Pseudo-trajectory inference for identifying essential regulations and molecules in cell fate decisions.

Development is a highly ordered process that institutes considerable changes in cell cycle structure of the different cells composing the embryo. Cell fate is decided at early stages of embryogenesis in embryonic stem cells (ESCs). These cells are derived from the inner cell mass of the primordial embryo and can give rise to all cell lineages of the 3 primary germ layers. The cell cycle of ESCs is uncommonly rapid compared with a wide range of somatic cells due to shorter gap phases, especially G1, resulting in the characteristic high proportion of cells in S phase. Previous observations have highlighted the importance of a rapid and contracted cell cycle for ESCs maintenance. A high proliferation rate was shown to be required for maintenance of ESC identity,1 and increased susceptibility to differentiate occurs during the contracted G1 phase.2 Very recently, the molecular mechanism underlying cell fate decisions of self-renewing ESCs was analyzed in more detail and shown to originate at specific points within this critical retracted G1 phase,3 establishing a connection between the mechanisms that control the cell cycle, cell fate decisions, and loss of pluripotency. ESCs differentiation induces massive changes in the nuclear protein landscape, surprisingly reported to be largely regulated at the post-transcriptional level rather than by direct regulation of gene expression levels by transcription factors,4 as previously postulated by various studies in diverse model organisms. One prominent protein post-translational modification is ubiquitylation, which regulates abundance and/or localization of target proteins altering their function, inasmuch regulating a multitude of cellular processes operating in most biological systems. Ubiquitylation is performed in 3 main consecutive steps catalyzed by E1, E2, and E3 enzymes, and in many cases the specificity of the reaction is provided by the E3 ligase complex, which conjugates activated ubiquitin to target substrates. The reverse reaction consists of removing ubiquitin from target proteins and is catalyzed by deubiquitylating enzymes (Dubs). Interestingly, converging evidences indicate that in ESCs, pluripotency is under tight control of the ubiquitin-dependent degradation pathways and proteasomal activity.5,6 In these studies, substrates and activity of the proteasome were shown to be different in ESCs compared with differentiated cells, reflecting differentiation-induced adaptations. For example, Nanog and Oct4, 2 key transcription factors involved in maintenance of pluripotency, were shown to have short half-lives in ESCs. Given that the activity of degradation pathways are commonly coupled to cell cycle stage and progression, it is not unlikely that both Nanog and Oct4 may be degraded in a cell cycle-dependent manner. The ubiquitin proteasome degradation pathway governs major cell cycle events including G1 to S transition and anaphase. It is interesting to note that both in human and mouse ESCs, substrates of the anaphase-promoting complex/cyclosome (APC/C) have recently been identified as critical players in pluripotency maintenance.3,6 APC/C activity is elevated in late anaphase and persists through progression of the G1 phase. In human ESCs, Cyclin D levels are maximal at the G1/S transition and inhibit endoderm differentiation by inducing CDK4/6-dependent phosphorylation of Smad2 and Smad3 transcription factors, clearing them from chromatin and blocking expression of endoderm genes. In mouse ESCs, we have reported that post-translational control of Cdc25A, a cell cycle master regulator, by the Dub3 deubiquitylase, is essential for pluripotency maintenance by control of Cdc25A protein abundance. Indeed, the balance of ubiquitylating and deubiquitylating activities on Cdc25A is tipped toward deubiquitylation due to high levels of Dub3. In addition failure to reduce Dub3 levels during differentiation does not affect the onset of differentiation; however, it results in cell lethality at a time of cell cycle remodelling. This provides one molecular mechanism of how the cell cycle impacts cell fate decisions through contraction of the G1 phase and relaxation of the G1/S checkpoint.6 In addition, the activity of SCF (Skip1/Cul1/F-box protein)-mediated degradation pathway, which directly controls abundance of 2 major cell cycle regulators, the p21 and p27 proteins, is itself under tight control of APC/CCdh1. Altogether, these observations emphasize the importance of the ubiquitin signaling pathways and associated Dub activities during development that remove ubiquitin from substrates and regulate proteasomal function. These new observations tentatively provide insight into the regulation of midblastula transition onset in Drosophila, in which, similarly to mESCs, post-translational modifications of Cdc25A and not changes of its mRNA expression have also appeared as main determinants of cell cycle changes and fate decisions.7 Altogether, these data converge to the concept that developmentally regulated, post-translational control of key cell cycle substrates and proteasomal activity are essential for regulating cell fate decisions of pluripotent stem cells and during development (Fig. 1). Given that the degradation pathway targeting substrates for degradation and associated proteasomal activity itself are cell cycle-dependent, it seems relevant to dissect the regulatory mechanisms that govern and institute these proteasome-dependent degradation pathways in an unperturbed pluripotent cell cycle. Figure 1. Schematic representation of developmental transitions mediated by protein post-translational modifications (PTMs). In Drosophila, PTMS are implicated in pre- to post-midblastula transition (MBT). The enzyme(s) responsible for Drosophila ...

The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes.

Cell fate transition is a fascinating process involving complex dynamics of three-dimensional (3D) chromatin organization and phase separation, which play an essential role in cell fate decision by regulating gene expression. Phase separation is increasingly being considered a driving force of chromatin folding. In this review, we have summarized the dynamic features of 3D chromatin and phase separation during physiological and pathological cell fate transitions and systematically analyzed recent evidence of phase separation facilitating the chromatin structure. In addition, we discuss current advances in understanding how phase separation contributes to physical and functional enhancer-promoter contacts. We highlight the functional roles of 3D chromatin organization and phase separation in cell fate transitions, and more explorations are required to study the regulatory relationship between 3D chromatin organization and phase separation.Graphical 3D chromatin organization (shown by Hi-C contact map) and phase separation are highly dynamic and play functional roles during early embryonic development, cell differentiation, somatic reprogramming, cell transdifferentiation and pathogenetic process. Phase separation can regulate 3D chromatin organization directly, but whether 3D chromatin organization regulates phase separation remains unclear.

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

In the preimplantation mammalian embryo, cells of the inner cell mass can differentiate into any cell type present in the more mature embryo. As of 1981, in mice and 1998 in humans, it has been recognized that embryonic stem cells (ESCs) with a prolonged proliferative capacity can be derived from the inner cell mass in vitro (Evans and Kaufman 1981; Thomson, Itskovitz-Eldor et al. 1998). ESCs are pluripotent cells that can contribute to all tissues in vivo and to the three primary germ layers as well as extraembryonic tissues in vitro. Because pluripotency is maintained in these cells even after prolonged periods of culture, human ESCs have great therapeutic potential for tissue regeneration. Indeed, embryonic and adult stem cells (SCs) hold great promise for regenerative medicine, tissue repair, and gene therapy. Careful molecular characterization of embryonic pluripotency should help to optimize and scale up the in vitro production of ESCs for clinical applications. The mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. As compared with differentiated cell types, stem cells express a significantly higher number of genes (represented by expressed sequence tags) of unknown function. The properties that distinguish stem cells from other cells are largely unknown, and the identification of signals that regulate stem cell differentiation remains fundamental to our understanding of cellular diversity. Embryonic and adult stem cells have many similarities at the transcriptional level. The overlapping set of expressed gene products represents a molecular signature of stem cells (Bhattacharya, Miura et al. 2004; Assou, Le Carrour et al. 2007). A list of human and mouse genes involved in stemness has been generated (Assou, Le Carrour et al. 2007) and includes 92 stemness genes known to be expressed in mouse or human ESCs, e.g., OCT3/4, NANOG, Cripto/TDGFI, Cx43 and Galanin (Richards, Tan et al. 2004). Work in the field of embryogenesis has also contributed to our understanding of the function of these pluripotency-associated genes. The four most significantly overexpressed genes in undifferentiated embryonic tissues are Galanin, POU5FI, NANOG and DPPA4 (Zeng, Miura et al. 2004). In most studies, galanin has been highlighted as the most abundant transcript in ES culture as well as human and rodent embryonic tissues (Anisimov, Tarasov et al. 2002; Zeng, Miura et al. 2004). Both galanin and galanin receptors are expressed in ES cells, indicating a potential functional role for this protein (Tarasov, Tarasova et al. 2002). This chapter will be devoted to a description of the galanin expression profiles in embryonic tissues and stem cells as well as its possible functional role.

Heterochromatin protein 1γ overexpression in P19 embryonal carcinoma cells elicits spontaneous differentiation into the three germ layers

Cell-cell communication coordinates signalling between cells to guide context-dependent cell fate decisions such as proliferation, differentiation, and lineage specification. Such communication mechanisms are poorly understood in regulating the stem cell states. In this study, we investigate how cell-cell communication regulates cell fate transitions in heterogeneous embryonic stem cell populations, with a particular focus on totipotent-like cells that resemble the two-cell stage embryo. Using single-cell RNA sequencing in combination with computational frameworks, we map ligand-receptor interactions and model downstream regulatory effects across various stem cell states. We functionally validate the predictions by selectively perturbing signalling pathways under specific culture conditions. Our data reveal the key roles of BMP and NODAL (TGF-β) signalling in mediating intercellular communication to shape stem cell identity and heterogeneity. These findings enhance our understanding of the signalling logic that governs early developmental cell fate decisions, providing new insights into stem cell biology with broad implications for regenerative medicine and developmental modelling.

Forms, forces, and stem cell fate

Redox heterogeneity in mouse embryonic stem cells individualizes cell fate decisions

The nucleosome remodeling and deacetylation (NuRD) complex is required for modulating the transcription of essential pluripotency genes in ESC self-renewal. MBD3 is considered a key player in the formation of a functional NuRD complex. The recruitment of MBD3 to methylated promoters may require other prerequisite factors. We show that cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), an essential gene for early embryonic development, plays a role in pluripotency of ESC by engaging MBD3 to the promoter region of Wnt signaling genes. The occupancy of MBD3 on several promoters of Wnt genes was significantly lost in the absence of CDK2AP1, resulting in hyperactivation of Wnt. We propose that the transcriptional modulation of the Wnt pathway mediated by NuRD requires the presence of essential auxiliary components such as CDK2AP1, which may aid the association of the complex with specific focal regions of the target promoters.

Radiotherapy is one of the most commonly prescribed anticancer modalities in the clinic, which is widely recognized as an effective and safe treatment for a broad spectrum of solid tumor indications. Interestingly, there is increasing evidence that the tumors can dynamically modulate cell fate decisions after ionizing radiation (IR) exposure, which is beneficial for escaping the radiation-induced antitumorigenic cell damaging and immunostimulatory impacts. Consequently, the regulatory network of cell fate determination could be a promising target for enhancing the susceptibility of tumor cells to various radiotherapeutic modalities. In this review, we provide a comprehensive account on the mechanisms of post-radiation cell fate control in tumor cells to escape programmed cell death (PCD) including apoptosis, necrosis, pyroptosis and ferroptosis, while special emphasis is placed on the development of synthetic agents for the therapeutic modulation of post-radiation tumor cell fate decisions to facilitate tumor cell eradication, focusing on their therapeutic utility for amplifying the RT-induced direct tumor cell damage as well as promoting the post-IR antitumor immunity. We envision that these synthetic cell fate regulatory technologies could provide new avenues for improving radiotherapeutic efficacy.

This study investigates cell fate selection in a single cell by carrying out theoretical analysis on a parametric 2-Dimensional model. The investigated model involves eight fixed parameters, and two tuneable parameters, r and m, where the tuneable parameters are the determinants of cell fate decision. Using these tuneable parameters as the axes, the concept of apoptogram, where the exact values of r and m pairs that lead to a certain cell fate decision are portrayed as regions, is introduced. The boundaries that depicts the regions are theoretically determined in a parametric fashion so that one can construct an apoptogram image given the values of fixed parameters of the model. The regions that correspond to a cell fate decision in the apoptogram provide valuable insights to investigate and manipulate cell fate decisions. Further studies may utilize such concept as a new tool to explore the complexity of cancer and cancer therapy.

Pluripotency in the early embryo is defined as the capacity of a single cell to generate all lineages of the adult organism. In vivo this property is possessed only transiently by the cells of the epiblast, but it can be indefinitely “captured” in vitro by deriving embryonic stem (ES) cells from the inner cell mass of the blastocyst. A second property that characterizes ES cells is self-renewal, the capacity to generate more stem cells. Self-renewal requires the coordination of cell proliferation and cell-fate choice (Orford and Scadden, 2008). Originally, mouse ES cells were expanded on mitotically inactivated fibroblasts, in a culture medium containing fetal bovine serum and the leukemia inhibitory factor (LIF). Under these conditions ES cells are metastable, since they show heterogeneity both in morphology and expression of the core pluripotency factors Oct4 and Nanog due to differentiation signals coming from the serum components (Nichols and Smith, 2009). In serum + LIF medium, ES cells depend on the transcription factor and proto‐oncogene Myc for pluripotency and self‐renewal (Smith et al., 2010). Myc proteins have been shown to repress the expression of the primitive endoderm master regulator Gata6, and control the cell cycle by regulating the mir-17-92 miRNA cluster. The laboratory of Austin Smith has recently shown that the culture in the absence of serum but in the presence of two inhibitors (2i + LIF) of the Erk and glycogen synthase kinase-3 (GSK3) pathways is sufficient to stabilize ES cells in a more naïve, so-called ground state of pluripotency, which more closely resembles the status of the inner cell mass of the blastocyst (Ying et al., 2008). Since different culture environments impose distinctive transcriptional and epigenetic properties on mouse ES cells (Marks et al., 2012), we re-evaluated the expression and requirement of the Myc proteins in the naïve state of pluripotency. Making use of ES cells expressing a Myc-GFP reporter from the endogenous c-myc locus, we found that c-Myc protein expression is significantly lower in naïve ES cells compared to cells cultured in serum + LIF. When both c- and N-myc genes are conditionally deleted (Myc dKO) using the Cre-loxP system, naïve mouse ES cells undergo cell cycle arrest. Although this type of cell cycle behavior with a decreased rate of cell division is often associated with differentiation, Myc dKO ES cells form smaller but undifferentiated colonies. Most surprisingly, Myc dKO ES cells maintain the expression of the core stem cell factor network including Oct4, Nanog and Sox2 both at the RNA and the protein level. To determine the molecular properties of Myc dKO ES cells, we performed RNA‐seq analysis 24 and 96 hours after Cre induction. Our results show that Myc is not exclusively regulating the cell cycle machinery but is directly and indirectly involved in multiple aspects of ES cell metabolism. In the absence of both Myc proteins, expression of genes controlling and executing ribosomal biogenesis as well as protein and DNA synthesis is strongly down-regulated, leading to a state of “metabolic dormancy”. The state of “metabolic dormancy” of ES cells in vitro resembled the state of embryonic diapause in vivo. Diapause is a poorly understood phenomenon of reversible arrest of embryonic development prior to implantation. In mice, facultative diapause occurs to delay implantation of newly formed embryos when the mother is feeding a previous litter. When we compared our data to reported global gene expression profiling of diapause embryos (Hamatani et al., 2004), we observed that dKO Myc ES cells possess surprising similarity with the dormant blastocyst, characterized by reduced DNA synthesis and cell division, low metabolic activity and activation of the insulin pathway (Given, 1988; Hamatani et al., 2004). These data suggest that, in the absence of differentiation cues, c-Myc and N-Myc control the cell cycle of ES cells and the transcriptional networks responsible for the entire metabolism and biosynthesis pathways while the pluripotency network is controlled by other means. Loss of Myc activity has dramatic consequences on the metabolic status of ES cells promoting their entry into a state of likely reversible dormancy closely resembling arrested diapause embryos.

How equipotent cells develop into complex tissues containing many diverse cell types is still a mystery. However, evidence is accumulating from different tissue systems in multiple organisms that many of the specific receptor families known to regulate cell fate decisions target conserved signaling pathways. A mechanism for preserving specificity in the cellular response that has emerged from these studies is one in which quantitative differences in receptor signaling regulate the cell fate decision. A signal strength model has recently gained support as a means to explain alphabeta/gammadelta lineage commitment. In this review, we compare the alphabeta/gammadelta fate decision with other cell fate decisions that occur outside of the lymphoid system to attain a better picture of the quantitative signaling mechanism for cell fate specification.

Adult stem cells reside in hypoxic niches, and embryonic stem cells (ESCs) are derived from a low oxygen environment. However, it is not clear whether hypoxia is critical for stem cell fate since for example human ESCs (hESCs) are able to self-renew in atmospheric oxygen concentrations as well. We now show that hypoxia can govern cell fate decisions since hypoxia alone can revert hESC- or iPSC-derived differentiated cells back to a stem cell-like state, as evidenced by re-activation of an Oct4-promoter reporter. Hypoxia-induced "de-differentiated" cells also mimic hESCs in their morphology, long-term self-renewal capacity, genome-wide mRNA and miRNA profiles, Oct4 promoter methylation state, cell surface markers TRA1-60 and SSEA4 expression, and capacity to form teratomas. These data demonstrate that hypoxia can influence cell fate decisions and could elucidate hypoxic niche function.