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

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

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

The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.
Animals ; Embryonic Stem Cells ; Fibroblasts ; Heart ; Mice ; Mouse Embryonic Stem Cells ; Paracrine Communication

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

BACKGROUND: Bone tissue engineering based on pluripotent stem cells (PSCs) is a new approach to deal with bone defects. Protocols have been developed to generate osteoblasts from PSCs. However, the low efficiency of this process is still an important issue that needs to be resolved. Many studies have aimed to improve efficiency, but developing accurate methods to determine efficacy is also critical. Studies using pluripotency to estimate efficacy are rare. Telomerase is highly associated with pluripotency. METHODS: We have described a quantitative method to measure telomerase activity, telomeric repeat elongation assay based on quartz crystal microbalance (QCM). To investigate whether this method could be used to determine the efficiency of in vitro osteogenic differentiation based on pluripotency, we measured the pluripotency pattern of cultures through stemness gene expression, proliferation ability and telomerase activity, measured by QCM. RESULTS: We showed that the pluripotency pattern determined by QCM was similar to the patterns of proliferation ability and gene expression, which showed a slight upregulation at the late stages, within the context of the general downregulation tendency during differentiation. Additionally, a comprehensive gene expression pattern covering nearly every stage of differentiation was identified. CONCLUSION: Therefore, this assay may be powerful tools for determining the efficiency of differentiation systems based on pluripotency. In this study, we not only introduce a new method for determining efficiency based on pluripotency, but also provide more information about the characteristics of osteogenic differentiation which help facilitate future development of more efficient protocols.
Bone and Bones ; Down-Regulation ; Gene Expression ; In Vitro Techniques ; Methods ; Mouse Embryonic Stem Cells ; Osteoblasts ; Pluripotent Stem Cells* ; Quartz Crystal Microbalance Techniques ; Telomerase* ; Up-Regulation

OBJECTIVE: To investigate the effect of Notch signaling pathways on differentiation of mouse embryonic stem cells(ESC) into haematopoietic stem cells or haematopoietic progenitors cells(HSC/HPC). METHODS: Mouse embryonic stem cells were proliferated in vitro to form embryoid bodies; the differentiation of embryoid bodies should be induced in vitro, the experiments were divided into BE, control, VEGF, DAPT and VEGF-DAPT groups; HSC/HPC ohenotype: CD117D34Sca1 was detected by flow cytometry; the related gene expression was detected by RT-PCR. RESULTS: The number of VEGF-induced HSC/HPC in VEGF group was significantey higher than that in the control and EB group (P<0.05), suggesting that VEGF promotes ESC differentiation to HSC/HPC; the number of DAPT-induced HSC/HPC in DAPT group was significanty higher than that in the Control and EB groups(P<0.05), suggesting that DAPT promotes ESC differentiation to HSC/HPC; the number of VEGF+DAPT-induced HSC/HPC in VEGF-DAPT group was significantly higher than that in VEGF and DAPT groups(P<0.05), suggesting that DAPT and VEGF play a synergistic role to promote differentiation of ESC into HSC/HPC. CONCLUSION Notch signal pathway inhibits differentiation of ESC into HSC / HPC by VEGF.
Animals ; Cell Differentiation ; Hematopoietic Stem Cells ; Mice ; Mouse Embryonic Stem Cells ; Receptors, Notch ; Signal Transduction ; Vascular Endothelial Growth Factor A

Pluripotent stem cells (PSCs) are a useful source of cells for exploring the role of genes related with early developmental processes and specific diseases due to their ability to differentiate into all somatic cell types. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system has proven to be a robust tool for targeted genetic modification. Here, we generated miR-451-deficient PSCs using the CRISPR/Cas9 system with PCR-based homologous recombination donor and investigated the impact of its deletion on self-renewal and hematopoietic development. CRISPR/Cas9-mediated miR-451 knockout did not alter the gene expressions of pluripotency, cellular morphology, and cell cycle, but led to impaired erythrocyte development. These findings propose that a combination of PSCs and CRISPR/Cas9 system could be useful to promote biomedical applications of PSCs by elucidating the function and manipulating of specific miRNAs during lineage specification and commitment.
Animals ; Cell Cycle ; Clustered Regularly Interspaced Short Palindromic Repeats* ; Erythrocytes ; Gene Expression ; Hematopoiesis ; Homologous Recombination ; Humans ; Mice* ; MicroRNAs ; Mouse Embryonic Stem Cells* ; Pluripotent Stem Cells ; Tissue Donors