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Fetal membranes as a source of multipotent stem cells
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Abstracts / Placenta 45 (2016) 63e133
Fig. 1. Selection of regions of interests. The images sites were constrained to midway between the chorionic and basal plates, an optimal transport location for maternal blood flow.
extremely difficult to maintain in culture which motivated investigators to generate villous and extravillous trophoblastic cell lines. Nevertheless, when aspiring to extrapolate the results they obtain in the trophoblastic cell lines to the trophoblasts in the placenta, the scientific community must be cautious as to whether these cell lines are truly representative of the physiologic setting. Aim: Our study raises questions regarding the validity of using the choriocarcinoma cell lines (BeWo, JEG-3, JAR) vs. the extravillous cell line (HTR-8/SVneo) as in vitro model systems for human trophoblast studies. Methods: Immunofluorescence staining was used to investigate the expression of CK7, e-cadherin and vimentin in BeWo, JEG-3, JAR and HTR8/SVneo cell lines. RT-PCR allowed the measurement of mRNA levels of CK7, epCAM and vimentin in BeWo, JEG-3, JAR and HTR-8/SVneo while western blots assessed the protein expression of CK7, e-cadherin and vimentin in BeWo, JEG-3, JAR and HTR-8/SVneo. Results: BeWo, JEG-3 and JAR cell lines all expressed CK7, e-cadherin and epCAM and did not express vimentin while HTR-8/SVneo cell line showed lower expression of CK7 and e-cadherin and expressed vimentin. Additionally, two populations were observed in HTR-8/SVneo cell line: a CK7+/ Vimentin- population vs. CK7-/Vimentin+ population. Conclusion: Our study indicated that even though BeWo, JEG-3 and JAR cell lines have proved to be epithelial trophoblastic cell lines, HTR-8/SVneo has been disqualified in that regard and results based on this cell line should be controlled in primary trophoblast models. P2.10 FETAL MEMBRANES AS A SOURCE OF MULTIPOTENT STEM CELLS ssica Borghesi, Nikolas Waak, Maria Angelica Phelipe Oliveira Favaron, Je Miglino. School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
Fig. 2. Original and post-processed images. a) Pre-processed image with a scale bar denoting 600mm. b) Post-processed image after background subtraction and holefilling.
P2.9 ASSESSMENT OF DIFFERENT TROPHOBLAST CELL LINES AS IN VITRO MODELS FOR PLACENTAL DEVELOPMENT Joanna Barrak, Hiba Msheik, Wassim Abou-Kheir, Daoud. American University of Beirut, Beirut, Lebanon
Fetal membranes are regarded to be abundant, ethically acceptable and easily accessible sources of stem cells. Especially the yolk sac (YS) and aminiotic membrane (AM) have been investigate in this field, and promising results have been obtained for several lineages of progenitor mesenchymal stem cells (MSCs) that cause only minor immunogenic troubles. In this context, for sharing with humans several characteristics related to placentation, special attention has been given to rodents and its relatives. Objectives: We verified and compared the immunophenotyping characteristics of YS and AM MSCs from rodent species and lagomorph, as well as the potential of cell differentiation. Methods: YS explants from Necromys lasiurus (Sigmodontinae, n¼10) and Cavia porcellus (Caviidae, guinea pig, n¼5), and AM from Oryctolagus cuniculus (rabbit, n¼5), all from mid-gestation were maintained in DMEM-High glucose medium with 10% fetal bovine serum. The cells were characterized by morphological analysis, immunophenotyping by flow citometry and immunofluorescence, cell differentiation and tumorigenicity assay. Results: The YS cultures were composed by fibroblast and epithelial-like cells, in contrast to the AM for which only fibroblast-like cells were identified. Fibroblast-like cells were small, elongated and had reduced cytoplasm, whereas the epithelial cells were large and rounded in shape with sparse cytoplasm and centrally nucleus. Both, YS and AM cells were positive to MSC (Stro1, CD90, CD105, and CD73), pluripotency (Oct4 and Nanog), and hematopoietic precursor cells (CD117) markers. In vitro, they showed successful osteogenic, adipogenic, and chondrogenic differentiation and did not develop tumors. Conclusion: Although YS and AM membranes are able to generate MSCs with satisfactory characteristics of growth, expansion, and in vitro differentiation, from the AM we were able to obtain a homogeneous cell culture. These cells resulted as potential stem cells with promising therapeutic values for cell therapy. Supported by FAPESP (Proc. 2012/51864-2 and 2009/53392-8).
Georges
Background: The placenta is a temporary organ of pregnancy that is involved in insuring optimal growth and development of the fetus. Since the 1980s, there has been a growing interest in the isolation of villous trophoblasts from human placenta for primary culture. Although very interesting, isolated primary trophoblasts have the disadvantage of being
P2.11 FATTY ACID AND LIPID PROFILES IN PRIMARY HUMAN TROPHOBLAST OVER 90 HOURS IN CULTURE Mickael C. Rudolph 2, Thomas Veronique Ferchaud-Roucher 1, 1 3 1 Jansson , Theresa L. Powell . Department of Obstetrics & Gynecology, Division of Reproductive Sciences, University of Colorado Anschutz Medical
Abstracts / Placenta 45 (2016) 63e133
Fig. 1. Selection of regions of interests. The images sites were constrained to midway between the chorionic and basal plates, an optimal transport location for maternal blood flow.
extremely difficult to maintain in culture which motivated investigators to generate villous and extravillous trophoblastic cell lines. Nevertheless, when aspiring to extrapolate the results they obtain in the trophoblastic cell lines to the trophoblasts in the placenta, the scientific community must be cautious as to whether these cell lines are truly representative of the physiologic setting. Aim: Our study raises questions regarding the validity of using the choriocarcinoma cell lines (BeWo, JEG-3, JAR) vs. the extravillous cell line (HTR-8/SVneo) as in vitro model systems for human trophoblast studies. Methods: Immunofluorescence staining was used to investigate the expression of CK7, e-cadherin and vimentin in BeWo, JEG-3, JAR and HTR8/SVneo cell lines. RT-PCR allowed the measurement of mRNA levels of CK7, epCAM and vimentin in BeWo, JEG-3, JAR and HTR-8/SVneo while western blots assessed the protein expression of CK7, e-cadherin and vimentin in BeWo, JEG-3, JAR and HTR-8/SVneo. Results: BeWo, JEG-3 and JAR cell lines all expressed CK7, e-cadherin and epCAM and did not express vimentin while HTR-8/SVneo cell line showed lower expression of CK7 and e-cadherin and expressed vimentin. Additionally, two populations were observed in HTR-8/SVneo cell line: a CK7+/ Vimentin- population vs. CK7-/Vimentin+ population. Conclusion: Our study indicated that even though BeWo, JEG-3 and JAR cell lines have proved to be epithelial trophoblastic cell lines, HTR-8/SVneo has been disqualified in that regard and results based on this cell line should be controlled in primary trophoblast models. P2.10 FETAL MEMBRANES AS A SOURCE OF MULTIPOTENT STEM CELLS ssica Borghesi, Nikolas Waak, Maria Angelica Phelipe Oliveira Favaron, Je Miglino. School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil
Fig. 2. Original and post-processed images. a) Pre-processed image with a scale bar denoting 600mm. b) Post-processed image after background subtraction and holefilling.
P2.9 ASSESSMENT OF DIFFERENT TROPHOBLAST CELL LINES AS IN VITRO MODELS FOR PLACENTAL DEVELOPMENT Joanna Barrak, Hiba Msheik, Wassim Abou-Kheir, Daoud. American University of Beirut, Beirut, Lebanon
Fetal membranes are regarded to be abundant, ethically acceptable and easily accessible sources of stem cells. Especially the yolk sac (YS) and aminiotic membrane (AM) have been investigate in this field, and promising results have been obtained for several lineages of progenitor mesenchymal stem cells (MSCs) that cause only minor immunogenic troubles. In this context, for sharing with humans several characteristics related to placentation, special attention has been given to rodents and its relatives. Objectives: We verified and compared the immunophenotyping characteristics of YS and AM MSCs from rodent species and lagomorph, as well as the potential of cell differentiation. Methods: YS explants from Necromys lasiurus (Sigmodontinae, n¼10) and Cavia porcellus (Caviidae, guinea pig, n¼5), and AM from Oryctolagus cuniculus (rabbit, n¼5), all from mid-gestation were maintained in DMEM-High glucose medium with 10% fetal bovine serum. The cells were characterized by morphological analysis, immunophenotyping by flow citometry and immunofluorescence, cell differentiation and tumorigenicity assay. Results: The YS cultures were composed by fibroblast and epithelial-like cells, in contrast to the AM for which only fibroblast-like cells were identified. Fibroblast-like cells were small, elongated and had reduced cytoplasm, whereas the epithelial cells were large and rounded in shape with sparse cytoplasm and centrally nucleus. Both, YS and AM cells were positive to MSC (Stro1, CD90, CD105, and CD73), pluripotency (Oct4 and Nanog), and hematopoietic precursor cells (CD117) markers. In vitro, they showed successful osteogenic, adipogenic, and chondrogenic differentiation and did not develop tumors. Conclusion: Although YS and AM membranes are able to generate MSCs with satisfactory characteristics of growth, expansion, and in vitro differentiation, from the AM we were able to obtain a homogeneous cell culture. These cells resulted as potential stem cells with promising therapeutic values for cell therapy. Supported by FAPESP (Proc. 2012/51864-2 and 2009/53392-8).
Georges
Background: The placenta is a temporary organ of pregnancy that is involved in insuring optimal growth and development of the fetus. Since the 1980s, there has been a growing interest in the isolation of villous trophoblasts from human placenta for primary culture. Although very interesting, isolated primary trophoblasts have the disadvantage of being
P2.11 FATTY ACID AND LIPID PROFILES IN PRIMARY HUMAN TROPHOBLAST OVER 90 HOURS IN CULTURE Mickael C. Rudolph 2, Thomas Veronique Ferchaud-Roucher 1, 1 3 1 Jansson , Theresa L. Powell . Department of Obstetrics & Gynecology, Division of Reproductive Sciences, University of Colorado Anschutz Medical