STEM CELLS最新文献

There is increasing interest in the use of marsupial models in research, for use in next-generation conservation by improving fitness through genetic modification, and in de-extinction efforts. Specifically, this includes dasyurid marsupials such as the Thylacine, Tasmanian devil, quolls, and the small rodent-like dunnarts. Technologies for generating genetically modified Australian marsupials remain to be established. Given the need to advance research in this space, the fat-tailed dunnart (Sminthopsis crassicaudata) is being established as a model for marsupial spermatogonial stem cell isolation, modification, and testicular transplantation. This species is small (60-90 mm body size), polyovulatory (8-12 pups per birth), and can breed in standard rodent facilities when housed in a 12:12 light cycle. To develop the fat-tailed dunnart as a model for next-generation marsupial conservation, this study aimed to enrich dunnart spermatogonial stem cells from whole testis digestions using a fluorescent dye technology and fluorescence-activated cell sorting. This approach is not dependent on antibodies or genetic reporter animals that are limiting factors when performing cell sorting on species separated from humans and mice by large evolutionary timescales. This study also assessed the development of spermatogonia and spermatogenesis in the fat-tailed dunnart, by making the first definition of the cycle of the seminiferous epithelium in any dasyurid. Overall, this is the first detailed study to assess the cycle of dasyurid spermatogenesis and provides a valuable method to enrich marsupial spermatogonial stem cells for cellular, functional, and molecular analysis.

Neural stem cells (NSCs) hold great potential in neurodegenerative disease therapy, drug screening, and disease modeling. However, current approaches for induced NSCs (iNSCs) generation from somatic cells are still slow and inefficient. Here we report the establishment of a rapid and efficient method of iNSCs generation from human and mouse fibroblasts by using single microRNAs (miR-302a). These iNSCs exhibited morphological, molecular and functional properties resembling those of adult human and mouse NSCs, respectively. Additionally, human iNSCs can be expanded for more than 20 passages in vitro. Furthermore, miR-302a alone was demonstrated to be sufficient to reprogram both human and mouse fibroblasts into iNSCs. Our results showed a method of direct conversion of autologous fibroblasts with miR-302a into iNSCs, providing a rapid and efficient strategy to generate iNSCs for both basic research and clinical applications.

In our aging society, the degeneration of the musculoskeletal system and adjacent tissues is a growing orthopedic concern. As bones age, they become more fragile, increasing the risk of fractures and injuries. Furthermore, tissues like cartilage accumulate damage, leading to widespread joint issues. Compounding this, the regenerative capacity of these tissues declines with age, exacerbating the consequences of fractures and cartilage deterioration. With rising demand for fracture and cartilage repair, bone-derived stem cells have attracted significant research interest. However, the therapeutic use of stem cells has produced inconsistent results, largely due to ongoing debates and uncertainties regarding the precise identity of the stem cells responsible for musculoskeletal growth, maintenance and repair. This review focuses on the potential to leverage endogenous skeletal stem cells (SSCs)-a well-defined population of stem cells with specific markers, reliable isolation techniques, and functional properties-in bone repair and cartilage regeneration. Understanding SSC behavior in response to injury, including their activation to a functional state, could provide insights into improving treatment outcomes. Techniques like microfracture surgery, which aim to stimulate SSC activity for cartilage repair, are of particular interest. Here, we explore the latest advances in how such interventions may modulate SSC function to enhance bone healing and cartilage regeneration.

Extracellular microvesicles (ExMVs) were one of the first communication platforms between cells that emerged early in evolution. Evidence indicates that all types of cells secrete these small circular structures surrounded by a lipid membrane that plays an important role in cellular physiology and some pathological processes. ExMVs interact with target cells and may stimulate them by ligands expressed on their surface and/or transfer to the target cells their cargo comprising various RNA species, proteins, bioactive lipids, and signaling nucleotides. These small vesicles can also hijack some organelles from the cells and, in particular, transfer mitochondria, which are currently the focus of scientific interest for their potential application in clinical settings. Different mechanisms exist for transferring mitochondria between cells, including their encapsulation in ExMVs or their uptake in a "naked" form. It has also been demonstrated that mitochondria transfer may involve direct cell-cell connections by signaling nanotubules. In addition, evidence accumulated that ExMVs could be enriched for regulatory molecules, including some miRNA species and proteins that regulate the function of mitochondria in the target cells. Recently, a new beneficial effect of mitochondrial transfer has been reported based on inducing the mitophagy process, removing damaged mitochondria in the recipient cells to improve their energetic state. Based on this novel role of ExMVs in powering the energetic state of target cells, we present a current point of view on this topic and review some selected most recent discoveries and recently published most relevant papers.

Human induced pluripotent stem cells (iPSCs) provide powerful cellular models of Alzheimer's disease (AD) and offer many advantages over non-human models, including the potential to reflect variation in individual-specific pathophysiology and clinical symptoms. Previous studies have demonstrated that iPSC-neurons from individuals with Alzheimer's disease (AD) reflect clinical markers, including β-amyloid (Aβ) levels and synaptic vulnerability. However, despite neuronal loss being a key hallmark of AD pathology, many risk genes are predominantly expressed in glia, highlighting them as potential therapeutic targets. In this work iPSC-derived astrocytes were generated from a cohort of individuals with high versus low levels of the inflammatory marker YKL-40, in their cerebrospinal fluid (CSF). iPSC-derived astrocytes were treated with exogenous Aβ oligomers and high content imaging demonstrated a correlation between astrocytes that underwent the greatest morphology change from patients with low levels of CSF-YKL-40 and more protective APOE genotypes. This finding was subsequently verified using similarity learning as an unbiased approach. This study shows that iPSC-derived astrocytes from AD patients reflect key aspects of the pathophysiological phenotype of those same patients, thereby offering a novel means of modelling AD, stratifying AD patients and conducting therapeutic screens.

Background: Premature osteoporosis caused by parathyroid hormone-related peptide (PTHrP) dysfunction presents significant bone health challenges. The role of p16-mediated cellular senescence in this condition remains unclear.

Methods: Using a Pthrp knock-in (KI) mouse model lacking the nuclear localization sequence and C-terminus of PTHrP, we generated p16⁻⁄⁻KI mice and compared them with wild-type, p16⁻⁄⁻, and KI mice. We analyzed survival, skeletal phenotypes, bone marrow mesenchymal stem cell (BM-MSC) function, and molecular markers of senescence.

Results: Genetic ablation of p16 in KI mice extended their lifespan, increased body size and weight, and improved skeletal growth. Micro-CT analysis revealed significantly increased bone volume, while histological studies showed enhanced chondrocyte proliferation and osteoblast function in p16⁻⁄⁻KI mice compared to KI mice. In vitro experiments demonstrated enhanced differentiation capacity and reduced senescence of BM-MSCs from p16⁻⁄⁻KI mice, as evidenced by increased colony formation and osteogenic marker expression. Molecular analyses indicated that p16 knockout partially reversed oxidative stress, DNA damage, and cellular senescence observed in KI mice, shown by upregulated antioxidant enzymes, reduced DNA damage markers, and decreased senescence markers.

Conclusions: p16-mediated cellular senescence plays a crucial role in premature osteoporosis caused by PTHrP dysfunction. Targeting cellular senescence pathways may offer a promising therapeutic strategy for treating premature osteoporosis and age-related bone loss.

iPSCs can serve as a renewable source of a consistent edited cell product, overcoming limitations of primary cells. While feeder-free generation of clinical grade iPSC-derived CD8 T cells has been achieved, differentiation of iPSC-derived CD4sp and regulatory T cells requires mouse stromal cells in an artificial thymic organoid. Here we report a serum- and feeder-free differentiation process suitable for large-scale production. Using an optimized concentration of PMA/Ionomycin, we generated iPSC-CD4sp T cells at high efficiency and converted them to Tregs using TGFβ and ATRA. Using genetic engineering, we demonstrated high, non-viral, targeted integration of an HLA-A2 CAR in iPSCs. iPSC-Tregs ± HLA-A2-targeted CAR phenotypically, transcriptionally and functionally resemble primary Tregs and suppress T-cell proliferation in vitro. Our work is the first to demonstrate an iPSC-based platform amenable to manufacturing CD4 T cells to complement iPSC-CD8 oncology products and functional iPSC-Tregs to deliver Treg cell therapies at scale.

Background: Amniotic mesenchymal stem cells (AMSCs) have been demonstrated as effective in tissue repair and regeneration. Trophoblast dysfunction is associated with several types of pregnancy complications. The aim of this study is to investigate the effects of AMSCs on the biological activities of human trophoblasts, as well as their molecular mechanisms.

Methods: Exosomes were isolated from AMSC supernatants, and characterized and quantified by transmission electron microscopy, nanoparticle tracking analysis and Western blotting assay. Immunofluorescence assay was performed to detect the uptake of AMSCs-derived exomes (AMSC-Exos) by human trophoblasts. Human trophoblasts were subjected to transcriptome analysis after being cocultured with AMSC-Exos. Lentiviral transfection was performed to construct the human trophoblast cell lines with stable HRK knockdown or overexpression. Immunohistochemistry was used to detect the HRK expression in preeclampsia (PE) patients. CCK8 and Transwell assays were, respectively, used to detect the trophoblast proliferation and migration. TUNEL flow cytometry assay was used to detect the apoptosis in trophoblasts. Quantitative real-time (qRT) PCR and Western blotting assays were used to detect the mRNA and protein levels of the genes. Dual luciferase reporter assays were used to detect the changes in gene-transcript levels.

Results: AMSC-Exos could be absorbed by human trophoblasts. Transcriptome analysis showed that HRK was significantly reduced in human trophoblasts cocultured with AMSC-Exos. HRK inhibited cell proliferation and migration in human trophoblasts and promoted their apoptosis via p53 upregulation. miR-18a-5p, present at high levels in AMSC-Exos, improved trophoblast proliferation and migration, and inhibited their apoptosis by inhibiting the HRK expression.

Conclusion: miR-18a-5p present in AMSC-Exos could be absorbed by trophoblasts, in turn, improved their proliferation and migration, and inhibited their apoptosis by HRK downregulation.

The miR-290 and miR-302 clusters of microRNAs are highly expressed in naïve and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell (ESC)-specific cell cycle regulating family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their ESC microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state.