether high throughput screening (HTS) methods using mouse pluripotent Embryonic Stem Cells (ESC) reporters can effectively identify embryo toxic and non-embryo toxic drugs. DESIGN: Experimental design MATERIALS AND METHODS: Mouse ESC with Pdgfra promoter-GFP reporter are regulated by endogenous factors of the PDGFRa promoters. Pdgfra -GFP ESCs were tested for stress-forced differentiation in the presence and absence of LIF-maintained potency. The key test was stress-forced differentiation in the presence of LIF, which maintains proliferation and potency. Microplate, Immunofluorescence and Immunoblot assays tested Pdgfra -GFP ESC responses and quantified with Simple PCI. Dose and kinetic responses were tested using hyperosmotic sorbitol as a positive stress-forced differentiation control. Retinoic acid (RA) is a first lineage inducer used as a normal differentiation control. These were compared to LIF+ normal potency culture control. Pdgfra-GFP was validated with other first lineage markers, Dab2, LRP2, and by anti-GFP by both immunofluorescence and quantitative immunoblots. A panel of 17 compounds previously used in a European multilab ESC cytoxicity assay validation were used here to test HTS1/2 ESC assays for potency decrease and differentiation increase. RESULTS: Hyperosmotic stress +/- LIF initiated decreased stem cell growth and potency and increased differentiation as demonstrated by Hoechst staining assayed by microplate reader, co-localization of PDGFRa-GFP and Dab2 co-marker for first differentiated lineage. Microplate reader assays for stress-induced PDGFRa-GFP differentiation were corroborated by Immunoblot for Dab2, LRP2, GFP protein and showed similar dose responses. In differentiating ESCs there are areas not co-localized indicating the salt-and-pepper effects of cell patterning known to occur during cell lineages intermediate between potent and differentiated states. Drugs like penicillin were nonembryotoxic and did cause Rex1-RFP loss of Pdgfra-GFP increase, but drugs like Methotrexate caused more potency loss than hyperosmotic stress and also cause Pdgfra-GFP increase. CONCLUSIONS: Stress decreases stem cell growth (runting) and forces differentiation of remaining ESCs hypothetically to provide sufficient nutrient-acquiring function from fewer cells. Previously, we reported Rex1-RFP ESC potency reporters decrease and now we report that PDGFRa-GFP ESC differentiation reporters increase with strong stress exposures. These combined results support HTS of mouse ESC with PDGFa/GFP as an effective method to detect effects of stress to first lineage which paves the way for rapid reproductive toxicity testing and may give clinical relevance to IVF stress exposures. Rex1-RFP is more sensitive for 70% of compounds than previous ESC cytotoxicity assays and is in good agreement with in vivo data for identifying gestational embryotoxic and nonembryotoxic compounds. P-229 Tuesday, October 31, 2017 A SIMPLE METHOD TO GENERATE GERM CELLS FROM MALE EMBRYONIC STEM CELLS. M. Irani, V. Lu, A. Parrella, S. Chow, B. Chin, C. O’Neill, S. Cheung, Z. Rosenwaks, G. D. Palermo. The Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medicine, New York, NY. OBJECTIVE: Many complex methods are available for creating and maintaining differentiated germ cells. We evaluated the hanging drop (HD) system to expeditely induce germ cell (GC) appearance in an embryonic stem cell culture. DESIGN: Embryoid bodies were induced and maintained in medium supplemented with differentiating factors in either HD or in culture dishes (CD). PGC appearance and differentiation was assessed for germ cell markers by microscopy staining, immunofluorescence, and molecular epigenetic assay by next-generation sequencing (NGS). MATERIALS AND METHODS: Male mESCs were maintained on mouse embryonic fibroblast (MEF) cells until confluency was achieved, after which

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