Assessment of Genome Stability on Full-Term Rat Amniotic Fluid Stem Cell Line (R3) Upon Prolonged Culture

Amran, Muhammad Muhtadee (2022) Assessment of Genome Stability on Full-Term Rat Amniotic Fluid Stem Cell Line (R3) Upon Prolonged Culture. [Project Paper] (Submitted)

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Abstract

Previous research has suggested that amniotic fluid stem cells (AFSCs) have a promising therapeutic application in regenerative medicine due to their broad multipotency status and less ethical concerns. Therefore, it is essential to obtain sufficient quantity and good quality of AFSCs when culturing them in-vitro. Stem cell genome instability due to prolonged culture has become a concern, as this can affect its therapeutic effectiveness in regenerative medicine applications. Until now, the referral parameter as an indicator and possibility for the AFSCs to undergo transformation upon prolonged cultures remains unclear, particularly for the full-term AFSC line. Objective: This study investigates the genome stability based on the chromosome number and the RNA expression levels of relevant markers (Nanog, p53, and p16) that could be used to indicate the genomic status of full-term rat AFSC line (R3) upon prolonged cultures. Methodology: The cryopreserved R3 (passages (P) 36 to 38) were revived in culture media containing Glasgow Minimum Essential Medium (GMEM), supplements, 10% foetal bovine serum (FBS) (Gibco) and 15 ng/mL leukaemia inhibitory factor (LIF). P37 cells were subjected to karyotyping to determine the chromosome number. A semi-quantification reverse transcription PCR (semi-q RT-PCR) analysis using Image J was performed to evaluate the relative expression levels of Nanog (a stem cell marker), p53 (a tumour-suppressor marker), and p16 (a marker for cell cycle regulator) of RNA extracted from the P36 and P38 of R3 cells against GAPDH (housekeeping genes). RNA from differentiated R3-derived neural stem cells (NSCs) was included as the positive control for stable cells (as the non-transformation baseline). Results: P36 and P37 R3 have similar morphological features (multiple nucleoli and fibroblastic shape). At the chromosomal level, R3 passage 37 retains its diploid chromosomal number (2n = 42). However, at a molecular level, only p53 was expressed in P36, P38 and NSC, while Nanog and p16 remained suppressed. Discussion: Freshly thawed R3 from prolonged cryopreservation and the subculturing may have induced stress factor on R3, causing the activation of p53 pathways, thus inhibiting Nanog expression. However, the stress is still tolerable by R3 as the p16 gene remained suppressed, indicating R3 has not entered malignant transformation. Conclusion: This finding could mark R3 as a sensitive, yet stable stem cell line that can be applied safely in the downstream application.

Item Type:	Project Paper
Faculty:	Faculty of Medicine and Health Science
Depositing User:	Ms. Nor Safa'aton Saidin
Date Deposited:	22 Aug 2023 04:37
Last Modified:	22 Aug 2023 04:37
URI:	http://psaspb.upm.edu.my/id/eprint/1383

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