Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-β/activin/nodal signalling inhibition

Koel, Mariann; Võsa, Urmo; Krjutškov, Kaarel; Einarsdottir, Elisabet; Kere, Juha; Tapanainen, Juha S.; Katayama, Shintaro; Ingerpuu, Sulev; Jaks, Viljar; Stenman, Ulf‐Håkan; Lundin, Karolina; Tuuri, Timo; Salumets, Andres

doi:10.1016/j.rbmo.2017.06.003

Cited by 14 publications

(8 citation statements)

References 33 publications

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“…On the other hand, taking advantage of the acquired high plasticity state, miR-200reprogrammed cells can be driven towards the TR lineage using an ad hoc induction protocol that favors the acquisition of a tight adherent epithelial morphology with a round shape and well-defined borders in both young and aged cells. This is consistent with previous observations that described the differentiation of human iPSCs and epigenetically converted cells towards the TR lineage with the same induction cocktail, containing BMP4, activin/nodal-, and FGF2-signaling inhibitors [24,27,[43][44][45][46][47][48][49][50][51][52], and confirms that, when present at adequate concentrations, these molecules are able to coax cells unidirectionally into the TR phenotype [27,49,53,54]. The activation of the molecular pathways distinctive of the newly acquired differentiation is also demonstrated by the active transcription of mature TR-related markers, such as GCM1, KRT19, PGF, CYP11A1, CGA, ESRRB, CGB, and HSD17B1, as well as by KRT19 immunopositivity which was detected in all TR-like cells, regardless of the donor's age.…”

Section: Discussionsupporting

confidence: 93%

Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues

Pennarossa,

Arcuri,

Gandolfi

et al. 2024

Cells

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In vitro-generated blastocyst-like structures are of great importance since they recapitulate specific features or processes of early embryogenesis, thus avoiding ethical concerns as well as increasing scalability and accessibility compared to the use of natural embryos. Here, we combine cell reprogramming and mechanical stimuli to create 3D spherical aggregates that are phenotypically similar to those of natural embryos. Specifically, dermal fibroblasts are reprogrammed, exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either driven towards the trophectoderm (TR) lineage using an ad hoc induction protocol or encapsulated into polytetrafluoroethylene micro-bioreactors to maintain and promote pluripotency, generating inner cell mass (ICM)-like spheroids. The obtained TR-like cells and ICM-like spheroids are then co-cultured in the same micro-bioreactor and, subsequently, transferred to microwells to encourage blastoid formation. Notably, the above protocol was applied to fibroblasts obtained from young as well as aged donors, with results that highlighted miR-200’s ability to successfully reprogram young and aged cells with comparable blastoid rates, regardless of the donor’s cell age. Overall, the approach here described represents a novel strategy for the creation of artificial blastoids to be used in the field of assisted reproduction technologies for the study of peri- and early post-implantation mechanisms.

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Section: Discussionsupporting

confidence: 93%

Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues

Pennarossa,

Arcuri,

Gandolfi

et al. 2024

Cells

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“…Taking advantage of the acquired high permissivity window, cells were readdressed toward the TR lineage, using a wellestablished induction protocol that has been previously shown to successfully allow for the differentiation of human ESCs toward TR cells (30,(46)(47)(48)(49)(50)(51)(52)(53)(54). In particular, epigenetically erased fibroblasts were induced with MEF conditioned medium supplemented with BMP4 and inhibitors of the activin/nodal and FGF2 signaling pathways.…”

Section: Discussionmentioning

confidence: 99%

Generation of Trophoblast-Like Cells From Hypomethylated Porcine Adult Dermal Fibroblasts

et al. 2021

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The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial components of the placenta, and which supports the fetus during the intrauterine life. However, the epigenetic and paracrine controls at work in trophectoderm differentiation are still to be fully elucidated and the creation of dedicated in vitro models is desirable to increase our understanding. Here we propose a novel approach based on the epigenetic conversion of adult dermal fibroblasts into trophoblast-like cells. The method combines the use of epigenetic erasing with an ad hoc differentiation protocol. Dermal fibroblasts are erased with 5-azacytidine (5-aza-CR) that confers cells a transient high plasticity state. They are then readdressed toward the trophoblast (TR) phenotype, using MEF conditioned medium, supplemented with bone morphogenetic protein 4 (BMP4) and inhibitors of the Activin/Nodal and FGF2 signaling pathways in low O2 conditions. The method here described allows the generation of TR-like cells from easily accessible material, such as dermal fibroblasts, that are very simply propagated in vitro. Furthermore, the strategy proposed is free of genetic modifications that make cells prone to instability and transformation. The TR model obtained may also find useful application in order to better characterize embryo implantation mechanisms and developmental disorders based on TR defects.

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“…Taking advantage of the acquired high permissivity window, cells were readdressed towards the TR lineage, using an induction cocktail containing BMP4 in combination with activin/nodal and FGF2 signaling inhibitors. This differentiation medium has been previously shown to drive cells towards the TR phenotype in both human [27,[50][51][52][53][54][55][56][57][58] and pig [14,28], with the acquisition of a tight adherent epithelial morphology, round shape, and nuclei, as well as well-defined borders. Consistent with this, immunocytochemical results indicated a high conversion efficiency (~ 80%), which is similar to that scored in human reprogrammed iPSCs differentiated towards TR lineage [27,56,59,60] as well as in TR-like cells obtained from epigenetically converted porcine adult dermal fibroblasts [28].…”

Section: Discussionmentioning

confidence: 99%

Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts

Pennarossa

Arcuri

Iorio

et al. 2023

J Assist Reprod Genet

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Purpose This study is to develop a new protocol that combines the use of epigenetic cues and mechanical stimuli to assemble 3D spherical structures, arbitrarily defined “epiBlastoids,” whose phenotype is remarkably similar to natural embryos. Methods A 3-step approach is used to generate epiBlastoids. In the first step, adult dermal fibroblasts are converted into trophoblast (TR)-like cells, combining the use of 5-azacytidine, to erase the original phenotype, with an ad hoc induction protocol, to drive cells towards TR lineage. In the second step, epigenetic erasing is applied once again, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like organoids. Specifically, erased cells are encapsulated into micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, TR-like cells are co-cultured with ICM-like spheroids in the same micro-bioreactors. Subsequently, the newly generated embryoids are transferred to microwells to favor epiBlastoid formation. Results Adult dermal fibroblasts are successfully readdressed towards TR lineage. Cells subjected to epigenetic erasing and encapsulated into micro-bioreactors rearrange in 3D ICM-like structures. Co-culture of TR-like cells and ICM-like spheroids into micro-bioreactors and microwells induces the formation of single structures with uniform shape reminiscent in vivo embryos. CDX2+ cells localized in the out layer of the spheroids, while OCT4+ cells in the inner of the structures. TROP2+ cells display YAP nuclear accumulation and actively transcribed for mature TR markers, while TROP2− cells showed YAP cytoplasmic compartmentalization and expressed pluripotency-related genes. Conclusion We describe the generation of epiBlastoids that may find useful application in the assisted reproduction field.

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Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-β/activin/nodal signalling inhibition

Cited by 14 publications

References 33 publications

Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues

Generation of Artificial Blastoids Combining miR-200-Mediated Reprogramming and Mechanical Cues

Generation of Trophoblast-Like Cells From Hypomethylated Porcine Adult Dermal Fibroblasts

Combination of epigenetic erasing and mechanical cues to generate human epiBlastoids from adult dermal fibroblasts

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