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

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

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.326 ◽

2017 ◽

Vol 4 (S) ◽

pp. 131

Author(s):

Fiona Macniesia Thomas ◽

Vijay Kumar ◽

Siti Fatimah Simat ◽

Helen Benedict Lasimbang

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Telomere Length ◽

Cellular Senescence ◽

Mutation Detection ◽

Telomerase Activity ◽

P53 Mutation ◽

Human Amnion ◽

Long Term Culture

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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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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Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

10.1101/364059 ◽

2018 ◽

Author(s):

Sanjay K. Kureel ◽

Pankaj Mogha ◽

Akshada Khadpekar ◽

Vardhman Kumar ◽

Rohit Joshi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Culture System ◽

Human Mesenchymal Stem Cells ◽

Population Doubling ◽

Differentiation Potential ◽

Lineage Differentiation ◽

Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

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Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1471-y ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 15

Author(s):

Jin Seok ◽

Hyun Sook Jung ◽

Sohae Park ◽

Jung Ok Lee ◽

Chong Jai Kim ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Fatty Acid ◽

Mitochondrial Dysfunction ◽

Fatty Acid Oxidation ◽

Replicative Senescence ◽

Acid Oxidation ◽

Differentiation Potential ◽

Passage Number

Abstract Background Human placenta-derived mesenchymal stem cells (PD-MSCs) are powerful sources for cell therapy in regenerative medicine. However, a limited lifespan by senescence through mechanisms that are well unknown is the greatest obstacle. In the present study, we first demonstrated the characterization of replicative senescent PD-MSCs and their possible mitochondrial functional alterations. Methods Human PD-MSCs were cultured to senescent cells for a long period of time. The cells of before passage number 8 were early cells and after passage number 14 were late cells. Also, immortalized cells of PD-MSCs (overexpressed hTERT gene into PD-MSCs) after passage number 14 were positive control of non-senescent cells. The characterization and mitochondria analysis of PD-MSCs were explored with long-term cultivation. Results Long-term cultivation of PD-MSCs exhibited increases of senescent markers such as SA-β-gal and p21 including apoptotic factor, and decreases of proliferation, differentiation potential, and survival factor. Mitochondrial dysfunction was also observed in membrane potential and metabolic flexibility with enlarged mitochondrial mass. Interestingly, we founded that fatty acid oxidation (FAO) is an important metabolism in PD-MSCs, and carnitine palmitoyltransferase1A (CPT1A) overexpressed in senescent PD-MSCs. The inhibition of CPT1A induced a change of energy metabolism and reversed senescence of PD-MSCs. Conclusions These findings suggest that alteration of FAO by increased CPT1A plays an important role in mitochondrial dysfunction and senescence of PD-MSCs during long-term cultivation.

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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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