key: cord-1008381-bl4zoxj8 authors: Arcuri, Sharon; Gandolfi, Fulvio; Somigliana, Edgardo; Brevini, Tiziana A. L. title: A Two-Step Protocol to Erase Human Skin Fibroblasts and Convert Them into Trophoblast-like Cells date: 2021-02-19 journal: Next Generation Culture Platforms for Reliable In Vitro Models DOI: 10.1007/978-1-0716-1246-0_10 sha: 67f8f81e1fb9357beb3cb6d57ff4cf554ea065b4 doc_id: 1008381 cord_uid: bl4zoxj8 The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial component of the placenta and supports the fetus during intrauterine life. Our understanding of these events is limited, particularly in human, because of ethical and legal restrictions and availability of adequate in vitro models would be very advantageous. Here we describe a method that converts human fibroblasts into trophoblast-like cells, combining the use of 5-azacytidine-CR (5-aza-CR) to erase the original cell phenotype and a cocktail containing bone morphogenetic protein 4 (BMP4) with inhibitors of the Activin/Nodal/ERK signaling pathways, to drive erased fibroblasts into the trophoblastic differentiation. This innovative method uses very easily accessible cells to derive trophoblast-like cells and it can be useful to study embryo implantation disorders related to aging. In mammals, trophectoderm is the progenitor tissue of the entire outer epithelial component of the placenta that supports the fetus during intrauterine life. Its primary function is to allow implant of the embryo to the uterine wall, to protect the fetus from immunological response, to secrete hormones for pregnancy maintenance and to allow gasses and nutrient exchange [1] . Trophectoderm is composed by two subpopulations: villous cytotrophoblast that layers the basement membrane surrounding placental villi and syncytiotrophoblast, a cell population that makes direct contact with maternal blood and is characterized by production of chorionic gonadotropin (CG) and other placental hormones [2] . Although placental dysfunction seems to be the main disorder of pregnancy, with immediate consequences for the mother and child, aging is also one of the main causes of infertility. In this perspective, it comes as no surprise that the percentage of infertility has increased dramatically in recent years, given the fact that women's empowerment causes the postponement of motherhood, with an increase in placental and embryonic implantation defect. Recent evidence has shown that aged uterine environment is decisively involved, causing a reduced uterus decidualization and increased severe placentation defects resulting in abnormal embryonic development [3] . At the moment, only few in vitro models are available to study the main mechanisms involved in reproductive disorders and, all of them, are obtained from embryonic stem cells (ESCs). In particular, Roberts et al. have demonstrated that human pluripotent stem cells (hPSCs), bone morphogenetic protein 4 (BMP4) and hypoxic environment allow to derive trophoblast cells [2] ; Wang et al. have described that hESC seems to be a robust model to acquire trophoblast [4] and Turco et al. have highlighted how hESC together with BMP4, 3D matrix and hypoxic environment permits to obtain trophoblast organoids [5] . Here we describe a model that uses a two-step protocol to derive trophoblast-like cells (Fig. 1) . Step 1 involves the use of epigenetic erasing to drive human adult somatic cells into, into a high plasticity state [6, 7] . Step 2 takes advantage of this induced high plasticity window and uses a cocktail of trophoblast inducers to encourage the acquisition of the trophoblastic phenotype [8] (Fig. 2) . More in details, Step 2 is based on the use of mouse embryonic fibroblasts (MEF) conditioned medium combined with a cocktail of BMP4 and inhibitors of the [2, 4] . This method allows the generation of trophoblast cells from easily accessible cells, such as dermal fibroblasts that can be simply propagated in vitro. It is free of any genetic modifications that make cells prone to instability and transformation. Because of their stable phenotype, the cells generated with this procedure are more easily applied in regenerative medicine. The in vitro model generated is efficient and reproducible. It can be used for the acquisition of useful information on the pathogenesis of developmental disorders based on trophoblast defects and aging, as well as drug discovery and regenerative medicine. 3. Tri-gas incubator. 4. Trophoblast induction medium: 10 mL of conditioned MEF inactivated medium, 10 μL of BMP4 (50 ng/mL), 10 μL A83-01 (1 μM), and 10 μL PD173074 (0.1 μM). 1. Thaw a cryo-vial containing MEFs using a 37 C water bath. 2. Add 2 mL of fibroblast culture medium in a 15 mL sterile tube. 3. Transfer MEFs in the 15 mL sterile tube containing the 2 mL of fibroblast culture medium. 4. Centrifuge at 300 Â g for 5 min. 5. Resuspend the pellet in 2 mL of fibroblast culture medium. 6. Seed in a 35 mm petri dish and incubate at 37 C in CO 2 incubator. 7. Change fibroblast culture medium every day. 8. When they reach confluency, remove culture medium from the dish. 9. Wash three times in sterile PBS supplemented with 1% antibiotic-antimycotic solution. 10. Add 500 μL of trypsin-EDTA solution and incubate at 37 C until cell monolayer begins to detach from the bottom of the tissue culture dish and cells dissociate. 11. Neutralize trypsin-EDTA using 1.5 mL of MEF culture medium. 12. Dislodge cells by repeatedly and gently pipetting. 13. Plate cells in a new culture dish using a 1:2 passage ratio and culture at 37 C in 5% CO 2 incubator. 14. Change fibroblast culture medium every day and passage every 2 days. 18. Collect conditioned medium, filter using 0.20 μm filter and storage at À20 C until use. 1. Isolate skin biopsy and transfer it in a 50 mL sterile tube containing sterile PBS with 2% antibiotics. 2. Prepare 2 mL of 0.1% of porcine gelatin in a 35 mm petri dish. Wait 45 min to coat the surface at room temperature. 3. Wash the biopsy in sterile PBS and cut it in small fragments of approximately 2 mm 3 (see Note 1). 5. Add 20 μL of fibroblast culture medium onto each fragment and incubate at 37 C in CO 2 incubator. 6. Add around 20-50 μL of fibroblast culture medium daily for the first 5 days. 7. Add 1 mL in each petri dish and culture until cells reach confluency. 8. When cells reach confluency, trypsinize (see Subheading 3.1, steps 9-12) and transfer cells in a new T25 flask. 9. Incubate at 37 C. 10. Passage every 3 days with a ratio of 1:2. 1. Add 0.5 mL of 0.1% of porcine gelatin in each well of 4-well multidish and wait 20 min to coat the surface at room temperature. 2. Eliminate gelatin and let it dry for 45 min. 3. Remove the medium from T25 flask, and ten trypsinized using 0.6 mL of Trypsin-EDTA (see Subheading 3.1, steps 9-12) (see Note 2). 4. Collect cell suspension in a 15 mL sterile tube. 5. Count cells using a counting chamber under an optical microscope at room temperature. Calculate the volume of medium needed to resuspend cells in order to obtain 7.8 Â 10 4 cells/ cm 2 . 6. Centrifuge cells at 300 Â g for 5 min. 7. Resuspend the pellet in a calculated volume by pipetting carefully. 8. Plate cells in 4-well multidish. 9. Incubate at 37 C for 24 h. 3. Prepare 5-aza-CR solution just before use. 5. It is very important to add differentiation factors just before use. Dilute 1 μL of 5-aza-CR stock solution in 1 mL of fibroblast culture medium Add 0.5 mL of fibroblast culture medium containing 5-aza-CR in three wells. Use one as a negative control Incubate at 37 C for 18 h in CO 2 incubator Remove 5-aza-CR Rinse cells three times in sterile PBS and add 0.5 mL of ESC medium for 3 h at 37 C in CO 2 incubator Trophoblastic Induction 1. Rinse cells three times in sterile PBS Expose cells to the trophoblast induction medium (see Notes 4-5) and incubate in a hypoxic environment (5% O 2 ) in the tri-gas incubator for 11 days is crucial to cut very small pieces of skin tissue in order to facilitate fibroblasts to grow out of the tissue fragments Before seeding cells for 5-aza-CR treatment, it is important that they are in subconfluency Human placenta and trophoblast development: key molecular mechanisms and model systems Human embryonic stem cells as models for trophoblast differentiation Introduction: the trophoblast lineage and its emergence Infertility around the globe: new thinking on gender, reproductive technologies and global movements in the 21st century In vitro differentiation of human mesenchymal stem cells into functional cardiomyocyte-like cells Trophoblast organoids as a model for maternal-fetal interactions during human placentation Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulinproducing cells are boosted by the use of low-stiffness substrate 5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts Paracrine and epigenetic control of Trophectoderm differentiation from human embryonic stem cells: the role of bone Morphogenic protein 4 and histone deacetylases