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The rostrocaudal (head-to-tail) axis is supplied by populations of progenitors at the caudal end of the embryo. Despite recent advances characterising one of these populations, the neuromesodermal progenitors, their nature and relationship to other populations remains unclear. Here we show that neuromesodermal progenitors are a single Sox2lowTlow entity whose choice of neural or mesodermal fate is dictated by their position in the progenitor region. The choice of mesoderm fate is Wnt/β-catenin dependent. Wnt/β-catenin signalling is also required for a previously unrecognised phase of progenitor expansion during mid-trunk formation. Lateral/ventral mesoderm progenitors represent a distinct committed state that is unable to differentiate to neural fates, even upon overexpression of the neural transcription factor Sox2. They do not require Wnt/β-catenin signalling for mesoderm differentiation. This information aids the correct interpretation of in vivo genetic studies and the development of in vitro protocols for generating physiologically-relevant cell populations of clinical interest. Our bodies, like those of all animals with a backbone, form during embryo development in a head-to-tail sequence. This process is fuelled by populations of proliferating cells called progenitor cells, which are found in an early embryonic structure called the primitive streak, and later at the tail-end of the embryo. One of these populations – known as the neuromesodermal progenitors (or NMPs) – produces the animal’s spinal cord, muscle and bone tissue. However, it is not clear how this cell population is maintained or what triggers these cells to specialise into the correct cell type. It is even unclear whether NMPs are a single cell type or a collection of several types of progenitor, each with a slightly different propensity to make spinal cord or muscle and bone. Answering these questions could inform the future development of cell-replacement therapies for conditions such as spinal injuries. Wymeersch et al. used a range of techniques to identify, map the fate, and assess the developmental potential of progenitors in the primitive streak. This revealed fine-grained differences in the fates adopted by cells in the progenitor region. However, these regional differences were found to result from the progenitor cells’ extensive ability to respond to signals they receive from their environment, rather than being hard-wired into the progenitor cells. In fact, Wymeersch et al. detected only two distinct cell types: the NMPs and a new cell population termed lateral/paraxial mesoderm progenitors (or LPMPs), which, unlike NMPs, do not form nerve cells. Further experiments investigated the molecular signals present in the environment of these progenitors that help to decide their fate. NMPs respond to an important developmental signal, called Wnt, by adopting a so-called mesoderm fate. This signal also induces NMPs to undergo a previously unknown phase of proliferation during the formation of the animal’s body. LPMPs, on the other hand, do not require Wnt to form mesoderm. These findings show that studies with embryos can identify new progenitor populations that might be clinically relevant, and reveal new ways in which a cell’s environment inside an embryo can determine its fate.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

TET-family dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and oxidized methylcytosines in DNA. Here, we show that mouse embryonic stem cells (mESCs), either lacking Tet3 alone or with triple deficiency of Tet1/2/3, displayed impaired adoption of neural cell fate and concomitantly skewed toward cardiac mesodermal fate. Conversely, ectopic expression of Tet3 enhanced neural differentiation and limited cardiac mesoderm specification. Genome-wide analyses showed that Tet3 mediates cell-fate decisions by inhibiting Wnt signaling, partly through promoter demethylation and transcriptional activation of the Wnt inhibitor secreted frizzled-related protein 4 (Sfrp4). Tet1/2/3-deficient embryos (embryonic day 8.0–8.5) showed hyperactivated Wnt signaling, as well as aberrant differentiation of bipotent neuromesodermal progenitors (NMPs) into mesoderm at the expense of neuroectoderm. Our data demonstrate a key role for TET proteins in modulating Wnt signaling and establishing the proper balance between neural and mesodermal cell fate determination in mouse embryos and ESCs.

In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage-committed progenitors during regression of the primitive streak (PS) and tail bud (TB). The identification by retrospective clonal analysis of a population of neuromesodermal progenitors (NMPs) contributing to both musculoskeletal precursors (paraxial mesoderm) and spinal cord during axis formation challenged these notions. However, classical fate mapping studies of the PS region in amniotes have so far failed to provide direct evidence for such bipotential cells at the single-cell level. Here, using lineage tracing and single-cell RNA sequencing in the chicken embryo, we identify a resident cell population of the anterior PS epiblast, which contributes to neural and mesodermal lineages in trunk and tail. These cells initially behave as monopotent progenitors as classically described and only acquire a bipotential fate later, in more posterior regions. We show that NMPs exhibit a conserved transcriptomic signature during axis elongation but lose their epithelial characteristicsin the TB. Posterior to anterior gradients of convergence speed and ingression along the PS lead to asymmetric exhaustion of PS mesodermal precursor territories. Through limited ingression and increased proliferation, NMPs are maintained and amplified as a cell population which constitute the main progenitors in the TB. Together, our studies provide a novel understanding of the PS and TB contribution through the NMPs to the formation of the body of amniote embryos.

Although cell-to-cell heterogeneity in gene and protein expression within cell populations has been widely documented, we know little about its biological functions. By studying progenitors of the posterior region of bird embryos, we found that expression levels of transcription factors Sox2 and Bra, respectively involved in neural tube (NT) and mesoderm specification, display a high degree of cell-to-cell heterogeneity. By combining forced expression and downregulation approaches with time-lapse imaging, we demonstrate that Sox2-to-Bra ratio guides progenitor’s motility and their ability to stay in or exit the progenitor zone to integrate neural or mesodermal tissues. Indeed, high Bra levels confer high motility that pushes cells to join the paraxial mesoderm, while high levels of Sox2 tend to inhibit cell movement forcing cells to integrate the NT. Mathematical modeling captures the importance of cell motility regulation in this process and further suggests that randomness in Sox2/Bra cell-to-cell distribution favors cell rearrangements and tissue shape conservation.

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body. Here we employ human embryonic stem cell differentiation to define how neuromesodermal progenitor (NMP)-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC/spinal cord regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. This initial posteriorisation event is succeeded by a second phase of trunk HOX gene control that marks the differentiation of NMPs toward their TBXT-negative NC/spinal cord derivatives and relies predominantly on FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.

Background Identification of novel cell-based therapy sources has been of great interest in recent years to provide alternative and available therapy options in clinics. Conditioned medium (CM) can be a valuable supply for growth factors, cytokines and chemokines as a source of stem cell secretome. Exploring the role of new CM sources for tissue regeneration might be a promising approach for therapeutic purposes. Methods and results In the current study, neuromesodermal progenitors (NMPs) derived from induced pluripotent stem cells (iPSCs) were used to collect CM. Fibroblast derived iPSCs were successfully differentiated into NMPs and NMPs were characterized by double positive T/Bra and Sox2 staining. CM was collected from NMPs, and the content was characterized by membrane analysis. In vitro wound healing assay was used as a model system to observe potential activity of CM on cell migration. Fibroblasts, keratinocytes and endothelial cells were used to evaluate the effect of NMP-derived CM (NMP-CM) on cell migration in vitro. Several important proteins related to wound healing such as ANGPT 1, ANGPT 2, MCP-1, PDGF-AA, SDF-1α, TIMP-1 and TIMP-2 were increased in NMP-CM. NMP-CM increased cell proliferation and migration in vitro. Conclusions In vitro data obtained from three distinct cell types suggest a promising role of NMP-CM on cell migration. NMP-CM can be used for wound management in the further future after detailed in vitro and in vivo research.

Vertebrate axis patterning requires precise control of the differentiation of neuromesodermal progenitors (NMPs), which generate spinal cord (SC) and presomitic mesoderm (PSM). Previously, we identified a gastrula-premarked posterior enhancer (p-Enh) that is essential for posterior tissue development by regulating somite and SC in organogenetic embryos, while its role in early NMPs cells remains elusive. Here, using a highly efficient in vitro differentiation system, we found that the genetic removal of p-Enh leads to the aberrantly up-regulated PSM-related genes during both PSM and SC differentiation. Time-resolved transcriptomic analysis and experimental characterization revealed the activated PSM transcriptomic signature arose from disorganized NMPs composition, with an over-representation of the T high SOX2 low NMPs subtype. Besides, through a newly developed bioinformatic tool, ST-Pheno, which effectively bridges the in vitro samples to in vivo embryonic phenotypes within spatiotemporal context , we determined that the over-produced T high SOX2 low NMPs subtype is predominantly enriched in the anterior primitive streak and adjacent mesoderm region at E7.5, which may disrupt the proper development of NMPs towards prospective PSM and SC, ultimately leading to the posterior development failure. In summary, this study demonstrates a critical role of p-Enh in regulating NMPs subtype composition, which will broaden the molecular understanding of mammalian embryogenesis.

Neuromesodermal progenitors (NMPs), serving as the common origin of neural and paraxial mesodermal development in a large part of the trunk, have recently gained significant attention because of their critical importance in the understanding of embryonic organogenesis and the design of in vitro models of organogenesis. However, the nature of NMPs at many essential points remains only vaguely understood or even incorrectly assumed. Here, we discuss the nature of NMPs, focusing on their dynamic migratory behavior during embryogenesis and the mechanisms underlying their neural vs. mesodermal fate choice. The discussion points include the following: (1) How the sinus rhomboidals is organized; the tissue where the neural or mesodermal fate choice of NMPs occurs. (2) NMPs originating from the broad posterior epiblast are associated with Sox2 N1 enhancer activity. (3) Tbx6-dependent Sox2 repression occurs during NMP-derived paraxial mesoderm development. (4) The nephric mesenchyme, a component of the intermediate mesoderm, was newly identified as an NMP derivative. (5) The transition of embryonic tissue development from tissue-specific progenitors in the anterior part to that from NMPs occurs at the forelimb bud axial level. (6) The coexpression of Sox2 and Bra in NMPs is conditional and is not a hallmark of NMPs. (7) The ability of the NMP pool to sustain axial embryo growth depends on Wnt3a signaling in the NMP population. Current in vitro models of NMPs are also critically reviewed.

Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases, inflammatory diseases, etc., due to their pleiotropic properties. However, largely incongruent data were obtained from different MSC-based clinical trials, which may be partially due to functional heterogeneity among MSC. Here, we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP), which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression, proliferation, migration, multipotency, immunomodulatory activity and global gene expression profile. Moreover, the therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC), and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly, NMP-MSC display much stronger immunomodulatory activity than BMSC and , as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies.