Full-term amniotic fluid stem cell line maintains its genome stability upon prolonged culture and cryopreservation. Malaysian Journal of Genetics (MJG), 2 (1). pp. 1-10. ISSN 2811-3594 (2022)
Abstract
Amniotic fluid stem cells (AFSCs) from full-term gestation pregnancy are broadly multipotent immune-privileged cells. They have fewer ethical issues with promising therapeutic potential, making them an ideal prospective stem cell type for therapy. Obtaining them in high quantity and quality in-vitro is essential for their use in bedside applications. One aspect of high-quality cells is maintaining their genome stability upon prolonged culture and cryopreservation. Genome instability may cause malignant transformation which may affect their effectiveness in therapy. Here, we aim to evaluate the genome stability of our in-house established rat full-term AFSC line (R3) based on the chromosomal number and RNA expression of selected markers upon prolonged culture in-vitro and cryopreservation. The cryopreserved R3 at passages (P) 36 to 38 were revived and cultured. Morphological features of newly revived P36 and P37 cells were compared before subjecting P37 cells to karyotyping. A semi-q RT-PCR analysis using Image J was performed to evaluate the relative expression level of markers for stemness (Nanog), tumour suppressor (p53), and senescence (p16) genes against the housekeeping gene, GAPDH, using RNA extracted from P36 and P38 cells. RNA extracted from differentiated R3-derived neural stem cells (NSCs) was included as a positive control for the non-transformational baseline. There was no significant difference in cell morphology between P36 and P37 cells. Karyotyping analysis revealed that the P37 cells retained the rat diploid chromosome number (2n=42). Only p53 was expressed in P36, P38 and NSCs, while Nanog and p16 remained suppressed. Freshly thawed R3 from prolonged cryopreservation may induce stress factor on R3, causing p53 pathway activation, thus inhibiting Nanog expression. However, the stress is still tolerable as the p16 gene remained suppressed, indicating R3 has not entered malignant transformation. This finding marks R3 as a sensitive yet stable stem cell line that can be applied safely in downstream applications.
| Item Type: | Article |
|---|---|
| Keywords: | Amniotic fluid stem cell, Full-term rat amniotic fluid stem cell, Genome stability, Transformation, Karyotyping, Cryopreservation |
| Taxonomy: | By Niche > Genome > Animal Genome Mapping By Niche > Genome > Genomes |
| Local Content Hub: | Niche > Genome |
| Depositing User: | Hazrul Amir Tomyang (Puncak Alam) |
| Date Deposited: | 27 May 2024 08:05 |
| Last Modified: | 27 May 2024 08:05 |
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