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Diana A, Setzu MD, Kokaia Z, Nat R, Maxia C, Murtas D. SmartFlare TM is a reliable method for assessing mRNA expression in single neural stem cells. World J Stem Cells 2021; 13:1918-1927. [PMID: 35069990 PMCID: PMC8727230 DOI: 10.4252/wjsc.v13.i12.1918] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 08/11/2021] [Accepted: 12/10/2021] [Indexed: 02/06/2023] Open
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.
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Affiliation(s)
- Andrea Diana
- Department of Biomedical Sciences, University of Cagliari, Monserrato 09042, Cagliari, Italy
| | - Maria Dolores Setzu
- Department of Biomedical Sciences, University of Cagliari, Monserrato 09042, Cagliari, Italy
| | - Zaal Kokaia
- Laboratory of Stem Cells & Restorative Neurology, Lund Stem Cell Center, Lund University, Lund SE-221 84, Lund, Sweden
| | - Roxana Nat
- Institute of Neuroscience, Medical University of Innsbruck, Innsbruck 6020, Austria
| | - Cristina Maxia
- Department of Biomedical Sciences, University of Cagliari, Monserrato 09042, Cagliari, Italy
| | - Daniela Murtas
- Department of Biomedical Sciences, University of Cagliari, Monserrato 09042, Cagliari, Italy
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Diana A, Gaido G, Maxia C, Murtas D. MicroRNAs at the Crossroad of the Dichotomic Pathway Cell Death vs. Stemness in Neural Somatic and Cancer Stem Cells: Implications and Therapeutic Strategies. Int J Mol Sci 2020; 21:E9630. [PMID: 33348804 PMCID: PMC7766058 DOI: 10.3390/ijms21249630] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Revised: 12/05/2020] [Accepted: 12/10/2020] [Indexed: 12/12/2022] Open
Abstract
Stemness and apoptosis may highlight the dichotomy between regeneration and demise in the complex pathway proceeding from ontogenesis to the end of life. In the last few years, the concept has emerged that the same microRNAs (miRNAs) can be concurrently implicated in both apoptosis-related mechanisms and cell differentiation. Whether the differentiation process gives rise to the architecture of brain areas, any long-lasting perturbation of miRNA expression can be related to the occurrence of neurodevelopmental/neuropathological conditions. Moreover, as a consequence of neural stem cell (NSC) transformation to cancer stem cells (CSCs), the fine modulation of distinct miRNAs becomes necessary. This event implies controlling the expression of pro/anti-apoptotic target genes, which is crucial for the management of neural/neural crest-derived CSCs in brain tumors, neuroblastoma, and melanoma. From a translational point of view, the current progress on the emerging miRNA-based neuropathology therapeutic applications and antitumor strategies will be disclosed and their advantages and shortcomings discussed.
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Affiliation(s)
- Andrea Diana
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy
| | | | - Cristina Maxia
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy
| | - Daniela Murtas
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy
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Zhang B, Xu H, Huang Y, Shu W, Feng H, Cai J, Zhong JF, Chen Y. Improving single-cell transcriptome sequencing efficiency with a microfluidic phase-switch device. Analyst 2019; 144:7185-7191. [PMID: 31688860 PMCID: PMC6925944 DOI: 10.1039/c9an00823c] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
In this paper, we present a novel method to improve the efficiency of single-cell transcriptome sequencing for analyzing valuable cell samples. The microfluidic device we designed integrates multiple single-cell isolation chambers with hydrodynamic traps and achieves a nearly 100% single-cell capture rate and minimal cell loss, making it particularly suitable for samples with limited numbers of cells. Single cells were encapsulated using a novel phase-switch method into picoliter-sized hydrogel droplets and easily recovered for subsequent reactions. Minimizing the reaction volume resulted in a high reverse transcription (RT) efficiency for RNA sequencing (RNA-Seq). With this novel microfluidic platform, we captured dozens of hESCs (H9) simultaneously and obtained live cells in individual picoliter volumes, thus allowing for the convenient construction of a high-quality library for deep single-cell RNA-Seq. Our single-cell RNA-Seq results confirmed that a spectrum of pluripotency existed within an H9 colony. This integrated microfluidic platform can be applied to various cell types for the investigation of rare cellular events, and the phase-switch single-cell processing strategy will improve the efficiency and accessibility of single-cell transcriptome sequencing analysis.
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Affiliation(s)
- Baoyue Zhang
- Key Lab for Health Informatics of Chinese Academy of Sciences, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
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Diana A, Gaido G, Murtas D. MicroRNA Signature in Human Normal and Tumoral Neural Stem Cells. Int J Mol Sci 2019; 20:ijms20174123. [PMID: 31450858 PMCID: PMC6747235 DOI: 10.3390/ijms20174123] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2019] [Revised: 08/16/2019] [Accepted: 08/20/2019] [Indexed: 12/11/2022] Open
Abstract
MicroRNAs, also called miRNAs or simply miR-, represent a unique class of non-coding RNAs that have gained exponential interest during recent years because of their determinant involvement in regulating the expression of several genes. Despite the increasing number of mature miRNAs recognized in the human species, only a limited proportion is engaged in the ontogeny of the central nervous system (CNS). miRNAs also play a pivotal role during the transition of normal neural stem cells (NSCs) into tumor-forming NSCs. More specifically, extensive studies have identified some shared miRNAs between NSCs and neural cancer stem cells (CSCs), namely miR-7, -124, -125, -181 and miR-9, -10, -130. In the context of NSCs, miRNAs are intercalated from embryonic stages throughout the differentiation pathway in order to achieve mature neuronal lineages. Within CSCs, under a different cellular context, miRNAs perform tumor suppressive or oncogenic functions that govern the homeostasis of brain tumors. This review will draw attention to the most characterizing studies dealing with miRNAs engaged in neurogenesis and in the tumoral neural stem cell context, offering the reader insight into the power of next generation miRNA-targeted therapies against brain malignances.
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Affiliation(s)
- Andrea Diana
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato (Cagliari), Italy.
| | - Giuseppe Gaido
- Department of Surgery, Cottolengo Mission Hospital Charia, 60200 Meru, Kenya
| | - Daniela Murtas
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato (Cagliari), Italy.
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Mimura S, Suga M, Liu Y, Kinehara M, Yanagihara K, Ohnuma K, Nikawa H, Furue MK. Synergistic effects of FGF-2 and Activin A on early neural differentiation of human pluripotent stem cells. In Vitro Cell Dev Biol Anim 2015; 51:769-75. [PMID: 25898826 DOI: 10.1007/s11626-015-9909-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2015] [Accepted: 04/01/2015] [Indexed: 10/23/2022]
Abstract
Neural differentiation is an important target of human embryonic stem cells, which provide a source for cell-based therapy, developmental biology, and pharmaceutical research. Previous studies revealed that inhibition of the bone morphogenetic protein is required for neural induction from human embryonic stem cells. On the contrary, the functions of fibroblast growth factors and Activin/Nodal signaling are controversial. Fibroblast growth factor-2 and Activin/Nodal pathways exert divergent influences on human embryonic stem cell concerning the maintenance of both pluripotency and cellular differentiation. We hypothesized that the combination of fibroblast growth factor-2 and Activin A at various concentrations synergistically exerts diverse effects on cell differentiation. To determine the effects of fibroblast growth factor-2 and Activin A on cellular differentiation into neural lineages, we examined the expression of neural differentiation markers in human embryonic stem cells treated with fibroblast growth factor-2 and/or Activin A at various concentrations in a growth factor-defined serum-free medium in short-term culture. In this study, we provide evidence that fibroblast growth factor-2 and Activin A synergistically regulated the initiation of human embryonic stem cell differentiation into neural cell lineages even though human embryonic stem cells autonomously differentiate into neural cell lineages.
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Affiliation(s)
- Sumiyo Mimura
- Laboratory of Stem Cell Cultures, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito Asagi, Ibaraki, Osaka, 567-0085, Japan.,Department of Oral Biology & Engineering, Integrated Health Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan
| | - Mika Suga
- Laboratory of Stem Cell Cultures, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito Asagi, Ibaraki, Osaka, 567-0085, Japan
| | - Yujung Liu
- Laboratory of Stem Cell Cultures, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito Asagi, Ibaraki, Osaka, 567-0085, Japan
| | - Masaki Kinehara
- Laboratory of Stem Cell Cultures, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito Asagi, Ibaraki, Osaka, 567-0085, Japan.,Department of Cellular and Molecular Biology, Basic Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan
| | - Kana Yanagihara
- Laboratory of Stem Cell Cultures, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito Asagi, Ibaraki, Osaka, 567-0085, Japan
| | - Kiyoshi Ohnuma
- Department of Bioengineering, Nagaoka University of Technology, Niigata, 940-2188, Japan
| | - Hiroki Nikawa
- Department of Oral Biology & Engineering, Integrated Health Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan
| | - Miho K Furue
- Laboratory of Stem Cell Cultures, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito Asagi, Ibaraki, Osaka, 567-0085, Japan.
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