Cellular senescence of the long term culture human amnion mesenchymal stem cells (hamcs)

• Main Author: Fiona Macniesia Thomas
• Format: Thesis
• Language: English; English
• Published: 2019
• Subjects: QH573-671 Cytology
• Online Access: https://eprints.ums.edu.my/id/eprint/41357/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/41357/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/41357/

A fundamental understanding of senescence in human amnion mesenchymal stem cells (HAMCs) is crucial for its application in cellular therapy. Cellular senescence is characterized by changes in cell morphology and the presence of senescence markers such as SA-β-Gal. Several genes such as p53, p21, pRB, p16 and GADD45 are commonly associated with the cellular senescence pathway. Also, telomerase activity which is important in the regulation of cell proliferation has been linked to cellular aging. At the same time, determining p53 sequence mutation is needed to assess the tumourigenicity risk in long-term cultured HAMCs. Thus, the aim of the study are: i) to determine the level of senescence in HAMCs at passage 5, 10 and 15 (P5, P10 and P15) through morphology changes of cells and the use of senescent-associated β-galactosidase (SA-β-Gal) assay; ii) to determine the expression of senescent-associated gene (p53, pRB, p21, p16 and GADD45) via reverse transcription polymerase chain reaction (RT-qPCR) at P5, P10 and P15; and iii)to determine the DNA damage level in HAMCs during long-term culture using comet assay, telomeric repeat amplification protocol (TRAP) and p53 mutation detection assay. The samples were obtained from amnion placentae of healthy mothers who underwent caesarean section at the Damai Specialist Hospital. After the isolation, HAMCs were cultured in vitro up to passage 15. They were assessed at passage 5, 10 and 15 and then analysed to correspond with the objectives of the study. The results show that HAMCs underwent morphological changes – from showing typical MSCs morphology at early passages to flattened and elongated shaped at late passages. The cells viability also decreased in percentage, i. e. 92.94±2.32% at P5, decreased to 87.15±1.48% at P10, and further decreased to 67.24±4.50% at P15. A larger number of cells were also tested positive for SA-β-Gal assay, with increasing percentage of senescent cells from P5 to P15 (P5: 0.03±0.01%, P10: 42.68±0.92%, P15: 82.61±1.40%). From the assessment of gene expression level at P5 to P15; it was found that p53 was up-regulated from 1 to 2.49 (0.27 to 1.3 fold); p21 was up-regulated from 1 to 5.45 (0.27 to 2.45 fold); pRB was up-regulated from 1 to 2.83 (0.38 to 1.39 fold); and p16 was up-regulated from 1 to 11.86 (0 to 0.35 fold). Meanwhile GADD45 was down-regulated from P5 to P15 (1 to 0.49 with 0.24 to 0.88 fold). p53/p21 and p53/pRB signalling pathway were activated in the senescence pathway of HAMCs. Genes expression level increased with increasing passage numbers. Comet assay showed that HAMCs at P15 have higher DNA damage compared to HAMCs at earlier passages (P5: 91±9.54 a.u., P10: 152.33±11.54 a.u., P15: 229±7.94 a.u.). Telomerase activity of HAMCs decreased between P5 to 15 (P5:103.75±37.89, P10: 64.67±34.96, P15: 35.03±13.98). DNA sequencing of p53 gene indicated that mutations had occurred after long-term culture with a higher presence of single nucleotide variants (SNVs) particularly in later passages. Assessment of senescence in HAMCs provided information that HAMCs at early passages have higher proliferative capacity and lower senescent cells. Thus, P5 and P10 are deemed as the most suitable for utilization in cellular therapy. Further study should be performed in vivo to investigate if long-term cultured HAMCs could cause malignant transformation.