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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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Related in: MedlinePlus

FT for human fibroblasts, 6 self-renewal passages and differentiation of hNF1-4C11 hiPSCs.The data are presented as mean ± SD. (n = 4/human fibroblast; 3/different passages; 3/differentiation). *P < 0.05 as determined by a Student's t-test.
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f4: FT for human fibroblasts, 6 self-renewal passages and differentiation of hNF1-4C11 hiPSCs.The data are presented as mean ± SD. (n = 4/human fibroblast; 3/different passages; 3/differentiation). *P < 0.05 as determined by a Student's t-test.

Mentions: An off-line telomerase activity detection is a method that recording frequency value only at the beginning and the end of QCM process. It means real-time consecutive QCM curve for displaying primers extension is not required. Thus, high-throughput TREAQ is feasible based on this off-line system. With suitable instruments, high-throughput TREAQ will provide us a more convenient way not only for the detection of telomerase activity but also for more applications like drug screening. With the polymer-coated QCM chip immobilized by oligonucleotides primers on its surface and placed in an open-top reaction chamber, we use the off-line TREAQ, as indicated above, to detect the human fibroblasts, HNF1-4C11 hiPSCs and differentiated HNF1-4C11 hiPSCs. As shown in Figure 4, about 36 Hz frequency shift triggered by extension of primers was detected by QCM, which indicated low telomerase activity of human fibroblasts. Evidently increasing frequency of hiPSCs indicated up-regulation of telomerase activity during reprogramming, in accordance with telomere elongation mediated by telomerase in the meantime36. High-level telomerase activity of hNF1-4C11 hiPSCs (FT between 70 Hz and 80 Hz) was maintained for 11 passages according to TREAQ. However, progressively decrease of frequency after differentiated for various periods (FT: 1 day for about 38 Hz, 3 days for about 29 Hz and 5 days for about 16 Hz) indicated progressively down-regulation of telomerase activity (Fig. 4). Morphological characteristics of hNF1-4C11 hiPSCs differentiation were shown in Figure 5.


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)

FT for human fibroblasts, 6 self-renewal passages and differentiation of hNF1-4C11 hiPSCs.The data are presented as mean ± SD. (n = 4/human fibroblast; 3/different passages; 3/differentiation). *P < 0.05 as determined by a Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: FT for human fibroblasts, 6 self-renewal passages and differentiation of hNF1-4C11 hiPSCs.The data are presented as mean ± SD. (n = 4/human fibroblast; 3/different passages; 3/differentiation). *P < 0.05 as determined by a Student's t-test.
Mentions: An off-line telomerase activity detection is a method that recording frequency value only at the beginning and the end of QCM process. It means real-time consecutive QCM curve for displaying primers extension is not required. Thus, high-throughput TREAQ is feasible based on this off-line system. With suitable instruments, high-throughput TREAQ will provide us a more convenient way not only for the detection of telomerase activity but also for more applications like drug screening. With the polymer-coated QCM chip immobilized by oligonucleotides primers on its surface and placed in an open-top reaction chamber, we use the off-line TREAQ, as indicated above, to detect the human fibroblasts, HNF1-4C11 hiPSCs and differentiated HNF1-4C11 hiPSCs. As shown in Figure 4, about 36 Hz frequency shift triggered by extension of primers was detected by QCM, which indicated low telomerase activity of human fibroblasts. Evidently increasing frequency of hiPSCs indicated up-regulation of telomerase activity during reprogramming, in accordance with telomere elongation mediated by telomerase in the meantime36. High-level telomerase activity of hNF1-4C11 hiPSCs (FT between 70 Hz and 80 Hz) was maintained for 11 passages according to TREAQ. However, progressively decrease of frequency after differentiated for various periods (FT: 1 day for about 38 Hz, 3 days for about 29 Hz and 5 days for about 16 Hz) indicated progressively down-regulation of telomerase activity (Fig. 4). Morphological characteristics of hNF1-4C11 hiPSCs differentiation were shown in Figure 5.

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