Embryonic Stem Cells Research Articles

Synergistic Roles of Non-Homologous End Joining and Homologous Recombination in Repair of Ionizing Radiation-Induced DNA Double Strand Breaks in Mouse Embryonic Stem Cells.

DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PKcs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PKcs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PKcs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PKcs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PKcs or both DNA-PKcs and Rad54 exhibit an increased nuclear size approximately 18-24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PKcs, we generated DNA-PKcs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PKcs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PKcs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PKcs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PKcs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes.

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Histone H3.3 lysine 9 and 27 control repressive chromatin at cryptic enhancers and bivalent promoters

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutate histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleash cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupts the deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal levels of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs.

Directed differentiation of human embryonic stem cells into conjunctival epithelial cells

Severe conjunctival damage can lead to extensive ocular cicatrisation, fornix shortening, and even ocular surface failure, resulting in significant vision impairment. Conjunctival reconstruction is the primary therapeutic strategy for these clinical conjunctival diseases. However, there have been limited studies on induced differentiation of conjunctival epithelial cells derived from stem cells. In this study, we established a chemical defined differentiation protocol from human embryonic stem cells (hESCs) into conjunctival epithelial cells. hES cell line H1 was used for differentiation, and RT-qPCR, immunofluorescence staining, Periodic-acid-Schiff staining (PAS), and transcriptome analysis were employed to identify the differentiated cells. Here, to imitate the development of the vertebrate conjunctiva, hESCs were induced using a three-step process involving first chetomin was used to induce ocular surface ectoderm, then nicotinamide was used to induce ocular surface epithelial progenitor cells, and finally epidermal growth factor, keratinocyte growth factor and other factors were used to differentiate mature conjunctival epithelial cells. hESC-derived conjunctival epithelial cells expressed mature conjunctival epithelial lineage markers (including PAX6, P63, K13). The presence of goblet cells was confirmed by positive PAS. Transcriptome analysis revealed that hESC-derived conjunctival epithelial cells possessed a more naïve phenotype, and exhibited greater proliferation capacity compared to mature human conjunctival epithelial cells, suggesting their potential as alternative seed cells for conjunctival reconstruction.

Differentiating Human Embryonic Stem Cells into Oligodendrocytes

Differentiating Human Embryonic Stem Cells into Oligodendrocytes

A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Differentiating Human Embryonic Stem Cells into Oligodendrocytes

Differentiating Human Embryonic Stem Cells into Oligodendrocytes

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Low Immunogenicity of Keratinocytes Derived from Human Embryonic Stem Cells.

Epidermal transplantation is a common and widely used surgical technique in clinical medicine. Derivatives of embryonic stem cells have the potential to serve as a source of transplantable cells. However, allograft rejection is one of the main challenges. To investigate the immunogenicity of keratinocytes derived from human embryonic stem cells (ESKCs), we conducted a series of in vivo and in vitro experiments. The results showed that ESKCs have low HLA molecule expression, limited antigen presentation capabilities, and a weak ability to stimulate the proliferation and secretion of inflammatory factors in allogeneic PBMCs in vitro. In humanized immune mouse models, ESKCs elicited weak transplant rejection responses in the host. Overall, we found that ESKCs have low immunogenicity and may have potential applications in the field of regenerative medicine.

Genome access is transcription factor-specific and defined by nucleosome position

Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable the binding of TFs with diverse tissue specificities, functions, and DNA-binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly phased nucleosome. The pluripotency factors OCT4-SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome-internal sites, a preference that also guides binding genome wide. By contrast, factors such as BANP, REST, or CTCF engage throughout, causing nucleosomal displacement. This supports that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF specific and influenced by nucleosome position in the cell.

USP31 promotes the inflammatory response of human lung organoids to lipopolysaccharide treatment.

Acute lung injury (ALI) is a severe condition characterized by a high mortality rate, driven by an uncontrolled inflammatory response. Emerging evidence has underscored the crucial role of the ubiquitin system in ALI. However, due to their vast number, the specific functions of individual ubiquitination regulators remain unclear. In this study, we established human lung organoids (HLOs) derived from human embryonic stem cells and subjected them to lipopolysaccharide (LPS) treatment to induce an inflammatory response, mimicking ALI. Subsequently, we detected the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interleukin 6, interleukin 18 (IL-18), and interleukin-1β (IL-1β), by qPCR experiments. We also detected changes in the mRNA expression of several USPs before and after HLOs treatment and thus screened for USPs that had significant changes in HLOs after LPS stimulation. After screening for USP, we silenced the USP in HLOs and then subjected them to LPS treatment, and TNF-α, IL-6, IL-18, and IL-1β expressions were detected using qPCR assays. Meanwhile, Western blot was used to detect changes in NOD-, LRR- and pyrin domain-containing 3 (NLRP3) and apoptosis-associated Speck-like protein containing a CARD (ASC) protein level in HLOs. Through screening the expression of 40 ubiquitin-specific proteases (USPs), which are responsible for removing ubiquitination, we identified several USPs that exhibited differential expression in LPS-treated HLOs compared to untreated HLOs. Notably, USP31 emerged as the most significantly upregulated USP, and the knockdown of USP31 markedly attenuated the inflammatory response of HLOs to LPS treatment. USP31 may play a facilitating role in the inflammatory response during ALI.

H3.3K122A results in a neomorphic phenotype in mouse embryonic stem cells.

The histone variant H3.3 acts in coordination with histone posttranslational modifications and other chromatin features to facilitate appropriate transcription. Canonical histone H3 and histone variant H3.3 are post-translationally modified with the genomic distribution of these marks denoting different features and with more recent evidence suggesting that these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the residue H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent, albeit with reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue of H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

Ubiquitination plays an important role during the formation of chicken primordial germ cells.

As an important posttranslational modification, ubiquitination plays an important role in regulating protein homeostasis in eukaryotic cells. In our previous studies, both the transcriptome and proteome suggested that ubiquitination is involved in the formation of chicken primordial germ cells (PGCs). Here, affinity enrichment combined with liquid chromatography-tandem mass spectrometry (MS/MS) was used to analyze the ubiquitome during the differentiation from embryonic stem cells to PGCs, and we identify that 724 lysine ubiquitinated sites were up-regulated in 558 proteins and 138 lysine ubiquitinated sites were down-regulated in 109 proteins. Furthermore, GO and KEGG enrichment analysis showed that ubiquitination regulates key proteins to participate in the progression of key events related to PGC formation and the transduction of key signals such as Wnt, MAPK, and insulin signals, followed by the detailed explanation of the specific regulatory mechanism of ubiquitination through the combined proteome and ubiquitome analysis. Moreover, both the activation and inhibition of neddylation were detrimental to the maintenance of the biological characteristics of PGCs, which also verified the importance of ubiquitination. In conclusion, this study provides a global view of the ubiquitome during the formation of PGCs by label-free quantitative ubiquitomics, which lays a theoretical foundation for the formation mechanism and specific application of chicken PGCs.

Large animal model species in pluripotent stem cell therapy research and development for retinal diseases: a systematic review.

Retinal cell therapy modalities, in the category of advanced therapy medicinal products (ATMPs), are being developed to target several retinal diseases. Testing in large animal models (LAMs) is a crucial step in translating retinal ATMPs into clinical practice. However, challenges including budgetary and infrastructure constraints can hinder LAM research design and execution. Here, to facilitate the comparison of the various LAMs in pluripotent retinal cell therapy research, we aimed to systematically evaluate the species distribution, reported scientific utility, and methodology of a range of LAMs. A systematic search using the words retina, stem cell, transplantation, large animal, pig, rabbit, dog, and nonhuman primate was conducted in the PubMed, Embase, Science Direct and GoogleScholar databases in February 2023. We included 22 studies involving pluripotent stem cells (induced pluripotent stem cells or human embryonic stem cells) in LAMs, including non-human primates (NHP), pigs, dogs, and rabbits. Nearly half of the studies utilized wild-type animal models. In other studies, retinal degeneration features were simulated via laser, chemical, or genetic insult. Transplants were delivered subretinally, either as cell suspensions or pre-formed monolayers (with or without biodegradable scaffolding). The transplanted cells dose per eye varied widely (40,000 - 4,000,000 per dose). Cells were delivered via vitrectomy surgery in 15 studies and by an "ab externo" approach in one study. Structural outcomes were assessed using confocal scanning laser ophthalmoscopy imaging. Functional outcomes included multifocal electroretinogram and, in one case, a measure of visual acuity. Generally, cell suspension transplants exhibited low intraretinal incorporation, while monolayer transplants incorporated more efficiently. Immune responses posed challenges for allogeneic transplants, suggesting that autologous iPSC-derived transplants may be required to decrease the likelihood of rejection. The use of appropriate LAMs helps to advance the development of retinal ATMPs. The anatomical similarity of LAM and human eyes allows the implementation of clinically-relevant surgical techniques. While the FDA Modernization Act 2.0 has provided a framework to consider alternative methods including tissue-on-a-chip and human cell culture models for pharmacologic studies, LAM testing remains useful for cell and tissue replacement studies to inform the development of clinical trial protocols.

Transcriptome and metabolome analyses reveal the effects of formula and breast milk on the growth and development of human small intestinal organoids

Breast milk is widely acknowledged as the ideal nutritional resource for infants and can well meet the nutritional requirements for baby’s growth and development. Infant formula is a substitute for breast milk, designed to closely mimic its composition and function for breast milk. Most of the previous studies used tumor colorectal cancer cell lines to study the nutritional potency of formula and its components, so realistic data closer to the baby could not be obtained. Small intestinal organoids, derived from differentiated human embryonic stem cells, can be used to simulate nutrient absorption and metabolism in vitro. In this experiment, we used small intestinal organoids to compare the nutrient absorption and metabolism of three infant formulae for 0–6 months with breast milk samples. Transcriptome and metabolome sequencing methods were used to analyze the differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs). The pathways related to DEGs, DEMs were enriched using GO, KEGG, GSEA and other methods to investigate their biological characteristics. We have found that both formula and breast milk promote the development of the infant’s immune system, nutrient absorption and intestinal development. In PMH1 we found that the addition of oligofructose to milk powder promoted lipid metabolism and absorption. In PMH2 we found that whey protein powder favours the development of the immune system in infants. In PMH3 we found that oligogalactans may act on the brain-gut axis by regulating the intestinal flora, thereby promoting axon formation and neural development. By linking these biological properties of the milk powder with its composition, we confirmed the effects of added ingredients on the growth and development of infants. Also, we demonstrated the validity of small intestine organoids as a model for absorption and digestion in vitro. Through the above analyses, the advantages and disadvantages of the roles of formula and breast milk in the growth and metabolism of infants were also compared.

Assessment of plant- and microbial-derived protein hydrolysates as sustainable for fetal bovine serum in seafood cell culture media

This study seeks to explore alternatives by substituting or reducing the conventional 10% serum concentration in zebrafish embryonic stem cell (ESC) growth media with protein hydrolysates sourced from peas, mushrooms, yeast, and algae. Notably, algae exhibited the highest protein content, optimal amino acid balance, and favorable functional properties. When applied at concentrations ranging from 1 to 10 mg/mL, all protein hydrolysates demonstrated pro-apoptotic effects and inhibited cell growth, particularly when used in conjunction with 10% serum. However, concentrations ranging from 0.001 to 0.1 mg/mL displayed anti-apoptotic properties and promoted cell proliferation. The study found that media containing 1% or 2.5% serum, along with 0.01 mg/mL of protein hydrolysates, supported cell growth effectively. Lactate Dehydrogenase (LDH) Activity served as an indicator of cell health and integrity under the specified conditions of protein hydrolysate supplementation. Cells cultured in serum-free media exhibited significantly decreased cell membrane integrity (P < 0.05) compared to those in regular media or media containing low serum (1% and 2.5%) along with low concentrations (0.01 mg/mL) of protein hydrolysates. Furthermore, analysis of Greenhouse Gas emissions (GHG) suggested that media formulations containing 1% serum combined with low concentrations of protein hydrolysates present a sustainable approach for cell-based seafood production.

Transcriptional Dynamics and Key Regulators of Adipogenesis in Mouse Embryonic Stem Cells: Insights from Robust Rank Aggregation Analysis.

Embryonic stem cells are crucial for studying developmental biology due to their self-renewal and pluripotency capabilities. This research investigates the differentiation of mouse ESCs into adipocytes, offering insights into obesity and metabolic disorders. Using a monolayer differentiation approach over 30 days, lipid accumulation and adipogenic markers, such as Cebpb, Pparg, and Fabp4, confirmed successful differentiation. RNA sequencing revealed extensive transcriptional changes, with over 15,000 differentially expressed genes linked to transcription regulation, cell cycle, and DNA repair. This study utilized Robust Rank Aggregation to identify critical regulatory genes like PPARG, CEBPA, and EP300. Network analysis further highlighted Atf5, Ccnd1, and Nr4a1 as potential key players in adipogenesis and its mature state, validated through RT-PCR. While key adipogenic factors showed plateaued expression levels, suggesting early differentiation events, this study underscores the value of ESCs in modeling adipogenesis. These findings contribute to our understanding of adipocyte differentiation and have significant implications for therapeutic strategies targeting metabolic diseases.