In vitro generation of mouse morula-like cells.

Generating cells with the molecular and functional properties of embryo cells and with full developmental potential is an aim with fundamental biological significance. Here we report the in vitro generation of mouse transient morula-like cells (MLCs) via the manipulation of signaling pathways. MLCs are molecularly distinct from embryonic stem cells (ESCs) and cluster instead with embryo 8- to 16-cell stage cells. A single MLC can generate a blastoid, and the efficiency increases to 80% when 8-10 MLCs are used. MLCs make embryoids directly, efficiently, and within 4 days. Transcriptomic analysis shows that day 4-5 MLC-derived embryoids contain the cell types found in natural embryos at early gastrulation. Furthermore, MLCs introduced into morulae segregate into epiblast (EPI), primitive endoderm (PrE), and trophectoderm (TE) fates in blastocyst chimeras and have a molecular signature indistinguishable from that of host embryo cells. These findings represent the generation of cells that are molecularly and functionally similar to the precursors of the first three cell lineages of the embryo.

Developmental cell fate choice in neural tube progenitors employs two distinct cis-regulatory strategies.

In many developing tissues, the patterns of gene expression that assign cell fate are organized by graded secreted signals. Cis-regulatory elements (CREs) interpret these signals to control gene expression, but how this is accomplished remains poorly understood. In the neural tube, a gradient of the morphogen sonic hedgehog (Shh) patterns neural progenitors. We identify two distinct ways in which CREs translate graded Shh into differential gene expression in mouse neural progenitors. In most progenitors, a common set of CREs control gene activity by integrating cell-type-specific inputs. By contrast, the most ventral progenitors use a unique set of CREs, established by the pioneer factor FOXA2. This parallels the role of FOXA2 in endoderm, where FOXA2 binds some of the same sites. Together, the data identify distinct cis-regulatory strategies for the interpretation of morphogen signaling and raise the possibility of an evolutionarily conserved role for FOXA2 across tissues.

3D ECM-rich environment sustains the identity of naive human iPSCs.

The establishment of in vitro naive human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways have been characterized during maintenance of human naive pluripotency. However, little is known about the role exerted by the extracellular matrix (ECM) and its three-dimensional (3D) organization. Here, using an unbiased and integrated approach combining microfluidic cultures with transcriptional, proteomic, and secretome analyses, we found that naive, but not primed, hiPSC colonies are characterized by a self-organized ECM-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naive hiPSCs and also allows direct and timely developmental morphogenesis simply by modulating the signaling environment. Our study opens new perspectives for future applications of naive hiPSCs to study critical stages of human development in 3D starting from a single cell.

Human Pluripotent Stem Cell-Derived Micropatterned Ectoderm Allows Cell Sorting of Meso-Endoderm Lineages.

The human developmental processes during the early post-implantation stage instruct the specification and organization of the lineage progenitors into a body plan. These processes, which include patterning, cell sorting, and establishment of the three germ layers, have been classically studied in non-human model organisms and only recently, through micropatterning technology, in a human-specific context. Micropatterning technology has unveiled mechanisms during patterning and germ layer specification; however, cell sorting and their segregation in specific germ layer combinations have not been investigated yet in a human-specific in vitro system. Here, we developed an in vitro model of human ectodermal patterning, in which human pluripotent stem cells (hPSCs) self-organize to form a radially regionalized neural and non-central nervous system (CNS) ectoderm. We showed that by using micropatterning technology and by modulating BMP and WNT signals, we can regulate the appearance and spatial distribution of the different ectodermal populations. This pre-patterned ectoderm can be used to investigate the cell sorting behavior of hPSC-derived meso-endoderm cells, with an endoderm that segregates from the neural ectoderm. Thus, the combination of micro-technology with germ layer cross-mixing enables the study of cell sorting of different germ layers in a human context.

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells.

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

Sox2 modulation increases naïve pluripotency plasticity.

Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity.

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.

Distinct Molecular Trajectories Converge to Induce Naive Pluripotency.

Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest. Here we show that acquisition of naive pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells (EpiSCs), one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency. These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature. We propose that fine-tuned regulation of this "transition factor" underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions.

Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males.

A hallmark of naive pluripotency is the presence of two active X chromosomes in females. It is not clear whether prevention of X chromosome inactivation (XCI) is mediated by gene networks that preserve the naive state. Here, we show that robust naive pluripotent stem cell (nPSC) self-renewal represses expression of Xist, the master regulator of XCI. We found that nPSCs accumulate Xist on the male X chromosome and on both female X chromosomes as they become NANOG negative at the onset of differentiation. This is accompanied by the appearance of a repressive chromatin signature and partial X-linked gene silencing, suggesting a transient and rapid XCI-like state in male nPSCs. In the embryo, Xist is transiently expressed in males and in females from both X chromosomes at the onset of naive epiblast differentiation. In conclusion, we propose that XCI initiation is gender independent and triggered by destabilization of naive identity, suggesting that gender-specific mechanisms follow, rather than precede, XCI initiation.

NANOG amplifies STAT3 activation and they synergistically induce the naive pluripotent program.

Reprogramming of a differentiated cell back to a naive pluripotent identity is thought to occur by several independent mechanisms. Two such mechanisms include NANOG and activated STAT3 (pSTAT3), known master regulators of naive pluripotency acquisition [1-5]. Here, we investigated the relationship between NANOG and pSTAT3 during the establishment and maintenance of naive pluripotency. Surprisingly, we found that NANOG enhances LIF signal transduction, resulting in elevated pSTAT3. This is mediated, at least in part, by suppression of the expression of the LIF/STAT3 negative regulator SOCS3. We also discovered NANOG to be limiting for the expression of KLF4, a canonical "Yamanaka" reprogramming factor [6] and key pSTAT3 target [2, 7, 8]. KLF4 expression resulted from the codependent and synergistic action of NANOG and pSTAT3 in embryonic stem cells and during initiation of reprogramming. Additionally, within 48 hr, the combined actions of NANOG and pSTAT3 in a reprogramming context resulted in reactivation of genes associated with naive pluripotency. Importantly, we show that NANOG can be bypassed during reprogramming by exogenous provision of its downstream effectors, namely pSTAT3 elevation and KLF4 expression. In conclusion, we propose that mechanisms of reprogramming are linked, rather than independent, and are centered on a small number of genes, including NANOG.

Genome-wide chromatin interactions of the Nanog locus in pluripotency, differentiation, and reprogramming.

The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin "interactome" for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.