A Case Study from the Past: “The RGC-5 vs. the 661W Cell Line: Similarities, Differences and Contradictions—Are They Really the Same?”
Abstract
:1. Introduction
- The origin and nature of the RGC-5 cell line: A fundamental research gap exists concerning the uncertain origin of the RGC-5 cell line. Lingering concerns regarding possible contamination with or genetic similarity to 661W cone photoreceptor cells have cast doubt on the lineage of RGC-5 cells. As such, there is a pressing need to investigate the true source and nature of the RGC-5 cell line to ensure its reliability as a retinal ganglion cell model.
- Clarifying differentiation status: Another key research gap involves determining the true differentiation status of the RGC-5 and 661W cell lines. A thorough examination is essential to ascertain whether these cell lines genuinely represent retinal ganglion cells or exhibit properties more akin to photoreceptor cells. Resolving this uncertainty is vital for accurate interpretation of experimental outcomes and targeted therapeutic studies.
- Evaluating research findings reliability: Previous studies utilizing RGC-5 cells have reported conflicting results, with some indicating ganglion cell-specific markers, while others suggested photoreceptor-like behavior. This discrepancy calls into question the reliability and validity of research findings based on RGC-5 cells. Addressing this limitation is crucial to ensure the credibility of ocular pathology studies and pave the way for robust scientific advancements.
- Lack of standardized differentiation protocols: The absence of standardized differentiation protocols for the RGC-5 and 661W cell lines has hindered accurate comparisons of their properties. Addressing this limitation by employing rigorous and consistent differentiation procedures will enhance the reliability and interpretability of experimental results.
- Concerns about contamination and cell properties: Persistent concerns regarding potential contamination and loss of cell properties have cast doubts on the trustworthiness of research conducted using RGC-5 cells. These concerns necessitate systematic investigations to ascertain the integrity and authenticity of the cell lines under study.
2. Results
2.1. Karyotyping
- 661W: 60~64,XY,−2,+der(3;3),−4,+5,+5,−6,−7,+10,−11,+12,+17,+17,+mar,+mar,+mar,+mar
- RGC-5: 55~59,X,−4,−5,−6,+10,+11,−12,−13,−13,−14,+15,−16,+17,−18,+mar,+mar,+mar,+mar,+mar
2.2. Morphology Changes in RGC-5 Cells and 661W Cells Due to TSA Treatment
2.3. Cell Amount and Viability of RGC-5 Cells and 661W Cells during TSA Incubation
2.4. The Effect of TSA on Apoptosis in RGC-5 Cells and 661W Cells
2.5. β-III Tubulin Expression in RGC-5 Cells and 661W Cells Caused by TSA Treatment
2.6. Relative Expression of Thy-1, PGP9.5, CRX- and GFAP-mRNA in RGC-5 and 661W Cells after TSA Incubation
3. Discussion
3.1. Characteristics of the RGC-5 Cells in the Present Study
3.2. The Reaction of 661W Cells to TSA Incubation
3.3. The Effects of TSA
3.4. The Problem of Cell Line Contamination
3.5. Contributions to Knowledge
4. Materials and Methods
4.1. Cell Culture
4.2. Chromosome Analysis
4.3. SDS-PAGE and Western Blot
4.4. Immunofluorescence Staining
4.5. qRT-PCR
4.6. MTS Viability Assay
4.7. Crystal Violet Staining
4.8. Caspase 3/7 Activity Assay
4.9. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Protocol | Schwechter et al. [16] | Wood et al. [14] |
---|---|---|
Morphology | RGC-5: Differentiation toward a neuronal phenotype after long-term incubation with TSA. 661W: Fewer cells changed morphologies, and neurites were fewer and shorter. Importantly, the majority of cells died during long-term incubation. | Both: Similar trend to that in Schwechter et al. |
β-III tubulin | RGC-5: In Western blot analysis as well as in the immunohistochemistry, maximum values for long-term incubation (96 h and 120 h) were with TSA. 661W: First, an increase in β-III tubulin expression could be observed. After 72 h, values dropped again. | RGC-5: Maximum increase could be detected after long-term incubation. Increase in expression was stronger than in treatment after Schwechter protocol. 661W: No increase in β-III tubulin expression under long-term incubation. |
MAP-2, Tau | Both: Expression increased with long-term incubation. | Both: Expression increased. |
Thy-1 mRNA | Both: No effect | RGC-5: Slight increase after incubation time of 120 h. 661W: No effect |
PGP9.5 mRNA | Both: No effect | 661W: Twofold increase in mRNA expression after 120 h. |
CRX mRNA | RGC-5: Sevenfold increase in mRNA expression after 120 h. 661W: No effect | RGC-5: Slight decrease in mRNA expression after 120 h. 661W: Fourfold increase after 120 h. |
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Hurst, J.; Attrodt, G.; Bartz-Schmidt, K.-U.; Mau-Holzmann, U.A.; Spitzer, M.S.; Schnichels, S. A Case Study from the Past: “The RGC-5 vs. the 661W Cell Line: Similarities, Differences and Contradictions—Are They Really the Same?”. Int. J. Mol. Sci. 2023, 24, 13801. https://doi.org/10.3390/ijms241813801
Hurst J, Attrodt G, Bartz-Schmidt K-U, Mau-Holzmann UA, Spitzer MS, Schnichels S. A Case Study from the Past: “The RGC-5 vs. the 661W Cell Line: Similarities, Differences and Contradictions—Are They Really the Same?”. International Journal of Molecular Sciences. 2023; 24(18):13801. https://doi.org/10.3390/ijms241813801
Chicago/Turabian StyleHurst, José, Gesine Attrodt, Karl-Ulrich Bartz-Schmidt, Ulrike Angelika Mau-Holzmann, Martin Stephan Spitzer, and Sven Schnichels. 2023. "A Case Study from the Past: “The RGC-5 vs. the 661W Cell Line: Similarities, Differences and Contradictions—Are They Really the Same?”" International Journal of Molecular Sciences 24, no. 18: 13801. https://doi.org/10.3390/ijms241813801