Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.
Neurons undergoing targeted photolytic cell death degenerate by apoptosis. Clonal, multipotent neural precursor cells were transplanted into regions of adult mouse neocortex undergoing selective degeneration of layer II͞III pyramidal neurons via targeted photolysis. These precursors integrated into the regions of selective neuronal death; 15 ؎ 7% differentiated into neurons with many characteristics of the degenerated pyramidal neurons. They extended axons and dendrites and established afferent synaptic contacts. In intact and kainic acid-lesioned control adult neocortex, transplanted precursors differentiated exclusively into glia. These results suggest that the microenvironmental alterations produced by this synchronous apoptotic neuronal degeneration in adult neocortex induced multipotent neural precursors to undergo neuronal differentiation which ordinarily occurs only during embryonic corticogenesis. Studying the effects of this defined microenvironmental perturbation on the differentiation of clonal neural precursors may facilitate identification of factors involved in commitment and differentiation during normal development. Because photolytic degeneration simulates some mechanisms underlying apoptotic neurodegenerative diseases, these results also suggest the possibility of neural precursor transplantation as a potential cell replacement or molecular support therapy for some diseases of neocortex, even in the adult.
Exosomes are 30-150nM membrane-bound secreted vesicles that are readily isolated from biological fluids such as urine (UEs). Exosomes contain proteins, micro RNA (miRNA), messenger RNA (mRNA), and long non-coding RNA (lncRNA) from their cells of origin. Although miRNA, protein and lncRNA have been isolated from serum as potential biomarkers for benign and malignant disease, it is unknown if lncRNAs in UEs from urothelial bladder cancer (UBC) patients can serve as biomarkers. lncRNAs are > 200 nucleotide long transcripts that do not encode protein and play critical roles in tumor biology. As the number of recognized tumor-associated lncRNAs continues to increase, there is a parallel need to include lncRNAs into biomarker discovery and therapeutic target algorithms. The lncRNA HOX transcript antisense RNA (HOTAIR) has been shown to facilitate tumor initiation and progression and is associated with poor prognosis in several cancers. The importance of HOTAIR in cancer biology has sparked interest in using HOTAIR as a biomarker and potential therapeutic target. Here we show HOTAIR and several tumor-associated lncRNAs are enriched in UEs from UBC patients with high-grade muscle-invasive disease (HGMI pT2-pT4). Knockdown of HOTAIR in UBC cell lines reduces in vitro migration and invasion. Importantly, loss of HOTAIR expression in UBC cell lines alters expression of epithelial-to-mesenchyme transition (EMT) genes including SNAI1, TWIST1, ZEB1, ZO1, MMP1 LAMB3, and LAMC2. Finally, we used RNA-sequencing to identify four additional lncRNAs enriched in UBC patient UEs. These data, suggest that UE-derived lncRNA may potentially serve as biomarkers and therapeutic targets.
Many central nervous system regions at all stages of life contain neural stem cells (NSCs). We explored how these disparate NSC pools might emerge. A traceable clone of human NSCs was implanted intraventricularly to allow its integration into cerebral germinal zones of Old World monkey fetuses. The NSCs distributed into two subpopulations: One contributed to corticogenesis by migrating along radial glia to temporally appropriate layers of the cortical plate and differentiating into lamina-appropriate neurons or glia; the other remained undifferentiated and contributed to a secondary germinal zone (the subventricular zone) with occasional members interspersed throughout brain parenchyma. An early neurogenetic program allocates the progeny of NSCs either immediately for organogenesis or to undifferentiated pools for later use in the "postdevelopmental" brain.
In humans, beta-hexosaminidase alpha-subunit deficiency prevents the formation of a functional beta-hexosaminidase A heterodimer resulting in the severe neurodegenerative disorder, Tay-Sachs disease. To explore the feasibility of using ex vivo gene transfer in this lysosomal storage disease, we produced ecotropic retroviruses encoding the human beta-hexosaminidase alpha-subunit cDNA and transduced multipotent neural cell lines. Transduced progenitors stably expressed and secreted high levels of biologically active beta-hexosaminidase A in vitro and cross-corrected the metabolic defect in a human Tay-Sachs fibroblasts cell line in vitro. These genetically engineered CNS progenitors were transplanted into the brains of both normal fetal and newborn mice. Engrafted brains, analyzed at various ages after transplant, produced substantial amounts of human beta-hexosaminidase alpha-subunit transcript and protein, which was enzymatically active throughout the brain at a level reported to be therapeutic in Tay-Sachs disease. These results have implications for treating neurologic diseases characterized by inherited single gene mutations.
To delineate the biochemical sequences of myelination in the human brain, we analyzed the protein and lipid composition of white matter in 18 baseline cases ranging in age from midgestation through infancy, the critical period in human myelination when the most rapid changes occur. Three adult cases were used as indices of maturity, and 4 cases with major disorders of CNS myelination (maple syrup urine disease, severe periventricular leukomalacia, idiopathic central hypomyelination, and metachromatic leukodystrophy) were analyzed. Brain samples were obtained < or = 24 hours after death. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance thin-layer chromatography were used to separate and identify proteins and polar and neutral lipids in an average of 10 sites/brain; computer-based densitometry was used to quantify polar lipids. Biochemical sequences, as manifested by the appearance of the myelin-associated lipids and myelin-specific proteins, closely followed previously described anatomic sequences both temporally and by region, and were identical in all sites sampled: sphingomyelin was followed simultaneously by cerebrosides, MBP, PLP, and nonhydroxy-sulfatide, followed by hydroxy-sulfatide. The onset and tempo of the expression of individual constituents, however, were quite variable among sites, suggesting a wide differential in vulnerable periods to insult in biochemically-specific pathways in early life. Cholesterol ester was transiently elevated during late gestation and early infancy, prior to and around the time of the appearance of cerebrosides, sulfatides, PLP, and MBP. Distinctive lipid and protein abnormalities were detected in idiopathic central hypomyelination and metachromatic leukodystrophy. This study underscores the feasibility of the combined biochemical approaches in pediatric brains and provides guidelines for the assessment of disorders of myelination in early human life.
flax 3,4 & Thomas R. Gaborski 2,4 ✉ extracellular vesicles (eVs) are membrane vesicles secreted by cells and can modulate biological activities by transferring their content following uptake into recipient cells. Labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. A reliable fluorescent technique needs to preserve the size of EVs since changes in size may alter their uptake and biodistribution. Lipophilic fluorescent dye molecules such as the PKH family have been widely used for EV labelling. Here, the effect of PKH labelling on the size of EVs was systematically evaluated using nanoparticle tracking analysis (NTA), which is a widely used technique for determining the size and concentration of nanoparticles. NTA analysis showed a size increase in all the PKH labelling conditions tested. As opposed to lipophilic dye molecules, no significant shift in the size of labelled EVs was detected with luminal binding dye molecules such as 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, hereinafter CFSE). This finding suggests that PKH labelling may not be a reliable technique for the tracking of EVs.
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