Cellular reprogramming最新文献

Spermatogonial stem cells (SSCs) play an essential role in the transfer of genetic information through generations, making studying their cellular and molecular mechanisms critical. However, since SSCs are few in mice, directly studying them is limited, requiring specialized in vitro cultivation. Feeder layers such as mouse embryonic fibroblasts (MEFs), SNL, neonate, and adult mouse testicular stromal feeder cells (TSCs) support in vitro survival and growth. To understand the effectiveness of these feeder layers on SSC proliferation, we compared MEF, SNL, neonatal, and adult TSCs. Furthermore, we identified hub genes and potential pathways in spermatogenesis. Two populations of differentiated and undifferentiated SSCs were compared for mouse SSC colony formation and proliferation effectiveness. Additionally, Cytoscape and STRING databases were employed for protein-protein interaction networks and functional gene enrichment. The expression of three hub genes, including Dazl, Zbtb16, and Stra8, was analyzed using dynamic array chips (Fluidigm) followed by statistical analysis. Our results indicated that undifferentiated SSCs favored MEF feeders, while differentiated SSCs thrived on SNL and primary TSC feeders for long-term culture. Functional enrichment results demonstrated hub genes involvement in cell differentiation, meiosis, regulation of meiotic nuclear division, cell development, and spermatogenesis. Furthermore, mRNA expression levels of Stra8, Zbtb16, and Dazl genes show different patterns among feeder layers and SSC differentiation phases.

From the first cloning of animals-salamanders-to the cloning of primates-monkeys-nuclear transfer research has spanned an extensive 96-year history. Over the course of nearly a century, it has addressed fundamental scientific questions and found applications across a wide range of practical fields. This review provides a comprehensive overview of the key milestones in its development, its practical applications, and the challenges it continues to face.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system is revolutionizing genome engineering and is expected to bring significant advancements in livestock traits, including the treatment of genetic diseases. This study focuses on CRISPR/Cas9-mediated modifications of the CYP19 gene, which encodes aromatase, an enzyme crucial for converting testosterone to estrogen and essential for steroid metabolism. Guide RNAs (gRNAs) were designed to target the CYP19 gene and cloned into the pX459 vector. The recombinant plasmid was then electrotransfected into fibroblast cells from a Lori-Bakhtiari buck, and these transfected cells were used for embryo production via somatic cell nuclear transfer (SCNT). The cloned embryos were evaluated for their progression through embryonic stages, showing no significant difference in blastocyst development between knock-out and unedited groups. The knockout efficiency was 78.4% in cells and 68.9% in goat blastocysts, demonstrating the successful depletion of CYP19. We successfully achieved a high rate of CYP19 gene-edited embryos through the combined application of cell electrotransfection and SCNT technologies, while maintaining the normal developmental rate of the embryos. These embryos can be used for transfer to generate knock-out goats, providing a foundation for further studies on CYP19's role in male fertility and production traits.

Stem cells are key to human tissue maintenance. Because tissue maintenance allows us to live and reproduce, stem cell control is fundamental for animal life and evolution. A team of researchers set out to explore the origins of transcription factors at the core of the induction and the maintenance of stemnss. They focus on the conservation of the Sry-related box 2 (Sox2) and the octamer-binding transcriptor factor 4 (Oct4) in the Pit-Oct-Unc (POU) family. While these have been thought as animal-specific, the authors identified SOX and POU in pre-animal organisms. In particular, the SOX protein from a very simple unicellular organism was functionally conserved enough to reprogram somatic mouse cells to induce pluripotent stem cells. To ponder on the importance of their findings, we first need to step back a couple of hundred million years.

Genome editing techniques have potential to revolutionize the field of life sciences. Several limitations associated with traditional gene editing techniques have been resolved with the development of prime editors that precisely edit the DNA without double-strand breaks (DSBs). To further improve the efficiency, several modified versions of prime editing (PE) system have been introduced. Bi-directional PE (Bi-PE), for example, uses two PE guide RNAs enabling broad and improved editing efficiency. It has the potential to alter, delete, integrate, and replace larger genome sequences and edit multiple bases at the same time. This review aims to discuss the typical gene editing methods that offer DSB-mediated repair mechanisms, followed by the latest advances in genome editing technologies with non-DSB-mediated repair. The review specifically focuses on Bi-PE being an efficient tool to edit the human genome. In addition, the review discusses the applications, limitations, and future perspectives of Bi-PE for gene editing.

Glioblastoma multiforme (GBM) is a highly invasive brain tumor, and traditional treatments combining surgery with radiochemotherapy have limited effects, with tumor recurrence being almost inevitable. Given the lack of proliferative capacity in neurons, inducing terminal differentiation of GBM cells or glioma stem cells (GSCs) into neuron-like cells has emerged as a promising strategy. This approach aims to suppress their proliferation and self-renewal capabilities through differentiation. This review summarizes the methods involved in recent research on the neuronal differentiation of GBM cells or GSCs, including the regulation of transcription factors, signaling pathways, miRNA, and the use of small molecule drugs, among various strategies. It also outlines the interconnections between the mechanisms studied, hoping to provide ideas for exploring new therapeutic avenues for GBM and the development of differentiation-inducing drugs for GBM.

This study explores the protective mechanism of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in diabetic foot ulcer (DFU). Human umbilical cord MSCs (HucMSCs) were identified via osteogenesis and adipogenic differentiation, as well as flow cytometry. EVs were isolated from HucMSCs and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Fluorescence microscopy revealed the uptake of PKH67-labeled EVs and Cy3-labeled microRNA-21-5p (miR-21-5p) by human skin fibroblasts (HSFs). EVs were cocultured with HSFs, and cell proliferation and migration were assessed using Cell Counting Kit-8, colony formation, scratch, and Transwell assays. miR-21-5p overexpression in EVs was evaluated for its role in promoting HSF functions. The expression levels of miR-21-5p, Krüppel-like factor 6 (KLF6), α-smooth muscle actin, and collagen type I alpha 1 chain were analyzed via quantitative real-time PCR and Western blotting. The interaction between miR-21-5p and KLF6 was confirmed through a dual-luciferase reporter gene assay. HucMSC-derived EVs enhanced the proliferation and migration of HSFs under high glucose by delivering miR-21-5p, which targeted and inhibited KLF6. Overexpression of KLF6 counteracted the pro-proliferative and migratory effects of EVs carrying miR-21-5p. Overall, these findings suggest that HucMSC-EVs promote HSF proliferation and migration by downregulating KLF6 via miR-21-5p delivery, offering a potential therapeutic strategy for DFU.