ID: 1051 Telomerase activity, telomere length and P53 mutation detection on cellular senescence of Human Amnion Mesenchymal Stem Cells (HAMCs)

A fundamental understanding of senescence in human amnion mesenchymal stem cells (HAMCs) is crucial for its application in cellular therapy. Previous findings strongly support that HAMCs undergoes cellular senescence after long term in-vitro culture, with evidence of significant morphological changes and the presence of the senescent associated β-galactosidase (SA-β-Gal) marker. The telomere length and the telomerase activity have been linked with cellular aging and they are important in regulating cell proliferation. In addition, p53 gene has been associated with cell senescence. The aim of this study was to investigate the telomerase activity, telomere length in senescent HAMCs, and to detect p53 mutations in these cells. Samples were obtained from amnion placenta and then cultured for long term. Prolong-cultured HAMCs was isolated at passages 5, 10 and 15 and then analysed via telomeric repeat amplification protocol (TRAP), telomere length assay and p53 mutation detection assay. The results showed that after long term culture of HAMCs, there was a decrease in telomere length and telomerase activity from passages 5, 10 to 15. Telomerase controls the telomere’s length which maintains the cells proliferation. The decrease of telomere length and telomerase activity may suggest that the proliferation of HAMCs has slowed down due to HAMCs entering senescence after long term culture. P53 mutation detection study indicated that HAMCs at all passage did not have altered sequences. Thus, the cells did not undergo uncontrollable replication due to the effect of long-term culture. Further studies on senescence in HAMCs will be assessed by investigating the expression level of p53, p21, p16, pRB and GADD45 genes in long term culture of HAMCs via RT-qPCR. The findings will help us understand the associations between gene expressions and the process of senescence

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ID: 1054 Comparison between fresh and cryopreserved Human Amnion Mesenchymal Stem Cells (HAMCs) in terms of serial passaging, morphology and differentiation potential during long term culture

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.329 ◽

2017 ◽

Vol 4 (S) ◽

pp. 134

Author(s):

Sandra Lisa Gumpil ◽

Kamaruzaman Ampon ◽

Helen Lasimbang ◽

Simat Siti Fatimah ◽

S.Vijay Kumar

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Mesenchymal Stem Cells ◽

Cellular Therapy ◽

Enzymatic Digestion ◽

Positive Staining ◽

Differentiation Potential ◽

Human Amnion ◽

Long Term Culture

The therapeutic efficiency of Human Amnion Mesenchymal stem cells (HAMCs) is significantly promising. However, its sustainability in long term sub-cultivation has yet to be identified, especially post-cryopreservation. There are concerns whether stem cells which are cryopreserved will be able to retain its functions and differentiation potential efficiently. The aim of this study is to determine the effects of cryopreservation on the serial passaging, morphology and differentiation potential of HAMCs culture post-cryopreservation compared to fresh HAMCs culture. HAMCs was isolated through serial enzymatic digestion of the amnion membrane of human term placenta after delivery. Fresh HAMCs cultures were sub-cultivated until passage 15 while cryopreserved HAMCs samples was preserved at passage 2 of the cell culture and revived after 3 months of cryopreservation period. Through microscopic observation, the cryopreserved HAMCs started to flatten and become larger in size, losing it fibroblastic feature as early as passage 6. The enlarged and flatten morphological features of the HAMCs indicate that the cells began to lose its proliferative activity at this point. In comparison, the HAMCs normally began to enlarge when reaching passage 15 in non-cryopreserved HAMCs cultures. Fresh HAMCs cultures were able to be sustained up to 15 passages while cryopreserved HAMCs were not able to survive pass 10 passages of cell culture. Furthermore, it was observed that both fresh and cryopreserved HAMCs were able to retain its differentiation potential through osteogenesis and adipogenesis medium induction. This was experimentally visualized through positive staining of the calcium composite and lipid droplet in the induced HAMCs culture using Von Kossa stain solution and Oil Red-O stain solution, respectively. Despite retaining it differentiation potential, cryopreserved HAMCs were only able to survive a few passages after being revived. The result suggests that fresh HAMCs is a more suitable candidate to be used in cellular therapy and various clinical application as it were able to retain all its function in long term culture.

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Senescence-Associated Secretory Phenotype Suppression Mediated by Small-Sized Mesenchymal Stem Cells Delays Cellular Senescence through TLR2 and TLR5 Signaling

Cells ◽

10.3390/cells10010063 ◽

2021 ◽

Vol 10 (1) ◽

pp. 63

Author(s):

Ji Hye Kwon ◽

Miyeon Kim ◽

Soyoun Um ◽

Hyang Ju Lee ◽

Yun Kyung Bae ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cellular Senescence ◽

Small Cells ◽

Critical Determinant ◽

Senescent Phenotype ◽

Toll Like Receptor 2 ◽

Secretory Phenotype ◽

Major Factors

In order to provide a sufficient number of cells for clinical use, mesenchymal stem cells (MSCs) must be cultured for long-term expansion, which inevitably triggers cellular senescence. Although the small size of MSCs is known as a critical determinant of their fate, the main regulators of stem cell senescence and the underlying signaling have not been addressed. Umbilical cord blood-derived MSCs (UCB-MSCs) were obtained using size-isolation methods and then cultured with control or small cells to investigate the major factors that modulate MSC senescence. Cytokine array data suggested that the secretion of interukin-8 (IL-8) or growth-regulated oncogene-alpha (GROa) by senescent cells was markedly inhibited during incubation of small cells along with suppression of cognate receptor (C-X-C motif chemokine receptor2, CXCR2) via blockade of the autocrine/paracrine positive loop. Moreover, signaling via toll-like receptor 2 (TLR2) and TLR5, both pattern recognition receptors, drove cellular senescence of MSCs, but was inhibited in small cells. The activation of TLRs (2 and 5) through ligand treatment induced a senescent phenotype in small cells. Collectively, our data suggest that small cell from UCB-MSCs exhibit delayed cellular senescence by inhibiting the process of TLR signaling-mediated senescence-associated secretory phenotype (SASP) activation.

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DNA Methylation Changes duringIn VitroPropagation of Human Mesenchymal Stem Cells: Implications for Their Genomic Stability?

Stem Cells International ◽

10.1155/2013/192425 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 33

Author(s):

Angela Bentivegna ◽

Mariarosaria Miloso ◽

Gabriele Riva ◽

Dana Foudah ◽

Valentina Butta ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Ex Vivo ◽

Genomic Stability ◽

Great Promise ◽

Human Mscs ◽

Low Oxygen ◽

Long Term Culture ◽

Functional Changes

Mesenchymal stem cells (MSCs) hold great promise for the treatment of numerous diseases. A major problem for MSC therapeutic use is represented by the very low amount of MSCs which can be isolated from different tissues; thusex vivoexpansion is indispensable. Long-term culture, however, is associated with extensive morphological and functional changes of MSCs. In addition, the concern that they may accumulate stochastic mutations which lead the risk of malignant transformation still remains. Overall, the genome of human MSCs (hMSCs) appears to be apparently stable throughout culture, though transient clonal aneuploidies have been detected. Particular attention should be given to the use of low-oxygen environment in order to increase the proliferative capacity of hMSCs, since data on the effect of hypoxic culture conditions on genomic stability are few and contradictory. Furthermore, specific and reproducible epigenetic changes were acquired by hMSCs duringex vivoexpansion, which may be connected and trigger all the biological changes observed. In this review we address current issues on long-term culture of hMSCs with a 360-degree view, starting from the genomic profiles and back, looking for an epigenetic interpretation of their genetic stability.

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Effects of cryopreservation and long-term culture on biological characteristics and proteomic profiles of human umbilical cord-derived mesenchymal stem cells

Clinical Proteomics ◽

10.1186/s12014-020-09279-6 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Xufeng Fu ◽

Bo Xu ◽

Jiang Jiang ◽

Xing Du ◽

Xiaoli Yu ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Umbilical Cord ◽

Biological Characteristics ◽

Human Umbilical Cord ◽

Long Term Culture

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A high-throughput polymer microarray approach for identifying defined substrates for mesenchymal stem cells

Biomaterials Science ◽

10.1039/c4bm00112e ◽

2014 ◽

Vol 2 (11) ◽

pp. 1683-1692 ◽

Cited By ~ 8

Author(s):

Cairnan R. E. Duffy ◽

Rong Zhang ◽

Siew-Eng How ◽

Annamaria Lilienkampf ◽

Guilhem Tourniaire ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

High Throughput ◽

Polymer Substrates ◽

Long Term Culture

Defined polymer substrates supported the growth of mesenchymal stem cells in long-term culture while maintaining their phenotype and lineage potential.

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218 DONOR-MATCHED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF CANINE MESENCHYMAL STEM CELLS DERIVED FROM DIFFERENT ORIGINS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab218 ◽

2012 ◽

Vol 24 (1) ◽

pp. 221

Author(s):

S. A. Ock ◽

G. H. Maeng ◽

Y. M. Lee ◽

T. H. Kim ◽

B. M. Kumar ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Transcription Factors ◽

Telomere Length ◽

Telomerase Activity ◽

Rt Pcr ◽

Cytochemical Staining ◽

Single Donor ◽

Cell Capacity

Canine mesenchymal stem cells (cMSC) have been successfully isolated from several adult tissue sources. However, differences in the biological properties of MSC have been shown to be associated with donor variability. Further, the stem cell capacity of cMSC of various tissues isolated from a single donor is currently unclear. Therefore, this study investigated the functional and molecular characteristics of cMSC derived from bone marrow (cBM-MSC), adipose tissue (cA-MSC) and dermal skin (cDS-MSC) of a single donor. Three kinds of cMSC were isolated by following previously published protocols. AP activity was assessed with a chromogen kit (Abcam Inc., Cambridge, MA, USA). Expression of CD markers (CD45, 90 and 105) and stem cell transcription factors (Oct3/4, Nanog and Sox2) was analysed by immunocytochemical staining. All cells were induced into osteogenesis and adipogenesis by following protocols described earlier and confirmed by cytochemical staining and the detection of lineage specific genes by RT-PCR. Chromosomal stability was assessed by a method described earlier (Ock and Rho 2008 J. Vet. Med. Sci. 70, 1165–1172) and cell cycle status was determined by a flow cytometry. Telomere length was analysed by Telo TAGGG Telomere Length Assay kit (Roche, Mannheim, Germany) and telomerase activity was evaluated by semiquantitative nested RT-PCR. Statistical analysis was performed by ANOVA using SPSS 12.0 and significance was tested when P < 0.05. Expressions of AP activity and the transcription factors, such as Oct3/4, Nanog and Sox2 were absent in all cMSC. All 3 types of cMSC positively expressed the surface markers CD90 and 105 but not CD45. Exposure of all cell lines to osteogenic and adipogenic induction medium resulted in the calcium deposition evidenced by Alizarin red S staining and the accumulation of fat globules indicated by Oil red O staining, respectively. Differentiation was further confirmed by the detection of marker genes, such as Runx2 and Pparγ. However, the degree of osteogenic or adipogenic differentiation among the 3 kinds of cMSC was different and particularly, cA-MSC had enhanced cytochemical staining associated with expression of specific genes, Runx2 and Pparγ. Ploidy analysis showed that the diploid rate was high with over 90% in all cMSC and indicated no noticeable chromosomal abnormalities. Further, less than 52% of cells were found at G1 phase in all cMSC, with lowest percentage observed in cDS-MSC (33.3%). Regardless of varied tissue sources, cMSC from a single donor showed no differences in telomere lengths (∼18–19 kbp), but the telomerase activity was different with significantly higher levels found in cBM-MSC. In conclusion, the above results suggest that tissue specific cMSC derived from a single donor possess differences in stem cell capacity and support the consideration of tissue source before judging the suitability of cells for therapeutic applications. This work was supported by grant from Basic Science Research Program through NRF funded by the Ministry of Education, Science and Technology (2009-0064229).

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