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

Axon regeneration is crucial for recovery from neurological diseases. Numerous studies have identified several genes, microRNAs (miRNAs), and transcription factors (TFs) that influence axon regeneration. However, the regulatory networks involved have not been fully elucidated. In the present study, we analyzed a regulatory network of 51 miRNAs, 27 TFs, and 59 target genes, which is involved in axon regeneration. We identified 359 pairs of feed-forward loops (FFLs), seven important genes (Nap1l1, Arhgef12, Sema6d, Akt3, Trim2, Rab11fip2, and Rps6ka3), six important miRNAs (hsa-miR-204-5p, hsa-miR-124-3p, hsa-miR-26a-5p, hsa-miR-16-5p, hsa-miR-17-5p, and hsa-miR-15b-5p), and eight important TFs (Smada2, Fli1, Wt1, Sp6, Sp3, Smad4, Smad5, and Creb1), which appear to play an important role in axon regeneration. Functional enrichment analysis revealed that axon-associated genes are involved mainly in the regulation of cellular component organization, axonogenesis, and cell morphogenesis during neuronal differentiation. However, these findings need to be validated by further studies.
Axons/physiology* ; Cell Differentiation ; Cluster Analysis ; Embryonic Stem Cells/cytology* ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Regulatory Networks ; Humans ; MicroRNAs/metabolism* ; Nerve Regeneration ; Neurons/metabolism* ; Software ; Transcription Factors/metabolism*

Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-κB activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-κB regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into various vascular cell derivatives to study how NF-κB modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating inflammation, survival, vasculogenesis, cell differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon tumor necrosis factor α (TNFα) stimulation. Lastly, further evaluation of gene expression patterns in IκBα knockout vascular cells demonstrated that IκBα acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-κB/RelA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.
Blood Vessels ; cytology ; metabolism ; CRISPR-Cas Systems ; Embryonic Stem Cells ; cytology ; Gene Knockout Techniques ; Homeostasis ; Humans ; NF-kappa B ; deficiency ; metabolism ; Transcription Factor RelA ; deficiency ; metabolism

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.
Activins ; metabolism ; Animals ; Cells, Cultured ; Embryonic Development ; Germ Layers ; metabolism ; Mice ; Pluripotent Stem Cells ; cytology ; metabolism

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential of differentiating into all types of adult cells. Today, mature functional sperm can be derived from mouse PSCs in vitro, and meanwhile primordial germ cells (PGCs) and meiotic prophase sperm cells can be generated from human ESCs/iPSCs (hESCs/hiPSCs). It is proposed that non-genetic azoospermia might be cured if functional sperm could be obtained from human PSCs (hPSCs) in vitro. It is also possible that healthy functional sperm could be derived from the patient with genetic factor-induced azoospermia by combining iPSCs and gene editing technology. IPSC-derived functional sperm have a higher clinical value for the avoidance of the sperm source and the issue of medical ethics. This article summarizes recent advances in the differentiation of PSCs into male germ cells in vitro, aiming to provide some reference for the treatment of male infertility with PSCs.
Animals ; Cell Differentiation ; Embryonic Stem Cells ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Infertility, Male ; therapy ; Male ; Meiosis ; Mice ; Pluripotent Stem Cells ; cytology ; Spermatozoa ; cytology

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
Animals ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Nanog Homeobox Protein ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Patch-Clamp Techniques ; Primary Cell Culture ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Thymosin ; pharmacology

Muscle atrophy caused by nerve injury is a common and difficult clinical problem. The development of stem cell researches has opened up a new way for the treatment of nerve injury-induced muscle atrophy. The induced pluripotent stem cells(iPSCs)can differentiate into various types of cells and have more advantages than embryonic stem cells (ESCs). After being transplanted into the damaged area, iPSCs are guided by neurogenic signals to the lesion sites, to repair the damaged nerve, promote generation of axon myelination, rebuild neural circuits and restore physiological function. Meanwhile, iPSCs can also differentiate into muscle cells and promote muscle tissue regeneration. Therefore, it would be possible to attenuate muscle atrophy caused by nerve injury with iPSCs treatment.
Animals ; Disease Models, Animal ; Embryonic Stem Cells ; Humans ; Induced Pluripotent Stem Cells ; cytology ; transplantation ; Muscular Atrophy ; therapy

Embryonic stem cells have unlimited proliferative capacity, which may provide a source of tendon stem/progenitor cells for tissue engineering. Experts of International Science and Technology Collaborative Program of Ministry of Science and Technology have developed a protocol consensus on differentiation of human embryonic stem cells into the tendon cells. The consensus recommends a protocol of two-step generation of human embryonic stem cells into tendon cells: the human embryonic stem cells are first differentiated into mesenchymal stem cells on different material surfaces; then with the scaffold-free tissue engineering tendon formed by high-density planting, the mesenchymal stem cells are induced into tendon cells under static or dynamic mechanical stimulation in vivo and in vitro. Tissue engineering tendon established in vitro by the protocol can be used as a model in toxicological analysis and safety evaluation of tendon-relevant small molecule compounds, medical materials and drugs.
Cell Differentiation ; Consensus ; Human Embryonic Stem Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Tendons ; cytology ; Tissue Engineering

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.
Animals ; Blastocyst ; Chimera ; Culture Media ; Embryo Culture Techniques ; veterinary ; Embryo, Mammalian ; Embryonic Development ; Induced Pluripotent Stem Cells ; cytology ; Mice ; Swine

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism