Microfluidic platform for 3D cell culture with live imaging and clone retrieval.

The role of EXOSC8 during mouse embryonic stem cells differentiation into neural precursor cells. Understanding the interplay between transcriptional and translational regulation is essential for deciphering stem cell differentiation. Here, we present a comprehensive study investigating the role of EXOSC8, a subunit of the core of the RNA exosome complex (EC), in mouse embryonic stem cell (mESC) differentiation toward neural precursor cells (NPC). By employing a controlled embryoid body (EB) formation protocol using diamond-bottom well plates and optimized cell seeding, we achieved synchronized neural induction. Transcriptional profiling confirmed progressive downregulation of pluripotency genes and upregulation of neuroectodermal markers across differentiation stages. We established and validated stable mESC lines expressing Ribo-STAMP and DOX-inducible shRNA constructs targeting EXOSC8, achieving 88% protein depletion. Integration of Ribo-STAMP with bulk and single-cell RNA-sequencing revealed dynamic shifts in both transcriptome and translatome, including genes regulated exclusively at the translational level. EXOSC8 knockdown led to altered expression of genes involved in protein ubiquitination and mRNA surveillance, affirming the RNA exosome&#8217s role in post-transcriptional control. Through immunofluorescence and subcellular fractionation, we observed stage-specific relocalization of EXOSC8, shifting from a balanced nuclear-cytoplasmic distribution in mESCs to predominant cytoplasmic enrichment during intermediate stages, and partial nuclear return in neural precursors. Co-immunoprecipitation and mass spectrometry revealed a differentiation-stage-specific EXOSC8 interactome, transitioning from nucleolar and ribosome biogenesis factors to chromatin regulators, mRNA transporters, and finally to cytoskeletal proteins involved in axon development. These findings suggest that EXOSC8 localization is functionally programmed, enabling context-specific interactions that support lineage progression and neuronal differentiation. Finally, single-cell transcriptome and translatome analysis uncovered heterogeneity and revealed dynamic regulatory changes during differentiation under EXOSC8 perturbation. Collectively, this study establishes a robust experimental framework to dissect transcriptional, translational, and spatial regulation in stem cell fate commitment and highlights the RNA exosome complex as a modular RNA regulator during development.

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Gene silencing by RNA interference (RNAi) using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool for silencing specific genes in various organisms. As compared with transgene-based RNAi using shRNA expression vectors, chemically synthesized siRNAs have an advantage in that they do not modify genome organization. This nature is ideal as a differentiation method for embryonic stem (ES) cells. The objective of this work was to develop an efficient method to repress a specific gene expression in mouse and monkey ES cells using chemically synthesized siRNA, and to investigate whether this transient gene silencing can be used as a new differentiation method of ES cells. First, we tried to knock down the expression of enhanced green fluorescent protein (EGFP) gene in GFP-expressing mouse ES cells. We found that lipofection was effective to deliver siRNA into mouse ES cells. Suppression of EGFP expression was observed by fluorescence microscopy after 24 h of transfection and the silencing continued at least for 5 days. FACS analysis revealed that 86% of cells showed suppression of EGFP at 48 h after transfection. Then we tried to suppress endogenous gene Oct4 that plays an important role in the maintenance of pluripotency and the lineage commitment in mouse ES cells. We performed RT-PCR analysis and western blotting to assay for Oct4 mRNA and protein at 24, 48, 72, and 96 h after transfection. Hand1 and Cdx2, transcription factors implicated in trophoblast differentiation, were also analyzed. RT-PCR analysis showed a reduced level of Oct4 mRNA at 24–96 h. Reduction of Oct4 protein was confirmed by western blotting and the reduced level was still maintained at 96 h after transfection. RT-PCR analysis also showed up-regulation of Hand1 and Cdx2 concomitant with the suppression of Oct4. Furthermore, ES cells changed their morphology into a large and flattened shape that is characteristic of trophoblast cells. These results suggested that the transient suppression of Oct4 induced differentiation of mouse ES cells to trophectoderm cells as expected. Therefore our data imply that the chemically synthesized siRNA can be used to differentiate ES cells. Next, we tried to suppress EGFP and Oct4 in monkey ES cells. In monkey ES cells, we found that the Sendai virus (hemagglutinating virus of Japan, HVJ) envelope was suitable to deliver siRNA into cells. With this method, we detected efficient silencing of EGFP and Oct4 by fluorescence microscopy, RT-PCR analysis, and western blotting. In the case of monkey ES cells, however, no morphological change was observed by Oct4 suppression at 96 h after transfection. These results suggest possible diversity between murine and primate ES cells in the differentiation process. This work was supported by grants from the Ministry of Education, Science, Sports, and Culture (14580798, 13358014 and 14380382).

The stemness maintenance of embryonic stem cells (ESCs) requires pluripotency transcription factors, including Oct4, Nanog, and Sox2. We have previously reported that protein arginine methyltransferase 7 (PRMT7), an epigenetic modifier, is an essential pluripotency factor that maintains the stemness of mouse ESCs, at least in part, by down-regulating the expression of the anti-stemness microRNA (miRNA) miR-24-2. To gain greater insight into the molecular basis underlying PRMT7-mediated maintenance of mouse ESC stemness, we searched for new PRMT7-down-regulated anti-stemness miRNAs. Here, we show that miR-221 gene-encoded miR-221-3p and miR-221-5p are anti-stemness miRNAs whose expression levels in mouse ESCs are directly repressed by PRMT7. Notably, both miR-221-3p and miR-221-5p targeted the 3' untranslated regions of mRNA transcripts of the major pluripotency factors Oct4, Nanog, and Sox2 to antagonize mouse ESC stemness. Moreover, miR-221-5p silenced also the expression of its own transcriptional repressor PRMT7. Transfection of miR-221-3p and miR-221-5p mimics induced spontaneous differentiation of mouse ESCs. CRISPR-mediated deletion of the miR-221 gene, as well as specific antisense inhibitors of miR-221-3p and miR-221-5p, inhibited the spontaneous differentiation of PRMT7-depleted mouse ESCs. Taken together, these findings reveal that the PRMT7-mediated repression of miR-221-3p and miR-221-5p expression plays a critical role in maintaining mouse ESC stemness. Our results also establish miR-221-3p and miR-221-5p as anti-stemness miRNAs that target Oct4, Nanog, and Sox2 mRNAs in mouse ESCs.

Endocrine disruptors (ED) may have adverse impacts on reproductive and immune systems in human and wild animals. It has been shown that octyl-phenol (OP) and nonyl-phenol (NP) have estrogenicity in estrogen-responding cells or tissues. In this study, we further investigated the effect(s) of OP and NP on the expression of undifferentiation and differentiation markers in mouse embryonic stem cells (ESC), which function as an important factor in the differentiation of ESC into cardiomyocytes. Mouse ESC were cultured in hanging drops to form embryoid bodies (EB). The medium was replaced with phenol red-free DMEM/F-12 supplemented with 5% charcoal-dextran-stripped FBS. The ESC were treated with OP, NP (1Ã-10-6 and 1Ã-10-7 M) or 17β-estradiol (E2; 1Ã-10-8 and 1Ã-10-9 M) in a time-dependent manner (1, 2 and 3 days), and EB were treated with identical concentrations for 4 and 8 days, respectively. High increasing doses of OP and NP were employed in this study because a binding affinity of ED to estrogen receptors (ER) is about 1000 less than that of E2. We determined the mRNA expression of undifferentiation markers (Oct4, Sox2 and Zfp206) and cardiomyocyte differentiation markers (cardiac alpha-MHC, beta-MHC and myosin light chain isoform-2V) using real-time PCR. In ESC, undifferentiation markers were identified. It is of interest that treatment with OP, NP or E2 induced a significant increase (1.4 5.5-fold) in Oct4 expression at the transcription levels according to a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2 and Zfp206 genes in ESC, suggesting that OP and NP may play a role as an Oct4 enhancer in ESC. In addition, both undifferentiation and cardiomyocyte differentiation markers were identified in EB. Treatment with OP and NP induced a significant increase in the expression of Oct4, Sox2 and Zfp206 genes at the transcription levels in a dose-dependent manner for 4 days, whereas Oct4 expression was only induced at these doses for 8 days. In contrast, cardiomyocyte differentiation markers were reduced by these ED in EB. Taken together, these results suggest that OP and NP play a role as a positive regulator in the undifferentiation process of ESC and EB, and maintenance and differentiation of mouse ESC.

The healing process of diabetic foot ulcers (DFUs) presents a slow pattern with an increased risk of infections, ischemia, and thrombosis correlated with high levels of reactive oxygen species production. Vascular injury is one of the factors contributing to the difficulty of wound healing in diabetic patients. Although the understanding of the pathophysiology of DFUs has significantly increased in recent years, associated treatments still have a high level of failure, leading to high morbidity rates, mortality, and amputations. Three-dimensional (3D) cell culture platforms offer a new approach to investigating and treating these wounds, as they can reproduce one or more physiological systems in a relevant microenvironment. This systematic review describes the advancements, challenges, and future implications of advanced 3D culture models in vascularization, encompassing pathophysiological understanding, treatment, and prognostic perspectives. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This work was registered under PROSPERO protocol number CRD42022336473. The eligibility criteria addressed studies related to chronic DFUs that analyzed vascularization during the healing process. The selected study designs involved 3D cultures and organ-on-a- chip (OoC) platforms, utilizing either primary or secondary human cell lines. Studies published more than 10 years ago or using only animal cells in 2D culture environments were excluded. The search was conducted in PubMed, LILACS, Embase, MEDLINE, IEEE, the BSV regional portal, ScienceDirect, Scopus, CINAHL, EBSCO, and Web of Science. A total of 2,539 relevant studies were identified as of June 1, 2025. After screening, only seven met the inclusion criteria. These studies reflect a growing interest in using hydrogel scaffolds and microfluidic systems to replicate diabetic skin environments; however, the field remains in an early stage of development. OoC platforms, in particular, stand out for their ability to recreate dynamic, tissue-like conditions and vascular function. While some promising attempts have been made to combine hydrogels with these technologies, the evidence is still limited and inconclusive for chronic wound modeling. This review underscores both the potential and the urgent need for more robust and translational research in this area—especially toward building personalized, clinically relevant models to support future drug testing and therapeutic innovation.

Developmental pluripotency-associated 2 (Dppa2) gene is one of the genes recently identified to be expressed specifically in pluripotent cells. To investigate the role of Dppa2 in mouse embryonic stem (ES) cells, we examined its expression during differentiation and performed knockdown of Dppa2 in mouse ES cells. Our results showed that the expression of Dppa2 decreased markedly in differentiated cells. Dppa2 knockdown induced the differentiation of mouse ES cells, as indicated by reduced alkaline phosphatase activity, slightly downregulated expression of the putative pluripotency marker genes Oct4 and Nanog and increased expression of early differentiation marker genes, such as Fst and Psx1. Moreover, reduced expression of Dppa2 also repressed cell proliferation activity as shown by the 5-bromo-2-deoxyuridine incorporation assay. Hence, Dppa2 might play a role in maintenance of the undifferentiated state and proliferation of ES cells.

ABSTRACTWe reported previously that EGAM1 homeoproteins (EGAM1 and EGAM1N), transcribed from the Crxos gene as splicing variants, are expressed in preimplantation mouse embryos and mouse embryonic stem (ES) cells. Exogenous expression of these proteins affects the maintenance of an undifferentiated state and the progression of differentiation in mouse ES cells. Human tetrapeptide-repeat homeobox 1 (TPRX1), a member of the eutherian totipotent cell homeobox (ETCHbox) genes, is an ortholog of Crxos. However, the roles of TPRX1 in the differentiation of human pluripotent cells are still unknown. Because the TPRX1 transcripts were undetectable in an undifferentiated state and during the progression of differentiation in wild-type human embryonal carcinoma NT2/D1 cells, it would be advantageous to clarify the relationship between the exogenous expression of TPRX1 and the induction of genes encoding lineage-specific transcription factors in pluripotent cells. The expression of GATA6 and FOXA2, crucial transcription factors for the formation of the primitive endoderm, was upregulated, whereas that of CDX2, a crucial transcription factor for the formation of the trophectoderm, was downregulated by enforced expression of TPRX1. Overall, we suggest that TPRX1 is capable of modulating the expression of lineage-specific transcription factors in pluripotent cells derived from humans.

Embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications to regenerative medicine for diabetes; however, a useful and safe way to generate pancreatic β cells has not been developed. In this study, we tried to establish an effective method of differentiation through the protein transduction of three transcription factors (Pdx1, NeuroD, and MafA) important to pancreatic β cell development. The method poses no risk of unexpected genetic modifications in target cells. Transduction of the three proteins induced the differentiation of mouse ES and mouse iPS cells into insulin-producing cells. Furthermore, a laminin-5-rich extracellular matrix efficiently induced differentiation under feeder-free conditions. Cell differentiation was confirmed with the expression of the insulin 1 gene in addition to marker genes in pancreatic β cells, the differentiated cells secreted glucose-responsive C-peptide, and their transplantation restored normoglycemia in diabetic mice. Moreover, Pdx1 protein transduction had facilitative effects on differentiation into pancreatic endocrine progenitors from human iPS cells. These results suggest the direct delivery of recombinant proteins and treatment with laminin-5-rich extracellular matrix to be useful for the generation of insulin-producing cells.

Inhibiting histone deacetylase (HDAC) activity modulates the epigenetic status of cells, resulting in an alteration of gene expression and cellular function. Here, we investigated the effects of HDAC inhibitors on mouse embryonic stem (ES) cells. The HDAC inhibitors trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate, and valproic acid induced early differentiation of mouse ES cells and triggered induction of heatshock protein (HSP)70. In contrast, class III HDAC inhibitors failed to induce differentiation or HSP70 expression. Transcriptional upregulation of HSP70 was confirmed by mRNA expression analysis, an inhibitor study, and chromatin immunoprecipitation. HSP70 induction was dependent on the SAPK/ JNK, p38, and PI3K/Akt pathways. Differentiation and induction of HSP70 by a subset of HDAC inhibitors was also examined in human ES cells, which suggests that the phenomenon generally occurs in ES cells. A better understanding of the effects of HDAC inhibitors may give more insight into their application in stem cell biology.

Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4, Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen, Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but, until now, its role in mammalian development and ESCs has not been explored. In this study, we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs, we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly, knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2, Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore, we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle.

A Small Molecule Primes Embryonic Stem Cells for Differentiation

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

Paternally expressed gene 10 (PEG10) is an imprinting gene. In addition to its known roles in placental development, as well as mouse embryonic stem cell and trophoblast stem cell differentiation, PEG10 has recently been shown to have significance in cancers. High expression of PEG10 is observed in various cancer types and is associated with poor prognosis. Of note, disruption of PEG10 expression leads to increased apoptosis, as well as decreased proliferation, invasion and migration of cancer cells. PEG10 is expected to become a target for cancer and neurodegenerative disorder therapy. This article reviewed the latest progress in the role of PEG10 in cancers.