Search

Human pluripotent stem cells can self-renew indefinitely or be induced to differentiate into the three embryonic germ lineages: endoderm, mesoderm, and ectoderm. Multiple studies show that reconfiguring metabolic pathway activities influences the fate of pluripotent stem cells through metabolite-regulated changes in intracellular signal transduction pathways and the epigenome. Through these and potentially additional mechanisms, the conversion of available nutrients into metabolite fluxes and steady-state levels can tip the balance between pluripotent stem cell self-renewal and differentiation, although which nutrients and metabolites favor specific cell fate decisions are largely unidentified.Glutamine participates in a wide variety of cellular processes beyond biomass and energy-producing reactions in pluripotent and adult-type stem cells. We showed that endoderm, mesoderm, and ectoderm all consume abundant glutamine supplied in cell culture media. Additionally, exogenous glutamine withdrawal biased spontaneous human pluripotent stem cell differentiation toward ectoderm and away from mesoderm. However, a mechanism for this nutrient-directed cell fate bias was, until recently, unknown.We uncovered that while all three germ lineages are capable of de novo glutamine synthesis, only ectoderm generates sufficient glutamine to sustain cell viability and differentiation, clarifying lineage fate restrictions observed with glutamine withdrawal. While exogenous glutamine is dispensable for ectoderm, complete glutamine starvation obliterates this lineage, suggesting a critical role for glutamine in ectoderm specification. We uncovered that glutamine fulfillment of mTORC1 activation, but not biosynthetic demands and protein translation, is critical for initiating ectoderm fate commitment. Glutamine acts as a signaling molecule to activate mTORC1 signaling in ectoderm that supersedes lineage-specifying cytokine induction. In contrast, glutamine in mesoderm and endoderm serves as the preferred precursor of -ketoglutarate without a direct role in cell fate signaling. Our findings show that a nutrient acts as a signaling molecule to control cell fate, raising a question about whether the nutrient environment functions directly in cell differentiation during development.For these reasons, we hypothesize that intracellular glutamine may help coordinate differentiation of the three embryonic germ layers. Differing patterns of nutrient availability occur during early embryonic germ lineage differentiation. Interestingly, transcriptome analysis of a gastrulation-stage human embryo shows unique glutamine enzyme-encoding gene expression patterns may also distinguish germ lineages in vivo.In summary, these findings reveal critical roles for metabolic regulation and nutrient signaling during early embryonic development.

Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) are valuable for the understanding/treatment of the deadly heart diseases and their drug screening. However, the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging. Here, it is discovered surprisingly that Rock inhibitor (RI), used ubiquitously to improve the survival/yield of human iPSCs, induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 μM). This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 μM for 3D culture, the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95 % in ~7 days (compared to less than ~50 % in 14 days for the 10 μM RI condition). Furthermore, the outset beating time (OBT) of all resultant cardiac spheroids (CSs) is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol (GelMA) hydrogel, showing high homogeneity (in terms of the OBT) in functional maturity of the CSs. Moreover, the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs. These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases., Graphical abstract Rock inhibitor (RI) at 10 μM induces early gastrulation-like heterogeneous differentiation of human iPSCs and compromises their cardiac differentiation in 3D. Importantly, reducing RI to 1 μM retains human iPSCs in solid inner cell mass-like spheroids with high pluripotency for efficient and homogeneous cardiac differentiation in 3D. This discovery may greatly facilitate human iPSCs-based therapy and modeling of heart diseases.Image 1, Highlights • Rock inhibitor (RI) at the widely used 10 μM induces early gastrulation-like differentiation of human iPSCs (hiPSCs) in 3D. • Reducing RI to 1 μM retains the high pluripotency of hiPSCs in inner cell mass-like solid spheroids in 3D. • Reducing RI to 1 μM shortens the time needed for 3D cardiac differentiation of hiPSCs by ~1 week. • Reducing RI to 1 μM doubles the percentage of beating cardiac spheroids after 3D cardiac differentiation of hiPSCs.

Elucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Computational gene regulatory network (GRN) reconstruction methods aim to map this control by inferring relationships from transcriptomic data. Unfortunately, existing methods are imprecise, may be computationally burdensome, and do not uncover how networks transition from one topology to another. Here we present Epoch, a computational network reconstruction tool that leverages single cell transcriptomics to infer dynamic network structures. Epoch performs favorably when benchmarked using data derived from in vivo, in vitro, and in silico sources. To illustrate the usefulness of Epoch, we applied it to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESC) guided by multiple primitive streak induction treatments. Our analysis demonstrates that modulating signaling pathways drives topological network changes that shape cell fate potential. We also find that Peg3 is a central contributor to the rewiring of the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRN structures, we traced how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we compare the networks established in in vitro directed differentiation of ESCs to those in in vivo gastrulation and mesoderm specification. The methods presented here are available in the R package Epoch, and provide a foundation for future work in understanding the biological implications of dynamic regulatory structures.

The endoderm, one of the three primary germ layers, gives rise to lung, liver, stomach, intestine, colon, pancreas, bladder, and thyroid. These endoderm-originated organs are subject to many life-threatening diseases. However, primary cells/tissues from endodermal organs are often difficult to grow in vitro. Human pluripotent stem cells (hPSCs), therefore, hold great promise for generating endodermal cells and their derivatives for the development of new therapeutics against these human diseases. Although a wealth of research has provided crucial information on the mechanisms underlying endoderm differentiation from hPSCs, increasing evidence has shown that metabolism, in connection with epigenetics, actively regulates endoderm differentiation in addition to the conventional endoderm inducing signals. Here we review recent advances in metabolic and epigenetic regulation of endoderm differentiation.

The basic body plan of the mammalian embryo is established through gastrulation, a pivotal early postimplantation event during which the three major germ layers (endoderm, ectoderm, and mesoderm) are specified with cellular and spatial diversity. Despite its basic and clinical importance, human embryo development from peri-implantation to gastrulation remains shrouded in mystery. Recent advances in the elongated in vitro culture of rodent and non-primate embryos and the construction of embryo-like structures have helped to improve understanding of the mechanisms of human early embryonic development. Here, we review the recent advances and possible future directions in the development of in vitro models to better understand human embryogenesis from peri-implantation to gastrulation.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Reactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4−/−) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4−/− cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4−/− iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells. Graphical Abstract

Pluripotent cells in the mammalian embryo undergo state transitions marked by changes in patterns of gene expression and developmental potential as they progress from pre-implantation through post-implantation stages of development. Recent studies of cultured mouse and human pluripotent stem cells (hPSCs) have identified cells representative of an intermediate stage (referred to as the formative state) between naive pluripotency (equivalent to pre-implantation epiblast) and primed pluripotency (equivalent to late post-implantation epiblast). We examine these recent findings in light of our knowledge of peri-implantation mouse and human development, and we consider the implications of this work for deriving human embryo models from pluripotent cells.

Summary Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers. Using a custom-built image analysis pipeline, we quantified the differentiation phenotypes of 13 iPSC lines that harbor nsSNVs in genes related to cell adhesion or germ layer development. All iPSC lines differentiated into the three germ layers; however, there was donor-specific variation in germ layer patterning. We identified one line that presented an outlier phenotype of expanded endodermal differentiation, which was associated with a nsSNV in ITGB1. Our study establishes a platform for investigating the impact of nsSNVs on differentiation., Graphical abstract, Highlights • Healthy donor iPSCs differentiate into radial germ layers on micropatterns • Differentiation can be quantified using high-content image analysis • iPSC lines show donor-specific variation in germ layer patterning • A deleterious nsSNV in ITGB1 is associated with expanded endoderm formation, In this study, Vickers and colleagues combine micropatterning and high-content image analysis of healthy donor iPSC lines to identify genetic variants that influence cellular differentiation. iPSC lines displayed donor-specific variation in germ layer patterning. Outlier cell lines were identified, including one line that displayed expanded endoderm differentiation and was associated with a deleterious nsSNV in ITGB1.

Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.

Background Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs). Methods The hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction. Results When SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased β-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells. Conclusion The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism. The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. β-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, β-catenin increases the expression of the endoderm markers Sox17 and CXCR4.

2,4,6-Tribromophenol (TBP, CAS No. 118-79-6), the most widely produced brominated phenol, is frequently detected in environmental components. The detection of TBP in human bodies has earned great concerns about its adverse effects on human beings, especially for early embryonic development. Here, we optimized the mouse embryo in vitro culture (IVC) system for early post-implantation embryos and employed it to determine the embryotoxicity of TBP. With this new research model, we revealed the dose-dependent toxic effects of TBP on mouse embryos from peri-implantation to egg cylinder stages. Furthermore, TBP exposure inhibited the differentiation and survival of epiblast (EPI) cells and extraembryonic endoderm (ExEn) cells, while those of extraembryonic ectoderm (ExEc) cells were not influenced. These results implied that TBP might inhibit embryonic development by influencing the generation of three primary germ layers and fetal membranes (the amnion, chorionic disk, umbilical cord, and yolk sac). In summary, we showed a proof of concept for applying mouse embryo IVC system as a novel research model for studying mammalian embryonic toxicology of environmental pollutants. This study also demonstrated the toxicity of TBP on early embryonic development of mammals.

Peripheral nerve injuries (PNIs) are some of the most common types of traumatic lesions affecting the nervous system. Although the peripheral nervous system has a higher regenerative ability than the central nervous system, delayed treatment is associated with disturbances in both distal sensory and functional abilities. Over the past decades, adult stem cell-based therapies for peripheral nerve injuries have drawn attention from researchers. This is because various stem cells can promote regeneration after peripheral nerve injuries by differentiating into neural-line cells, secreting various neurotrophic factors, and regulating the activity of in situ Schwann cells (SCs). This article reviewed research from the past 10 years on the role of stem cells in the repair of PNIs. We concluded that adult stem cell-based therapies promote the regeneration of PNI in various ways.

Background Induced pluripotent stem cell (iPSC)-based technology provides limitless resources for customized development of organs without any ethical concerns. In theory, iPSCs generated from terminally differentiated cells can be induced to further differentiate into all types of organs that are derived from the embryonic germ layers. Since iPSC reprogramming technology is relatively new, extensive efforts by the researchers have been put together to optimize the protocols to establish in vitro differentiation of human iPSCs (hiPSCs) into various desirable cell types/organs. Highlights In the present study, we review the potential application of iPSCs as an efficient alternative to primary cells for modulating signal molecules. Furthermore, an efficient culture system that promotes the differentiation of cell lineages and tissue formation has been reviewed. We also summarize the recent studies wherein tissue engineering of the three germ layers has been explored. Particularly, we focus on the current research strategies for iPSC-based tooth regeneration via molecular modulation. Conclusion In recent decades, robust knowledge regarding the hiPSC-based regenerative therapy has been accumulated, especially focusing on cellular modulation. This review provides the optimization of the procedures designed to regenerate specific organs.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA dictates human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA targeting of FOXA1 and FOXA2 in human hepatic (HepG2) cells and during hepatic differentiation significantly downregulated albumin (p < 0.05) and GRN TF gene expression (HNF4A, HEX, HNF1B, TBX3) (p < 0.05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid (GSC), FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < 0.05), all consistent with a partial/transient cell reprogramming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, and accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During hPSC-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate complex effects of FOXA1/2 on hepatic GRN effecting de-differentiation and metabolism with applications in studies of cancer, differentiation, and organogenesis.

Graphic abstract Limitations of monolayer culture conditions have motivated scientists to explore new models that can recapitulate the architecture and function of human organs more accurately. Recent advances in the improvement of protocols have resulted in establishing three-dimensional (3D) organ-like architectures called ‘organoids’ that can display the characteristics of their corresponding real organs, including morphological features, functional activities, and personalized responses to specific pathogens. We discuss different organoid-based 3D models herein, which are classified based on their original germinal layer. Studies of organoids simulating the complexity of real tissues could provide novel platforms and opportunities for generating practical knowledge along with preclinical studies, including drug screening, toxicology, and molecular pathophysiology of diseases. This paper also outlines the key challenges, advantages, and prospects of current organoid systems.

The neural crest is said to be the fourth germ layer in addition to the ectoderm, mesoderm and endoderm because of its ability to differentiate into a variety of cells that contribute to the various tissues of the vertebrate body. Neural crest cells (NCCs) can be divided into three functional groups: cranial NCCs, cardiac NCCs and trunk NCCs. Defects related to NCCs can contribute to a broad spectrum of syndromes known as neurocristopathies. Studies on the neural crest have been carried out using animal models such as Xenopus, chicks, and mice. However, the precise control of human NCC development has not been elucidated in detail due to species differences. Using induced pluripotent stem cell (iPSC) technology, we developed an in vitro disease model of neurocristopathy by inducing the differentiation of patient-derived iPSCs into NCCs and/or neural crest derivatives. It is now possible to address complicated questions regarding the pathogenetic mechanisms of neurocristopathies by characterizing cellular biological features and transcriptomes and by transplanting patient-derived NCCs in vivo. Here, we provide some examples that elucidate the pathophysiology of neurocristopathies using disease modeling via iPSCs.