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Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay.

Zhou Y, Zhou P, Xin Y, Wang J, Zhu Z, Hu J, Wei S, Ma H - Sci Rep (2014)

Bottom Line: Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres.The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR.Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P. R. China.

ABSTRACT
Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

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TREAQ measurement of HeLa Cells.(A) FT for HeLa cells at four different total Protein Concentrations. (B) A linear relationship (y = 185.22x + 10.6) between FT and the cell lysate concentration. (C) FT for HeLa cells on polymer surface. Samples which were heated in 90°C for 30 min were used as negative control.
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f3: TREAQ measurement of HeLa Cells.(A) FT for HeLa cells at four different total Protein Concentrations. (B) A linear relationship (y = 185.22x + 10.6) between FT and the cell lysate concentration. (C) FT for HeLa cells on polymer surface. Samples which were heated in 90°C for 30 min were used as negative control.

Mentions: We first used common cancer cell line HeLa as model to estimate a linear dynamic range between the telomerase-containing cell lysate concentration and the Δf value of TREAQ (FT). As shown in Figure. 3A, FT of HeLa lysates (ranging from 0.1 g/L to 0.6 g/L) was steadily changing with the concentration increased. An equal volume of 1 × CHAPS Lysis buffer was also measured by TREAQ as a control. We found a linear relationship between FT and the cell lysate concentration indicating that QCM was able to quantitatively measure telomerase activity (Fig. 3B). For cell lysate (active telomerase) treated by heat inactivation (90°C for 30 min), a FT tending to zero indicated eliminated telomerase activity (Fig. 3C). The same samples from HeLa cell lines are measured with a standard TRAP assay for verification (see Supplementary Fig. S1 online). Also, the relationship between different cell lysate concentrations and Total Product Generated (TPG) was investigated for the comparison with traditional methods (see Supplementary Fig. S2 online).


Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay.

Zhou Y, Zhou P, Xin Y, Wang J, Zhu Z, Hu J, Wei S, Ma H - Sci Rep (2014)

TREAQ measurement of HeLa Cells.(A) FT for HeLa cells at four different total Protein Concentrations. (B) A linear relationship (y = 185.22x + 10.6) between FT and the cell lysate concentration. (C) FT for HeLa cells on polymer surface. Samples which were heated in 90°C for 30 min were used as negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225532&req=5

f3: TREAQ measurement of HeLa Cells.(A) FT for HeLa cells at four different total Protein Concentrations. (B) A linear relationship (y = 185.22x + 10.6) between FT and the cell lysate concentration. (C) FT for HeLa cells on polymer surface. Samples which were heated in 90°C for 30 min were used as negative control.
Mentions: We first used common cancer cell line HeLa as model to estimate a linear dynamic range between the telomerase-containing cell lysate concentration and the Δf value of TREAQ (FT). As shown in Figure. 3A, FT of HeLa lysates (ranging from 0.1 g/L to 0.6 g/L) was steadily changing with the concentration increased. An equal volume of 1 × CHAPS Lysis buffer was also measured by TREAQ as a control. We found a linear relationship between FT and the cell lysate concentration indicating that QCM was able to quantitatively measure telomerase activity (Fig. 3B). For cell lysate (active telomerase) treated by heat inactivation (90°C for 30 min), a FT tending to zero indicated eliminated telomerase activity (Fig. 3C). The same samples from HeLa cell lines are measured with a standard TRAP assay for verification (see Supplementary Fig. S1 online). Also, the relationship between different cell lysate concentrations and Total Product Generated (TPG) was investigated for the comparison with traditional methods (see Supplementary Fig. S2 online).

Bottom Line: Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres.The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR.Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P. R. China.

ABSTRACT
Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

Show MeSH
Related in: MedlinePlus