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

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

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

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*

OBJECTIVE: To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD). METHODS: ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting. RESULTS: We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6. CONCLUSIONS 7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.
Cell Cycle Proteins ; Cell Proliferation ; Embryonic Stem Cells/metabolism* ; HeLa Cells ; Humans ; Nuclear Proteins ; Positive Transcriptional Elongation Factor B/metabolism* ; RNA, Long Noncoding/genetics* ; RNA-Binding Proteins ; Ribonucleoproteins ; Transcription Factors

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.
Adenosine Monophosphate/therapeutic use* ; Alanine/therapeutic use* ; Alveolar Epithelial Cells/virology* ; Antibodies, Neutralizing/therapeutic use* ; COVID-19/virology* ; Down-Regulation ; Drug Discovery ; Human Embryonic Stem Cells/metabolism* ; Humans ; Immunity ; Lipid Metabolism ; Lung/virology* ; RNA, Viral/metabolism* ; SARS-CoV-2/physiology* ; Virus Replication/drug effects*

Parthenogenetic embryos, created by activation and diploidization of oocytes, arrest at mid-gestation for defective paternal imprints, which impair placental development. Also, viable offspring has not been obtained without genetic manipulation from parthenogenetic embryonic stem cells (pESCs) derived from parthenogenetic embryos, presumably attributable to their aberrant imprinting. We show that an unlimited number of oocytes can be derived from pESCs and produce healthy offspring. Moreover, normal expression of imprinted genes is found in the germ cells and the mice. pESCs exhibited imprinting consistent with exclusively maternal lineage, and higher X-chromosome activation compared to female ESCs derived from the same mouse genetic background. pESCs differentiated into primordial germ cell-like cells (PGCLCs) and formed oocytes following in vivo transplantation into kidney capsule that produced fertile pups and reconstituted ovarian endocrine function. The transcriptome and methylation of imprinted and X-linked genes in pESC-PGCLCs closely resembled those of in vivo produced PGCs, consistent with efficient reprogramming of methylation and genomic imprinting. These results demonstrate that amplification of germ cells through parthenogenesis faithfully maintains maternal imprinting, offering a promising route for deriving functional oocytes and having potential in rebuilding ovarian endocrine function.
Animals ; Female ; Mice ; Mice, Transgenic ; Mouse Embryonic Stem Cells/metabolism* ; Oocytes/metabolism* ; Parthenogenesis

OBJECTIVE: To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs). METHODS: Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation;immunoprecipitation was used to detect the interaction between 1,5,5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot. RESULTS: Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (<0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (<0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (<0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (<0.01). CONCLUSIONS Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.
Animals ; Calcium Signaling ; genetics ; Gene Expression Regulation ; genetics ; Gene Knockdown Techniques ; Mice ; Mitochondria ; physiology ; Mouse Embryonic Stem Cells ; Myocytes, Cardiac ; physiology ; Protein Transport ; Rapamycin-Insensitive Companion of mTOR Protein ; genetics ; metabolism

BACKGROUND AND OBJECTIVES: Embryonic stem (ES) cells have pluripotent ability to differentiate into multiple tissue lineages. SIRT1 is a class III histone deacetylase which modulates chromatin remodeling, gene silencing, cell survival, metabolism, and development. In this study, we examined the effects of SIRT1 inhibitors on the hematopoietic differentiation of mouse ES cells. METHODS AND RESULTS: Treatment with the SIRT1 inhibitors, nicotinamide and splitomicin, during the hematopoietic differentiation of ES cells enhanced the production of hematopoietic progenitors and slightly up-regulated erythroid and myeloid specific gene expression. Furthermore, treatment with splitomicin increased the percentage of erythroid and myeloid lineage cells. CONCLUSIONS: Application of the SIRT1 inhibitor splitomicin during ES cell differentiation to hematopoietic cells enhanced the yield of specific hematopoietic lineage cells from ES cells. This result suggests that SIRT1 is involved in the regulation of hematopoietic differentiation of specific lineages and that the modulation of the SIRT1 activity can be a strategy to enhance the efficiency of hematopoietic differentiation.
Animals ; Cell Differentiation ; Cell Survival ; Chromatin Assembly and Disassembly ; Gene Expression ; Gene Silencing ; Histone Deacetylases ; Metabolism ; Mice ; Mouse Embryonic Stem Cells ; Niacinamide