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

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.

Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

“…Germ cells has high telomerase activity, while it disappears when they change into differentiated cells during differentiation [12]. Consistently, we have con rmed that the telomerase activities of hPSCs, human bone marrow mesenchymal stem cells (hBMSCs) and MG63 osteoblasts decreased successively [12]. Therefore, cell telomerase activity can be applied as one of the important quantitative markers to monitor the in vitro osteogenic differentiation process of hPSCs.…”

“…As we know, cell telomerase activity plays a key role in the self-renewal of cells, and it is gradually downregulated during embryonic development [9]. Germ cells has high telomerase activity, while it disappears when they change into differentiated cells during differentiation [12]. Consistently, we have con rmed that the telomerase activities of hPSCs, human bone marrow mesenchymal stem cells (hBMSCs) and MG63 osteoblasts decreased successively [12].…”

“…The protein concentration of the sample was adjusted by DPBS with the minimum protein concentration as a reference. To measure the cell telomerase activity, the NHSS/EDC activated QCM chip was immediately incubated with the primers (5 -NH2(CH2)6 ⊤ ⊤ ⊤ ⊤ ⊤ ∀ TℂGTCGAGCAGAG ⊤ -3) and the DNA assembly solution for 3 h. The pre-treated chips were placed into a QCM reactor and then underwent the same processes to detect the frequency changes relating to cell telomerase activity as we previously described [12].…”

Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

“…The telomerase activity of the cell samples throughout the osteogenic differentiation was quantitatively measured using a previously reported method based on a quartz crystal microbalance (QCM) [12]. Brie y, each of 1 million single cells was lysed in 150 µL CHAPS lysis buffer for 30 min on ice.…”

The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance

“…Telomerase activity may thus be useful in assessing iPSC reprogramming efficiency and as a hallmark of non-transformed somatic cells, which should contain low telomerase expression and activity. 61 Moreover, upregulation of pluripotency genes is strongly connected with expression of TERT, the overexpression of which enhances reprogramming efficiency. 62,63 For example, porcine iPSCs can only maintain telomeres with continued expression of exogenous reprogramming factors to maintain TERT levels, 31 and partially reprogrammed mouse embryonic fibroblasts (MEFs), and porcine embryonic fibroblasts (PEFs) show little TERT promoter activation and poor telomere elongation.…”