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Mapping of the human amniotic membrane
Università degli studi di Chieti
Medicine and Aging Sciences, Via dei Vestini, 31, G. d’Annunzio University of Chieti-Pescara, 66013, Madonna delle Piane (CH), Italy
Roberta Di Pietro
Roberta Di Pietro
English, Italian, Spanish, Portuguese, French
Type of Research Project
- Basic science
What is the background of the project?
The human placenta is a discoid-shaped organ made up of both fetal and maternal components. The fetal components include the placental disc, the amniotic and chorionic membranes (often referred to as the fetal membranes), the chorionic villi, and the umbilical cord; the maternal side is comprised of the decidua. The human placenta is an important source of stem cells that can be easily collected without ethical concerns since it is usually discarded after childbirth. Of particular interest is the human amniotic membrane (AM) that has a long history of clinical applications. The AM has proven to be a precious resource for tissue engineering and has been shown to possess anti-inflammatory properties and low immunogenicity, making it an attractive and widely studied tissue for diverse therapeutic applications. More recently, decellularized AM has been employed for postinfarct left ventricular remodeling and for the repair of an oroantral communication, thus generating new questions as to the therapeutic potential of the different regions and/or cells in the AM. In spite of the number of clinical studies, only a handful of reports have characterized and studied the phenotype of the cells comprising the AM in situ.
What is the aim of the project?
The aim of this project is to identify the presence of different cell populations of amniotic epithelial cells (AECs) within different regions of human amniotic membrane obtained from healthy women in order to map the region/regions where AECs express the highest stemness potential/characteristics useful for regenerative medicine purposes.
What techniques and methods are used?
1. Collection of human term placentas from healthy women after vaginal delivery or caesarean section at the at the SS. Annunziata Hospital, Chieti. Samples will be collected after obtaining informed written consent according to the guidelines set by the Ethics Committee for the G. d’Annunzio University of Chieti–Pescara. 2. Dissection of the placentas to isolate samples of AM from 4 different regions according to their position relative to the umbilical cord. 3. Preparation of samples for electron microscopy analysis. Samples will be fixed with 2.5% or 1.25% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4, for 2 to 3 h, at 4°C. After washing in cacodylate buffer samples will be postfixed in 1% osmium tetroxide for 1 to 2 h, at 4°C. After dehydration in increasing alcohol gradients, samples will be embedded in Spurr or Unicryl resin and cut at the ultramicrotome. Semithin sections will be stained with 1% toluidine blue for light microscopy analysis or with uranyl acetate and lead citrate for electron microscopy analysis. Observations will be carried out under a ZEISS Axioskop 40 (Carl Zeiss) light microscope equipped with a Coolsnap videocamera (Photometrics) or a ZEISS EM 108 equipped with a Gatan videocamera. 4. Preparation of samples for immunofluorescence. Samples of AM will be transferred to 30% sucrose solution in PBS (pH 7.4) for 24 h. The samples will be embedded in Shandon Cryomatrix-TM placed in cryo-molds by flash freezing on dry ice. Leica CM3050S cryostat (Leica, Wetzlar) will be used to obtain 7 μm thick sections of the embedded tissues. Routine hematoxylin/eosin (H&E) as well as Masson trichrome staining will be performed on serial frozen sections to analyze morphological details. Light microscopy images will be obtained using a ZEISS Axioskop 40 (Carl Zeiss) light microscope, equipped with a Coolsnap videocamera (Photometrics). MetaMorph Software System (Universal Imaging Inc., Bedford Hills, NY, USA; Molecular Devices Sunnyvale, CA, USA; Crisel Instruments, Rome, Italy) will be used to acquire, visualize, and analyze digital images. 5. Detection of the expression of different markers of pluripotency and proliferation (TROP1, TROP2, CREB, pCREB, OCT3/4 etc.) of the 4 amniotic membrane regions by means of immunofluorescence and immunohistochemistry techniques. 6. Data analysis will be performed with Metamorph image analysis software and Excel statistic analysis.
What is the role of the student?
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The student will be involved in the collection of the human placentas at the S.S. Annunziata Hospital, Chieti and in the isolation of the different regions of the human amniotic membrane from the collected placentas. The student will be able to prepare solutions and samples of human amniotic membrane for light and electron microscopy analysis. He/she will observe the cutting of samples with the cryostat or ultramicrotome and analyse them under light and electron microscope with the help of an older student as a tutor assistant. He/she will be involved in image and data analysis if there will be any.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
The tutor will personally introduce the topic and provide materials to the student before starting lab practice in the presence of PhD students expert in the field. Each Monday we will have a lab meeting to design experiments, discuss data and supervise the work done or observed by the student. The student will be invited to participate at all the relevant seminars and meetings that will be organized during the stage by our University, Department, School of Medicine and Health Sciences, School of Doctorate.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Knowledge of English language (and if possibile Italian language) Precision Patience Subjects passed: histology, anatomy, biochemistry Previous experience with: laboratories of basic research
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system
- Mapping of the Human Placenta: Experimental Evidence of Amniotic Epithelial Cell Heterogeneity. Centurione L; Passaretta F; Centurione MA; Munari S; Vertua E; Silini A; Liberati M; Parolini O; Di Pietro R. Cell Transplant. 2018 Jan;27(1):12-22. doi: 10.1177/0963689717725078.
- Shaping the Future of Perinatal Cells: Lessons From the Past and Interpretations of the Present. Silini AR; Masserdotti A; Papait A; Parolini O. Front Bioeng Biotechnol. 2019 Apr 10;7:75. doi: 10.3389/fbioe.2019.00075. eCollection 2019.
- Placental Cells and Derivatives: Advancing Clinical Translation. Silini AR; Parolini O. Cell Transplant. 2018 Jan;27(1):1-2. doi: 10.1177/0963689717745332.
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