While Mek1/2 and Gsk3β inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.
Male ; Animals ; Female ; Mice ; Mouse Embryonic Stem Cells ; Embryonic Stem Cells ; Genomic Instability ; Lipids ; DNA/metabolism* ; Cell Differentiation

Interleukin 6 (IL-6), an important component of cardiac microenvironment, favors cardiac repair by improving cardiomyocyte regeneration in different models. This study aimed to investigate the effects of IL-6 on stemness maintenances and cardiac differentiation of mouse embryonic stem cells (mESCs). The mESCs were treated with IL-6 for two days, and then subjected to CCK-8 essay for proliferation analysis and quantitative real-time PCR (qPCR) to evaluate the mRNA expression of genes related to stemness and germinal layers differentiation. Phosphorylation levels of stem cell-related signal pathways were detected by Western blot. siRNA was used to interfere the function of STAT3 phosphorylation. Cardiac differentiation was investigated by the percentage of beating embryoid bodies (EBs) and qPCR analysis of cardiac progenitor markers and cardiac ion channels. IL-6 neutralization antibody was applied to block the endogenous IL-6 effects since the onset of cardiac differentiation (embryonic day of 0, EB0). The EBs were collected on EB7, EB10 and EB15 to investigate the cardiac differentiation by qPCR. On EB15, Western blot was applied to investigate the phosphorylation of several signaling pathways, and immunochemistry staining was adopted to trace the cardiomyocytes. IL-6 antibody was administered for two days (short term) on EB4, EB7, EB10 or EB15, and percentages of beating EBs at late developmental stage were recorded. The results showed that exogenous IL-6 promoted mESCs proliferation and favored maintenances of pluripotency, evidenced by up-regulated mRNA expression of oncogenes (c-fos, c-jun) and stemness markers (oct4, nanog), down-regulated mRNA expression of germ layer genes (branchyury, FLK-1, pecam, ncam, sox17), and increased phosphorylation of ERK1/2 and STAT3. siRNA targeting JAK/STAT3 partially attenuated the effects of IL-6 on cell proliferation and mRNA expression of c-fos and c-jun. During differentiation, long term IL-6 neutralization antibody application decreased the percentage of beating EBs, down-regulated mRNA expression of ISL1, GATA4, α-MHC, cTnT, kir2.1, cav1.2, and declined the fluorescence intensity of cardiac α actinin in EBs and single cell. Long term IL-6 antibody treatment decreased the phosphorylation of STAT3. In addition, short term (2 d) IL-6 antibody treatment starting from EB4 significantly reduced the percentage of beating EBs in late development stage, while short term IL-6 antibody treatment starting from EB10 significantly increased the percentage of beating EBs on EB16. These results suggest that exogenous IL-6 promotes mESCs proliferation and favors stemness maintenance. Endogenous IL-6 regulates mESC cardiac differentiation in a development-dependent manner. These findings provide important basis for the study of microenvironment on cell replacement therapy, as well as a new perspective for understanding the pathophysiology of heart diseases.
Animals ; Mice ; Interleukin-6 ; Mouse Embryonic Stem Cells ; Cell Differentiation ; Proto-Oncogene Proteins c-fos ; RNA, Messenger

Infertility has become a serious disease since it affects 10%-15% of couples worldwide, and male infertility contributes to about 50% of the cases. Notably, a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China. As such, it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and/or the overexpression of germ cell-related genes. In this review, we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us, and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.
Humans ; Infant, Newborn ; Male ; Germ Cells ; Embryonic Stem Cells ; Cell Differentiation ; Infertility, Male ; Induced Pluripotent Stem Cells

To understand how the nervous system develops from a small pool of progenitors during early embryonic development, it is fundamentally important to identify the diversity of neuronal subtypes, decode the origin of neuronal diversity, and uncover the principles governing neuronal specification across different regions. Recent single-cell analyses have systematically identified neuronal diversity at unprecedented scale and speed, leaving the deconstruction of spatiotemporal mechanisms for generating neuronal diversity an imperative and paramount challenge. In this review, we highlight three distinct strategies deployed by neural progenitors to produce diverse neuronal subtypes, including predetermined, stochastic, and cascade diversifying models, and elaborate how these strategies are implemented in distinct regions such as the neocortex, spinal cord, retina, and hypothalamus. Importantly, the identity of neural progenitors is defined by their spatial position and temporal patterning factors, and each type of progenitor cell gives rise to distinguishable cohorts of neuronal subtypes. Microenvironmental cues, spontaneous activity, and connectional pattern further reshape and diversify the fate of unspecialized neurons in particular regions. The illumination of how neuronal diversity is generated will pave the way for producing specific brain organoids to model human disease and desired neuronal subtypes for cell therapy, as well as understanding the organization of functional neural circuits and the evolution of the nervous system.
Humans ; Neural Stem Cells/physiology* ; Neurons/physiology* ; Brain ; Spinal Cord ; Embryonic Development ; Cell Differentiation/physiology*

Human pluripotent stem cells provide an inexhaustible model to study human embryogenesis in vitro. Recent studies have provided diverse models to generate human blastoids by self-organization of different pluripotent stem cells or somatic reprogramming intermediates. However, whether blastoids can be generated from other cell types or whether they can recapitulate postimplantation development in vitro is unknown. Here, we develop a strategy to generate human blastoids from heterogeneous intermediates with epiblast, trophectoderm, and primitive endoderm signatures of the primed-to-naïve conversion process, which resemble natural blastocysts in morphological architecture, composition of cell lineages, transcriptome, and lineage differentiation potential. In addition, these blastoids reflect many features of human peri-implantation and pregastrulation development when further cultured in an in vitro 3D culture system. In summary, our study provides an alternative strategy to generate human blastoids and offers insights into human early embryogenesis by modeling peri- and postimplantation development in vitro.
Humans ; Pluripotent Stem Cells/metabolism* ; Embryo, Mammalian/metabolism* ; Cell Differentiation ; Blastocyst ; Cell Lineage ; Embryonic Development

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.
Animals ; Cell Differentiation ; Embryo, Mammalian/metabolism* ; Embryonic Development ; Mice ; Pluripotent Stem Cells/metabolism* ; RNA, Messenger/genetics* ; RNA, Ribosomal ; RNA-Binding Proteins/metabolism* ; Transcription Factors/metabolism* ; Zygote/metabolism*

In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.
Animals ; Apoptosis ; Cell Cycle ; Cell Differentiation ; Embryoid Bodies ; Induced Pluripotent Stem Cells/metabolism* ; Mice ; Transforming Growth Factor beta/pharmacology*

OBJECTIVE: To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. METHODS: EBs in suspension culture were divided into normoxic (21% O₂) and hypoxic (5% O₂) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. RESULTS: The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). CONCLUSION Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.
Echinomycin/metabolism* ; Embryoid Bodies/metabolism* ; Humans ; Hypoxia/metabolism* ; Induced Pluripotent Stem Cells/metabolism* ; beta Catenin/metabolism*

Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro. In this study, we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation (BiFC) platform for protein-protein interaction screens and epiblast-like cell (EpiLC)-induction assays using reporter mouse embryonic stem cells (mESCs). Investigation of candidate interaction partners of core human pluripotent factors OCT4, NANOG, KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell (PGC)-inducing factors including BEN-domain (BEND/Bend) family members. Through RNA-seq, ChIP-seq, and ATAC-seq analyses, we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro. Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.
Animals ; Cell Differentiation/genetics* ; Chromatin/metabolism* ; Embryonic Stem Cells ; Germ Cells/metabolism* ; Germ Layers/metabolism* ; Mice

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3β and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.
Animals ; Bone Morphogenetic Protein 4/metabolism* ; Cell Differentiation ; Chromosomal Instability ; Endosomal Sorting Complexes Required for Transport ; Mice ; Mitogen-Activated Protein Kinase Kinases/metabolism* ; Mouse Embryonic Stem Cells/cytology* ; Pluripotent Stem Cells/cytology* ; Signal Transduction ; Ubiquitin-Conjugating Enzymes