- Email: [email protected]
Pluripotent stem cells derived from adult testis tissue after introduction of OCT4, SOX2, KLF4, and C-MYC
STEM CELLS P-286 Tuesday, October 20, 2009 INSULIN-LIKE GROWTH FACTOR-1 IS REQUIRED FOR PLURIPOTENCY OF MOUSE SPERMATOGONIAL STEM CELLS. S.-F. Cheng, H.-N. Ho, C.-R. Tzeng, C.-C. Chin, T.-Y. Ling, Y.-H. Huang. Department of Biochemistry, Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan; Center for Reproductive Medicine, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan; Department of Obstetrics and Gynecology, Division of Endocrinology and Infertility, National Taiwan University and the Hospital, Taipei, Taiwan; Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan. OBJECTIVE: This study aims to identify the novel endocrine factors which regulated the self-renewal and pluripotent of mouse germline stem cells. DESIGN: We established a serum-free testicular niche-stem cell co-culture system to clonally derive pluripotent stem cells from wildtype neonatal mouse testis. In this study, the cell biology, molecular biology, in conjunction of biochemistry were used to identify the endocrinal signaling of IGF-1 in germ cell pluripotency. MATERIALS AND METHODS: The technical supports include primary cell culture, immunostaining, image analysis by confocal microscopy, animal model for stem cell transplantation, cytokine antibody array analysis, and signal tranduction approaches. RESULTS: We successfully generated the pluripotent mouse germline stem cells which presented strong alkaline phosphatase activity (designated as APþ GSCs). In vitro, the APþGSCs were able to differentiate into different cell lineages, including neuron-like, hepatocyte-like, and c-kitþ germ cells. In vivo, the APþGSCs successfully contributed into spermatogenesis and embryonic development. By using dissection of exogenous cytokine factors and cytokine antibody array, we identify that IGF-1 is an important factor in maintenance of APþGSCs self-renewal and pluripotency. Furthermore, we demonstrated that IGF-1and PI3K were involved in GSCs self-renewal/ proliferation by using IGF1R neutralizing assay and specific inhibitors which target on IGF-1R mediated signalings. In addition, testicular Leydig cells and myoid cells serve as niche feeders to support APþGSCs formation. CONCLUSIONS: In this study, we presented a serum-free testicular stroma niche to demonstrate the role of IGF-1/IGF-1R-mediated PI3K/Akt signaling in pluripotency of testicular GSCs. Supported by: This search project was supported by NSC research program NSC97-3111-B-038-001.
and 42.2% (19/45 drops), respectively. The expanded colonies of SSCs/SG in microdrops continued to expand after being transferred to regular culture dishes. CONCLUSIONS: We succeeded in making a limited number of SSCs/SG expand in the small environment of microdrop. This would be particularly useful in future applications when human SSCs need to be expanded from biopsy samples.
P-288 Tuesday, October 20, 2009 EPIGENETIC STABILITY OF HUMAN EMBRYONIC STEM CELLS CAN BE INFLUENCED BY CULTURE CONDITIONS. Y. Choi, J. Ding, G. D. Smith. Obstetrics and Gynecology, Urology, and Physiology, University of Michigan, Ann Arbor, MI. OBJECTIVE: Key properties of pluripotency conferred upon human embryonic stem cells (hESCs) contribute to their promise for regenerative medicine and for modeling early human development. However, derivation and culture conditions of hESCs may induce epigenetic alterations, which can have long lasting effects on gene expression and phenotype. Objectives of these studies were to examine hESC expression of imprinted genes under different culture conditions and states of differentiation. DESIGN: Laboratory study on NIH-approved hESCs. MATERIALS AND METHODS: H9 hESCs were culture on mouse embryonic fibroblasts (MEFs) or Matrigel and differentiated into embryonic bodies (EBs). Gene expression of pluripotency markers, 5 imprinted genes (IGF2, SNRPN, IPW, PEG3, H19), and DNA methyltransferases (DNMTs) were assessed by quantitative real time RT-PCR and DNA methylation assays. Statistics included ANOVA with significance defined at p<0.05. RESULTS: Proliferation and differentiation of hESCs were similar on MEFS and Matrigel and displayed similar expression patterns of pluripotency/differentiation markers and morphology dependent upon state of differentiation. Expression of IGF2, SNRPN and IPW were unstable and significantly increased under differentiation conditions on both matrices. IPW and PEG3 were highly expression during differentiation, and significantly elevated on MEFs, while H19 gene expression on Matrigel showed a significant decreased expression. Moreover, DNMT1, de novo DNMT3a and DNMT3b were significantly increased when hESC differentiation occurred on both conditions, and DNMT3b had different expression patterns on each matrix. CONCLUSIONS: Expression of imprinted genes change dramatically during hESC differentiation. While hESC proliferation and morphology are similar during differentiation on MEF and Matrigel, epigenetic gene expression is different. These studies emphasize importance of culture conditions on epigenetic stability of hESCs.
P-287 Tuesday, October 20, 2009 MICRODROP CAN INCREASE A FEW LIMITED NUMBER OF SPERMATOGONIAL STEM CELLS. Y. Araki, K. Katagiri, T. Ogawa, Y. Araki. The Institute for Advanced Reproductive Medical Technology, Seta, Gunma, Japan; Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan. OBJECTIVE: It recently became possible to expand spermatogonial stem cells (SSCs) of rodents in vitro. Therefore, the cultured cells include not only SSCs but also spermatogonia (SG) at different stages of differentiation. They have been cultured in a dish/well with medium covering the whole base as is the standard practice for other cells. In this case, however, it is difficult to track each single cell or colony of SSCs/SG during the term of the experiments. In this study, we applied a microdrop technique to culture SSCs/SG to see if these cells can grow when started from a limited numbers of cells. DESIGN: Stem cell research. MATERIALS AND METHODS: A few SSCs/SG were collected by micromanipulator and transferred to a microdrop containing mouse embryonic fibroblast (MEF) as a feeder with medium of about 2.5 ml. In experiment (A), a cluster of SSCs/SG of 2-12 cells was transferred into a microdrop. In experiment (B), SSCs/SG dissociated into single cells by trypsin treatment were used. 10 cells, and 20 cells of SSCs/SG were transferred into each microdrop. In both experiments, the cells were cultured for one month in the drops with regular medium changes to investigate the expansion of the cell colonies. RESULTS: In experiment (A), the SSCs/SG colony expanded to the size of about 200-300 cells. The expansion efficiency of SSCs/SG colony was 17.8% (8/45 drops). In experiment (B), in the groups with 10 cells/drop and 20 cells/drop, the expansion efficiencies were 18.3% (11/60 drops)
FERTILITY & STERILITYÒ
P-289 Tuesday, October 20, 2009 PLURIPOTENT STEM CELLS DERIVED FROM ADULT TESTIS TISSUE AFTER INTRODUCTION OF OCT4, SOX2, KLF4, AND C-MYC. H. Kobayashi, K. Nagao, Y. Oka, K. Miura, N. Ishii. Department of Urology, Toho University, School of Medicine, Tokyo, Japan. OBJECTIVE: Spermatogonial stem cells (SSCs) are one of the most important stem cell systems in the body. Recent research has shown that pluripotent stem cells can be generated from adult human testis, and it has been suggested that culture conditions are important for SSCs or precursors after reprogramming of pluripotent stem cells. We examined whether pluripotent stem cells derived from adult testis tissue could be obtained with a specific gene delivery system. DESIGN: Cell culture study. MATERIALS AND METHODS: Testis tissue was collected while examining patients for suspected male infertility and was used in the present research after obtaining informed consent. Tissues were mechanically dissected and dissociated by enzymatic treatment. The cells were incubated with 10% FBS and DMEM for 4 to 5 days. Either OCT4, SOX2, NANOG, and LIN28 or OCT4, SOX2, KLF4, and C-MYC were delivered to testis cells by using a lentiviral vector system. Testis cells were then cultured with 10% FBS and DMEM for 4 days. Then, testis cells were transferred to a mouse embryonic fibroblast feeder layer and incubated with ES medium and basic fibroblast growth factor. After 16 days, a small number of colonies were
S169
visible by microscopy. After 20 days, ES-like colonies were visible. The characteristics of ES-like colonies were investigated. RESULTS: ES-like colonies derived from adult testis tissues were obtained with OCT4, SOX2, KLF4, and C-MYC, but not with OCT4, SOX2, NANOG, and LIN28. Short tandem repeat analysis confirmed that these colonies were derived from testis tissue. The colonies expressed OCT4, NANOG, and STELLA, but did not express the germ cell markers VASA and DAZL. CONCLUSIONS: We found that pluripotent stem cells derived from adult testis tissues could be obtained with OCT4, SOX2, KLF4, and CMYC, and that these cells could be reprogrammed. Obtaining pluripotent stem cells from patients with Klinefelter syndrome or nonobstructive azoospermia may result in an effective treatment for male infertility.
P-290 Tuesday, October 20, 2009 GERM LINE AND PLURIPOTENCY GENE EXPRESSION MARKERS IN ADULT OVARIAN CORTEX. E. Novella-Maestre, J. V. Medrano, M. Sa´nchez-Serrano, J. Teruel, C. Simon, A. Pellicer. Hospital Universitario La Fe, Valencia, Spain; Valencia Stem Cell Bank, Centro de Investigacio´n Principe Felipe, Valencia, Spain; Hospital Universitario Dr. Peset, Valencia, Spain; Instituto Valenciano de Infertilidad (IVI)-Universidad de Valencia, Valencia, Spain. OBJECTIVE: Oocyte population is believed not to be renewed in mammalian ovaries after birth, but rodents experiments have arise a controversy over the existence of stem cells in adult ovaries that extends to humans. The aim of this study was to evaluate the presence/absence of stem cells in adult human ovarian cortex (HOC). DESIGN: HOC biopsies from 14 oncologic patients (24.41.7 yrs) requesting fertility preservation were recovered during laparoscopy. None received oncologic treatment prior HOC extraction. MATERIALS AND METHODS: Germ line (GL: VASA, c-kit) and pluripotency (PL: Oct-4, Nanog, Sox-2) markers expression were analized by real time RT-PCR and 2-DDCt method. For follicular density (FD) optical microscopy and morphometry were employed. Immunofluorescence was used to detect VASA and Nanog expression. RESULTS: Cluster analysis identified 3 groups with significantly different expression of markers.
Up-regulated PL
Up-regulated GL
Down-regulated
22.23.6 1.50.6* 0.70.1* 3.41.4* 4.51.1* 2.50.4* 0.90.5
24.02.9 5.51.5* 3.71.2* 0.70.2* 0.70.1* 1.00.4* 1.130.4
27.52.5 0.20.1* 0.40.1* 0.40.2* 0.40.1* 0.30.1* 0.80.6
* P value < 0.05, Data expressed as Mean SEM There was a trend towards decreased expression of GL (VASA, c-kit) and PL (Nanog, Sox-2) markers as aged increased. Higher GL expression markers in Hodgkin’s HOC (VASA 3.51.3; c-kit 3.11.8) as compared to breast cancer HOC (VASA 0.80.4; c-kit 1.40.6) was observed. VASA and Nanog expression was found in follicles in all cases, but not in the ovarian cortical stroma. CONCLUSIONS: These results show that GL and PL markers expression are conditioned by FD which decreases with age, and suggest that the ovarian reservoir can be affected by the type of disease. There was no evidence to support the hypothesis that GL stem cells may reside in adult HOC.
P-291 Tuesday, October 20, 2009 WITHDRAWN
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Abstracts
FEASIBILITY OF ESTABLISHING A PAID OOCYTE DONATION PROGRAM FOR HUMAN EMBRONIC STEM CELL RESEARCH. L. Li, M. Thornton, M. Sauer. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Columbia University Medical Center, New York, NY. OBJECTIVE: Should women be compensated for donating oocytes for human embryonic stem cell (HESC) research? There has been much debate but relatively little data on this topic. In reality, few women donate eggs altruistically. We aim to identify donor’s views on oocyte donation for HESC research and barriers to donate, and to assess the feasibility of establishing a successful HESC oocyte donation program. DESIGN: Prospective survey. MATERIALS AND METHODS: Women in our donor registry were approached to complete the survey regarding their views on oocyte donation for HESC research and barriers to donate. Data from the survey were used as framework by which we designed the protocol for patient recruitment and compensation for HESC donation, as well as obtained approval and support from the institutional stem cell, ethics, and IRB committees. RESULTS: 447 potential donors were surveyed, making this the largest existing data set on this topic. Our survey showed that the absence of financial compensation, rather than ethical concerns, would be the major deterrent to oocyte donation for HESC research. 79% of donors believed that they should be paid the same for donating to research as for reproduction. 36% indicated that they would donate for research if payment were limited to cost reimbursements and only 1.3% would do so for free. Using this data, we pursued the establishment of a paid oocyte donation program for HESC research. Over the past 6 months, 7 paid donors have completed IVF cycles leading to ooctye retrieval, whereas no donors were recruited during the past 5 years when payments were not offered. Harvest oocytes underwent somatic nuclear transfer. Experiments are still in progress to establish a viable stem cell line. CONCLUSIONS: Our experience shows that providing financial compensation is a feasible method of rapid donor recruitment. We suggest that policy discussions shift focus from whether to pay, to how much would be appropriate; and that education be increased to heighten understanding of these issues.
P-293 Tuesday, October 20, 2009
TABLE 1.
Age VASA c-kit Oct-4 Nanog Sox-2 FD (follicles/mm2)
P-292 Tuesday, October 20, 2009
PARTHENOGENIC ACTIVATION OF DISCARDED IN VITRO MATURED, VITRIFIED AND REWARMED HUMAN OOCYTES FOR STEM CELL PRODUCTION. L. Chuang, J. Barritt, L. Agapova, N. Turovets, L. Grunfeld, A. B. Copperman. Reproductive Medicine Associates of New York, New York, NY; International Stem Cell Corporation, Oceanside, CA; Department of OBGYN and Reproductive Science, Mount Sinai School of Medicine, New York, NY. OBJECTIVE: Oocyte parthenogenic activation (PA) is a potential source of embryonic stem cells. We performed PA on discarded human oocytes that were in vitro matured, vitrified and warmed, to assess the developmental potential of parthenotes for stem cell production. DESIGN: Prospective analysis of parthenogenically activated human oocytes. MATERIALS AND METHODS: Discarded oocytes were obtained from consenting patients. Immature oocytes were cultured in 5% Human Serum Albumin in Quinn’s Cleavage Media for 20 hours to mature in vitro. All mature oocytes were vitrified and warmed using either the Medicult or Irvine Scientific systems (Medicult Vitrification Cooling and Warming kits of Medicult, Denmark; Irvine Scientific Vitrification Freeze and Warm kits of Irvine Scientific, Santa Ana, CA). Oocytes surviving 2 hours post-warming were used for PA. Oocytes were exposed to 5mM calcium ionomysin (CI) then puromycin dihydrochloride (puro, 10mg/ml) or 6-dimethylaminopurine (6DMAP, 1mM). Activated oocytes were cultured in 10% Synthetic Serum Substitute in Quinn’s Cleavage Media. Chi-square analysis was performed. RESULTS: See table.
Vol. 92., No. 3, Supplement, September 2009
and 42.2% (19/45 drops), respectively. The expanded colonies of SSCs/SG in microdrops continued to expand after being transferred to regular culture dishes. CONCLUSIONS: We succeeded in making a limited number of SSCs/SG expand in the small environment of microdrop. This would be particularly useful in future applications when human SSCs need to be expanded from biopsy samples.
P-288 Tuesday, October 20, 2009 EPIGENETIC STABILITY OF HUMAN EMBRYONIC STEM CELLS CAN BE INFLUENCED BY CULTURE CONDITIONS. Y. Choi, J. Ding, G. D. Smith. Obstetrics and Gynecology, Urology, and Physiology, University of Michigan, Ann Arbor, MI. OBJECTIVE: Key properties of pluripotency conferred upon human embryonic stem cells (hESCs) contribute to their promise for regenerative medicine and for modeling early human development. However, derivation and culture conditions of hESCs may induce epigenetic alterations, which can have long lasting effects on gene expression and phenotype. Objectives of these studies were to examine hESC expression of imprinted genes under different culture conditions and states of differentiation. DESIGN: Laboratory study on NIH-approved hESCs. MATERIALS AND METHODS: H9 hESCs were culture on mouse embryonic fibroblasts (MEFs) or Matrigel and differentiated into embryonic bodies (EBs). Gene expression of pluripotency markers, 5 imprinted genes (IGF2, SNRPN, IPW, PEG3, H19), and DNA methyltransferases (DNMTs) were assessed by quantitative real time RT-PCR and DNA methylation assays. Statistics included ANOVA with significance defined at p<0.05. RESULTS: Proliferation and differentiation of hESCs were similar on MEFS and Matrigel and displayed similar expression patterns of pluripotency/differentiation markers and morphology dependent upon state of differentiation. Expression of IGF2, SNRPN and IPW were unstable and significantly increased under differentiation conditions on both matrices. IPW and PEG3 were highly expression during differentiation, and significantly elevated on MEFs, while H19 gene expression on Matrigel showed a significant decreased expression. Moreover, DNMT1, de novo DNMT3a and DNMT3b were significantly increased when hESC differentiation occurred on both conditions, and DNMT3b had different expression patterns on each matrix. CONCLUSIONS: Expression of imprinted genes change dramatically during hESC differentiation. While hESC proliferation and morphology are similar during differentiation on MEF and Matrigel, epigenetic gene expression is different. These studies emphasize importance of culture conditions on epigenetic stability of hESCs.
P-287 Tuesday, October 20, 2009 MICRODROP CAN INCREASE A FEW LIMITED NUMBER OF SPERMATOGONIAL STEM CELLS. Y. Araki, K. Katagiri, T. Ogawa, Y. Araki. The Institute for Advanced Reproductive Medical Technology, Seta, Gunma, Japan; Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan. OBJECTIVE: It recently became possible to expand spermatogonial stem cells (SSCs) of rodents in vitro. Therefore, the cultured cells include not only SSCs but also spermatogonia (SG) at different stages of differentiation. They have been cultured in a dish/well with medium covering the whole base as is the standard practice for other cells. In this case, however, it is difficult to track each single cell or colony of SSCs/SG during the term of the experiments. In this study, we applied a microdrop technique to culture SSCs/SG to see if these cells can grow when started from a limited numbers of cells. DESIGN: Stem cell research. MATERIALS AND METHODS: A few SSCs/SG were collected by micromanipulator and transferred to a microdrop containing mouse embryonic fibroblast (MEF) as a feeder with medium of about 2.5 ml. In experiment (A), a cluster of SSCs/SG of 2-12 cells was transferred into a microdrop. In experiment (B), SSCs/SG dissociated into single cells by trypsin treatment were used. 10 cells, and 20 cells of SSCs/SG were transferred into each microdrop. In both experiments, the cells were cultured for one month in the drops with regular medium changes to investigate the expansion of the cell colonies. RESULTS: In experiment (A), the SSCs/SG colony expanded to the size of about 200-300 cells. The expansion efficiency of SSCs/SG colony was 17.8% (8/45 drops). In experiment (B), in the groups with 10 cells/drop and 20 cells/drop, the expansion efficiencies were 18.3% (11/60 drops)
FERTILITY & STERILITYÒ
P-289 Tuesday, October 20, 2009 PLURIPOTENT STEM CELLS DERIVED FROM ADULT TESTIS TISSUE AFTER INTRODUCTION OF OCT4, SOX2, KLF4, AND C-MYC. H. Kobayashi, K. Nagao, Y. Oka, K. Miura, N. Ishii. Department of Urology, Toho University, School of Medicine, Tokyo, Japan. OBJECTIVE: Spermatogonial stem cells (SSCs) are one of the most important stem cell systems in the body. Recent research has shown that pluripotent stem cells can be generated from adult human testis, and it has been suggested that culture conditions are important for SSCs or precursors after reprogramming of pluripotent stem cells. We examined whether pluripotent stem cells derived from adult testis tissue could be obtained with a specific gene delivery system. DESIGN: Cell culture study. MATERIALS AND METHODS: Testis tissue was collected while examining patients for suspected male infertility and was used in the present research after obtaining informed consent. Tissues were mechanically dissected and dissociated by enzymatic treatment. The cells were incubated with 10% FBS and DMEM for 4 to 5 days. Either OCT4, SOX2, NANOG, and LIN28 or OCT4, SOX2, KLF4, and C-MYC were delivered to testis cells by using a lentiviral vector system. Testis cells were then cultured with 10% FBS and DMEM for 4 days. Then, testis cells were transferred to a mouse embryonic fibroblast feeder layer and incubated with ES medium and basic fibroblast growth factor. After 16 days, a small number of colonies were
S169
visible by microscopy. After 20 days, ES-like colonies were visible. The characteristics of ES-like colonies were investigated. RESULTS: ES-like colonies derived from adult testis tissues were obtained with OCT4, SOX2, KLF4, and C-MYC, but not with OCT4, SOX2, NANOG, and LIN28. Short tandem repeat analysis confirmed that these colonies were derived from testis tissue. The colonies expressed OCT4, NANOG, and STELLA, but did not express the germ cell markers VASA and DAZL. CONCLUSIONS: We found that pluripotent stem cells derived from adult testis tissues could be obtained with OCT4, SOX2, KLF4, and CMYC, and that these cells could be reprogrammed. Obtaining pluripotent stem cells from patients with Klinefelter syndrome or nonobstructive azoospermia may result in an effective treatment for male infertility.
P-290 Tuesday, October 20, 2009 GERM LINE AND PLURIPOTENCY GENE EXPRESSION MARKERS IN ADULT OVARIAN CORTEX. E. Novella-Maestre, J. V. Medrano, M. Sa´nchez-Serrano, J. Teruel, C. Simon, A. Pellicer. Hospital Universitario La Fe, Valencia, Spain; Valencia Stem Cell Bank, Centro de Investigacio´n Principe Felipe, Valencia, Spain; Hospital Universitario Dr. Peset, Valencia, Spain; Instituto Valenciano de Infertilidad (IVI)-Universidad de Valencia, Valencia, Spain. OBJECTIVE: Oocyte population is believed not to be renewed in mammalian ovaries after birth, but rodents experiments have arise a controversy over the existence of stem cells in adult ovaries that extends to humans. The aim of this study was to evaluate the presence/absence of stem cells in adult human ovarian cortex (HOC). DESIGN: HOC biopsies from 14 oncologic patients (24.41.7 yrs) requesting fertility preservation were recovered during laparoscopy. None received oncologic treatment prior HOC extraction. MATERIALS AND METHODS: Germ line (GL: VASA, c-kit) and pluripotency (PL: Oct-4, Nanog, Sox-2) markers expression were analized by real time RT-PCR and 2-DDCt method. For follicular density (FD) optical microscopy and morphometry were employed. Immunofluorescence was used to detect VASA and Nanog expression. RESULTS: Cluster analysis identified 3 groups with significantly different expression of markers.
Up-regulated PL
Up-regulated GL
Down-regulated
22.23.6 1.50.6* 0.70.1* 3.41.4* 4.51.1* 2.50.4* 0.90.5
24.02.9 5.51.5* 3.71.2* 0.70.2* 0.70.1* 1.00.4* 1.130.4
27.52.5 0.20.1* 0.40.1* 0.40.2* 0.40.1* 0.30.1* 0.80.6
* P value < 0.05, Data expressed as Mean SEM There was a trend towards decreased expression of GL (VASA, c-kit) and PL (Nanog, Sox-2) markers as aged increased. Higher GL expression markers in Hodgkin’s HOC (VASA 3.51.3; c-kit 3.11.8) as compared to breast cancer HOC (VASA 0.80.4; c-kit 1.40.6) was observed. VASA and Nanog expression was found in follicles in all cases, but not in the ovarian cortical stroma. CONCLUSIONS: These results show that GL and PL markers expression are conditioned by FD which decreases with age, and suggest that the ovarian reservoir can be affected by the type of disease. There was no evidence to support the hypothesis that GL stem cells may reside in adult HOC.
P-291 Tuesday, October 20, 2009 WITHDRAWN
S170
Abstracts
FEASIBILITY OF ESTABLISHING A PAID OOCYTE DONATION PROGRAM FOR HUMAN EMBRONIC STEM CELL RESEARCH. L. Li, M. Thornton, M. Sauer. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Columbia University Medical Center, New York, NY. OBJECTIVE: Should women be compensated for donating oocytes for human embryonic stem cell (HESC) research? There has been much debate but relatively little data on this topic. In reality, few women donate eggs altruistically. We aim to identify donor’s views on oocyte donation for HESC research and barriers to donate, and to assess the feasibility of establishing a successful HESC oocyte donation program. DESIGN: Prospective survey. MATERIALS AND METHODS: Women in our donor registry were approached to complete the survey regarding their views on oocyte donation for HESC research and barriers to donate. Data from the survey were used as framework by which we designed the protocol for patient recruitment and compensation for HESC donation, as well as obtained approval and support from the institutional stem cell, ethics, and IRB committees. RESULTS: 447 potential donors were surveyed, making this the largest existing data set on this topic. Our survey showed that the absence of financial compensation, rather than ethical concerns, would be the major deterrent to oocyte donation for HESC research. 79% of donors believed that they should be paid the same for donating to research as for reproduction. 36% indicated that they would donate for research if payment were limited to cost reimbursements and only 1.3% would do so for free. Using this data, we pursued the establishment of a paid oocyte donation program for HESC research. Over the past 6 months, 7 paid donors have completed IVF cycles leading to ooctye retrieval, whereas no donors were recruited during the past 5 years when payments were not offered. Harvest oocytes underwent somatic nuclear transfer. Experiments are still in progress to establish a viable stem cell line. CONCLUSIONS: Our experience shows that providing financial compensation is a feasible method of rapid donor recruitment. We suggest that policy discussions shift focus from whether to pay, to how much would be appropriate; and that education be increased to heighten understanding of these issues.
P-293 Tuesday, October 20, 2009
TABLE 1.
Age VASA c-kit Oct-4 Nanog Sox-2 FD (follicles/mm2)
P-292 Tuesday, October 20, 2009
PARTHENOGENIC ACTIVATION OF DISCARDED IN VITRO MATURED, VITRIFIED AND REWARMED HUMAN OOCYTES FOR STEM CELL PRODUCTION. L. Chuang, J. Barritt, L. Agapova, N. Turovets, L. Grunfeld, A. B. Copperman. Reproductive Medicine Associates of New York, New York, NY; International Stem Cell Corporation, Oceanside, CA; Department of OBGYN and Reproductive Science, Mount Sinai School of Medicine, New York, NY. OBJECTIVE: Oocyte parthenogenic activation (PA) is a potential source of embryonic stem cells. We performed PA on discarded human oocytes that were in vitro matured, vitrified and warmed, to assess the developmental potential of parthenotes for stem cell production. DESIGN: Prospective analysis of parthenogenically activated human oocytes. MATERIALS AND METHODS: Discarded oocytes were obtained from consenting patients. Immature oocytes were cultured in 5% Human Serum Albumin in Quinn’s Cleavage Media for 20 hours to mature in vitro. All mature oocytes were vitrified and warmed using either the Medicult or Irvine Scientific systems (Medicult Vitrification Cooling and Warming kits of Medicult, Denmark; Irvine Scientific Vitrification Freeze and Warm kits of Irvine Scientific, Santa Ana, CA). Oocytes surviving 2 hours post-warming were used for PA. Oocytes were exposed to 5mM calcium ionomysin (CI) then puromycin dihydrochloride (puro, 10mg/ml) or 6-dimethylaminopurine (6DMAP, 1mM). Activated oocytes were cultured in 10% Synthetic Serum Substitute in Quinn’s Cleavage Media. Chi-square analysis was performed. RESULTS: See table.
Vol. 92., No. 3, Supplement, September 2009