| 1. |
- Diana, Andrea, et al.
(author)
-
SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells
- 2021
-
In: World Journal of Stem Cells. - : Baishideng Publishing Group Inc.. - 1948-0210. ; 13:12, s. 1918-1927
-
Journal article (peer-reviewed)abstract
- BACKGROUND One of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders. AIM To investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation. METHODS Brain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133 and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells. RESULTS In agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes. CONCLUSION SmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.
|
|
| 2. |
- Hanson, C., et al.
(author)
-
Transplanting embryonic stem cells onto damaged human corneal endothelium
- 2017
-
In: World Journal of Stem Cells. - : Baishideng Publishing Group Inc.. - 1948-0210. ; 9:8, s. 127-132
-
Journal article (peer-reviewed)abstract
- AIM To investigate whether human embryonic stem cells (hESCs) could be made to attach, grow and differentiate on a human Descemet's membrane (DM). Spontaneously differentiated hESCs were transferred onto a human corneal button with the endothelial layer removed using ocular sticks. The cells were cultured on a DM for up to 15 d. The genetically engineered hESC line expressed green fluorescent protein, which facilitated identification during the culture experiments, tissue preparation, and analysis. To detect any differentiation into human corneal endothelial-like cells, we analysed the transplanted cells by immunohistochemistry using specific antibodies. We found transplanted cells form a single layer of cells with a hexagonal shape in the periphery of the DM. The majority of the cells were negative for octamer-binding transcription factor 4 but positive for paired box 6 protein, sodium potassium adenosine triphosphatase (NaKATPase), and Zona Occludens protein 1. In four of the 18 trials, the transplanted cells were found to express CK3, which indicates that the stem cells differentiated into corneal epithelial cells in these cases. It is possible to get cells originating from hESCs to become established on a human DM, where they grow and differentiate into corneal endothelial-like cells in vitro.
|
|
| 3. |
|
|
| 4. |
- Martin-Rubio, Paula, et al.
(author)
-
Metabolic determinants of stemness in medulloblastoma
- 2022
-
In: World Journal of Stem Cells. - : BAISHIDENG PUBLISHING GROUP INC. - 1948-0210. ; 14:8, s. 587-598
-
Research review (peer-reviewed)abstract
- Medulloblastomas (MBs) are the most prevalent brain tumours in children. They are classified as grade IV, the highest in malignancy, with about 30% metastatic tumours at the time of diagnosis. Cancer stem cells (CSCs) are a small subset of tumour cells that can initiate and support tumour growth. In MB, CSCs contribute to tumour initiation, metastasis, and therapy resistance. Metabolic differences among the different MB groups have started to emerge. Sonic hedgehog tumours show enriched lipid and nucleic acid metabolism pathways, whereas Group 3 MBs upregulate glycolysis, gluconeogenesis, glutamine anabolism, and glut athione-mediated anti-oxidant pathways. Such differences impact the clinical behaviour of MB tumours and can be exploited therapeutically. In this review, we summarise the existing knowledge about metabolic rewiring in MB, with a particular focus on MB-CSCs. Finally, we highlight some of the emerging metabolism-based therapeutic strategies for MB.
|
|
