Abstract
Retrospective clonal analysis in the mouse has demonstrated that the posterior spinal cord neurectoderm and paraxial mesoderm share a common bipotent progenitor. These neuromesodermal progenitors (NMPs) are the source of new axial structures during embryonic rostrocaudal axis elongation and are marked by the simultaneous co-expression of the transcription factors T(Brachyury) (T(Bra)) and Sox2. NMP-like cells have recently been derived from pluripotent stem cells in vitro following combined stimulation of Wnt and fibroblast growth factor (FGF) signaling. Under these conditions the majority of cultures consist of T(Bra)/Sox2 co-expressing cells after 48-72 hours of differentiation. Although the capacity of these cells to generate posterior neural and paraxial mesoderm derivatives has been demonstrated at the population level, it is unknown whether a single in vitro-derived NMP can give rise to both neural and mesodermal cells. Here we demonstrate that T(Bra) positive cells obtained from mouse epiblast stem cells (EpiSCs) after culture in NMP-inducing conditions can generate both neural and mesodermal clones. This finding suggests that, similar to their embryonic counterparts, in vitro-derived NMPs are truly bipotent and can thus be exploited as a model for studying the molecular basis of developmental cell fate decisions.
Highlights
Axis elongation in vertebrate embryos proceeds in a rostral-to-caudal sequence and involves the coordinated production of spinal cord neurectoderm and paraxial mesoderm/somites from a population of neuromesodermal progenitors (NMPs)
To track the emergence of NMPs in vitro we employed a T(Bra) reporter epiblast stem cells (EpiSCs) line (TGFP) generated from embryonic stem (ES) cells carrying a green fluorescent protein (GFP) transgene knocked into the T(Bra) locus[14]
In line with our previous findings[10], culture of T(Bra)-green fluorescent protein (GFP) reporter (TGFP) EpiSCs in the presence of FGF2/CHIR for 48 or 72 hours gave rise to a significant number of TGFP+ cells, many of which were positive for Sox[2] expression (55% of the total TGFP+ population at 48 hours and 65% at 72 hours) as revealed by antibody staining and image analysis (Figure 1). This result indicates that at least half of the TGFP+ cells emerging in the presence of FGF2/CHIR are NMP-like and we used TGFP expression under these conditions to enrich for cells with NMP identity
Summary
Axis elongation in vertebrate embryos proceeds in a rostral-to-caudal sequence and involves the coordinated production of spinal cord neurectoderm and paraxial mesoderm/somites from a population of neuromesodermal progenitors (NMPs) (for a review see[1]). We and others have recently shown that mouse and human pluripotent stem cells cultured for 48–72 hours in the presence of FGF2 and the Wnt signaling agonist CHIRON99021 (CHIR) yield a high percentage of T(Bra)+Sox2+ double-positive cells that transcriptionally resemble embryonic NMPs10,11 These NMP-like cells were shown to efficiently differentiate exclusively into paraxial mesoderm and posterior neurectoderm both in vitro and in vivo upon grafting into cultured mouse and chick embryos[10] suggesting an NM bipotent character. These studies were carried out at the population level and it would be important to test the NM potency of single cells.
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