Maintenance of functional embryoid bodies in cryopreservable, microfluidic chips: a platform for personalized medicine

2014CB943203), the National Natural Science Funds for general program (31371521 and 81370766), and Ph.D. Programs Foundation of Ministry of Education of China (20110001120008). P-318 Tuesday, October 21, 2014 DEFINING DIFFERENTIATED METHYLATION REGIONS ESPECIAL FOR PLURIPOTENCY ACQUISITION AND MAINTENANCE IN HUMAN STEM CELL VIA HIGH PROBE DENSITY MICROARRAY. Y. Fan, W. He, X. Sun. The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China. OBJECTIVE: Epigenetic regulation is a critical event in the maintenance of human pluripotent stem cells. It had been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appeared hyper-methylated status compared to differentiated cells. However, epigenetic mechanisms of ‘‘stemness maintaining’’ and ‘‘reprogramming’’ remained confused since the limitation of former detection platform. MATERIALS AND METHODS: we determined the DNA methylation profiles of 12 human cell lines, including 2 ESC lines, 4 virally-delivered iPSC lines, 2 episomal-delivered iPSC lines, and 2 parent cell lines that iPSCs derived from using Illumina’s Infinium HumanMethylation450. RESULTS: The iPSCs exhibited a hypermethylation status similarly to ESCs but distinct differences from the parent cells. Genes of common methylation pattern between iPSCs and ESCs were regarded as critical factors for stemness, while differences existing between iPSCs and ESCs implied that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cell reprogramming. No significant variant was identified between virally- and episomal- systems. Based on microarray platform of higher probe density and greater genome coverage, de novo differentiated methylation regions as the signature of particular stem cell lines were emerged in detail. CONCLUSION: This study figured out DNA methylation profiles of human iPSCs generated in virally- and episomal-system, the responding somatic cells as well as hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined in a high resolution. Knowledge of epigenetic information could be used as a signature for ‘‘stemness’’ and ‘‘self-renewal’’ and provided potential of selecting optimum pluripotent stem cells for human regenerative medicine .

P-319 Tuesday, October 21, 2014 TROPHOBLAST CELLS GENERATED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS (iPSC) DERIVED FROM UMBILICAL CORD BY USING BMP4 CAN BE DISEASE MODELS FOR T. Ezashi,b EARLY PLACENTAL DISORDERS. M. Amita,a,b L. C. Schulz,c D. J. Schust,c R. M. Roberts.b aObstetrics, Gynecology, Yamagata University Faculty of Medicine, Yamagata, Japan; bBond Life Sciences Center, University of Missouri, Columbia, MO; cUniversity of Missouri, Columbia, MO. OBJECTIVE: Poor placentation in early pregnancy leads to placental pathogenesis, such as preeclampsia (PE). Understanding the pathogenesis of the disease has been hampered by inaccessibility to early stage placental tissue where the initial causal changes take place. Recently we reported that human embryonic stem cells (hESC) treated with inhibitors of activin A (A83-01) and FGF2 (PD173074) signaling in presence of BMP4 (termed BAP) differentiate unidirectionally into trophoblast (TB) cells, which provide potential in vitro models of early placental development1. In this study, we derived iPSC by two procedures from umbilical cord and compared their responses to BMP4/BAP with hESC. DESIGN: We demonstrate the ability to differentiate iPSC from human umbilical cord to TB cells with BAP. MATERIALS AND METHODS: We performed the following experiments using H1 hESC and iPSC derived from human umbilical cord by either viral integration (V) or plasmid transfection (P). (1) Monitoring cellular morphological changes in cultured hESC and iPSC in the presence of BMP4 and BAP. (2) Immunoassay for daily accumulation of syncytiotrophoblast (STB) markers, hCG, P4, in medium (3) Immunohistochemistry (IHC) by using anti-KRT7, hCG and GATA2 antibodies. . (4) Fluorescence activated cell sorting (FACS) analysis to measure the proportion of KRT7 positive cells in the cells differentiated with BMP4 or BAP. (5) Western blotting analysis to detect proteins characteristic of extravillous trophoblast (EVT) cells. RESULTS: A morphological switch to larger epitheloid-type cells began by day 2; STB appeared around d 6 in both V lines and P lines. STB markers

FERTILITY & STERILITYÒ

were significantly (P<0.01) lower in V lines than P lines treated with BMP4 alone, whereas no differences were observed in response to BAP. IHC showed that both the V lines and P lines treated with BMP4/BAP expressed TB markers. FACS analysis revealed that KRT7 positive cells had emerged more slowly in V lines treated with BMP4 than in P lines by d 2, whereas no differences were observed in response to BAP, with almost all cells converting to KRT7+ by d 2. BAP also drove differentiation of both lines to TB without expression of mesoderm markers. CONCLUSION: iPSC from umbilical cords of newborn babies treated with BAP, differentiate into TB cells and provide a potential model system to uncover the mechanisms of PE. Supported by: NIH Grant NIH: 1R01HD067759 & HD077108. P-320 Tuesday, October 21, 2014 MAINTENANCE OF FUNCTIONAL EMBRYOID BODIES IN CRYOPRESERVABLE, MICROFLUIDIC CHIPS: A PLATFORM FOR PERSONALIZED MEDICINE. R. M. Anchan,a S. Guven,b J. Lindsey,a M. Nickerson,a,b S. Chinthala,a B. Gerami-Naini,a,c U. Demirci.b,d aCenter for Infertility and Reproductive Surgery, Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; bBio-Acoustic-MEMS in Medicine Laboratories, Division of Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; cDepartment of Oral and Maxillofacial Pathology, Oral Medicine and Craniofacial Pain, School of Dental Medicine, Tufts University, Boston, MA; dHarvard-MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA. OBJECTIVE: Employ microfluidic cassettes as a novel platform for longterm culture and cryopreservation of functional, differentiated mouse embryoid bodies. DESIGN: Basic Research Study. MATERIALS AND METHODS: Embryoid bodies (EBs), grown in suspension from mouse embryonic stem cells (ESCs), were embedded in Matrigelcoated channels with a constant 1 ml/min flow of culture media for 21 days. EB viability, differentiation, and functionality were assayed as measures of the culture system’s efficacy. Viability was assessed with Live/Dead stains and BrdU proliferation assays. Differentiation was analyzed with immunocytochemistry (ICC) for markers of endoderm, ectoderm, and mesoderm, as well as ovarian tissue. Hormone synthesis served as an indicator of EB differentiation and functionality. Conditioned media collected over 24 hr interval period was assayed by ELISA for estradiol (E2), progesterone (P4), and testosterone (T) synthesis. We also slow-froze sealed microfluidic cassettes in isopropanol, thawed these, and measured viability and functionality of the EBs. RESULTS: 1. EBs grown in microfluidic cassettes maintain long-term viability and proliferation after 21 days. 2. Differentiation of EBs in the microfluidic system was verified, as shown by ICC of cell markers from all three germ layers and expression of ovarian cell markers (inhibin, Cyp19a1, and AMHR). 3. Functional analyses show increasing synthesis of E2 (15 pg/ml on Day 1 to 31 pg/ml on Day 20). 4. Cryopreserved EB-laden microfluidic chips recovered upon thawing and continued hormone synthesis. CONCLUSION: 1. Microfluidic culture of functional EBs is a promising system that can maintain EB viability, differentiation, and functionality, even after recovery from cryopreservation. 2. This system affords an opportunity to develop patient-specific cassettes of differentiated human ESCs that may be stored, used in drug testing, or harvested for hormones. Supported by: NIH 1 R01 EB015776-01A1 (SG, RMA, UD), Michael Cassidy and Caroline Wang Stem Cell Research Fund (RMA). SEXUALITY P-321 Tuesday, October 21, 2014 ENVIRONMENTAL PHTHALATE EXPOSURE IS ASSOCIATED WITH LOW INTEREST IN SEXUAL ACTIVITY IN PREMENOPAUSAL WOMEN. E. S. Barrett,a L. E. Parlett,b S. H. Swan.c aObstetrics and Gynecology, University of Rochester School of Medicine and Dentistry, Rochester, NY; bEpidemiology, Johns Hopkins University, Baltimore, MD; cPreventive Medicine, Icahn School of Medicine at Mount Sinai, New York, NY. OBJECTIVE: Several studies have found that occupational exposure to high levels of endocrine disrupting chemicals can interfere with sexual

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