Detection of small numbers of iPSCs in different heterogeneous cell mixtures with highly sensitive droplet digital PCR

Artyuhov, Alexander; Дашинимаев, Э. Б.; Mescheryakova, Natalia; Ashikhmina, A.A.; Vorotelyak, E. A.; Vasiliev, A. V.

doi:10.1007/s11033-019-05100-2

Cited by 9 publications

(10 citation statements)

References 28 publications

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“…The detection methods and efficiency are listed in Table 4 . 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 Flow cytometry is one of the most common strategies for the detection of residual undifferentiated hPSCs using antibodies against hPSC‐specific transcription factors and/or cell surface markers. Kuroda et al assessed the detection efficiency of hPSCs using five antibodies that recognize stem cell marker antigens (OCT4, NANOG, SOX2, TRA‐1‐60, and TRA‐1‐81) and showed that anti‐OCT4, anti‐SOX2, and anti‐TRA‐1‐60 antibodies distinguished hiPSCs from hiPSC‐derived retinal pigment epithelial cells (hiPSC‐RPE).…”

Section: In Vitro Tumorigenicity Tests For Hpsc ...mentioning

confidence: 99%

“…Artyuhov et al showed that the detection limit of OCT4 , TGDF , and LIN28 was 0.01%, whereas that with ddPCR was 0.002%. 94 Watanabe et al reported a detection efficiency of 0.00002% when ddPCR was performed after enrichment of stem cell markers using magnetic beads. 97 In addition, it has been reported that the detection efficiency of hPSCs in hPSC‐CMs suggests that ddPCR is more suitable than qPCR for the assessment of residual undifferentiated hPSCs.…”

Section: In Vitro Tumorigenicity Tests For Hpsc ...mentioning

confidence: 99%

“…Therefore, it is crucial to develop or select highly quantitative methods and suitable factors for detection. The detection methods and efficiency are listed in Table 4 88–95 . Flow cytometry is one of the most common strategies for the detection of residual undifferentiated hPSCs using antibodies against hPSC‐specific transcription factors and/or cell surface markers.…”

Section: In Vitro Tumorigenicity Tests For Hpsc‐derived Productsmentioning

confidence: 99%

“…ddPCR analysis with LIN28 probes detected as low as 0.001% residual undifferentiated hPSCs in primary CMs. Artyuhov et al showed that the detection limit of OCT4 , TGDF , and LIN28 was 0.01%, whereas that with ddPCR was 0.002% 94 . Watanabe et al reported a detection efficiency of 0.00002% when ddPCR was performed after enrichment of stem cell markers using magnetic beads 97 .…”

Section: In Vitro Tumorigenicity Tests For Hpsc‐derived Productsmentioning

confidence: 99%

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Scalable manufacturing of clinical‐grade differentiated cardiomyocytes derived from human‐induced pluripotent stem cells for regenerative therapy

et al. 2022

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Basic research on human pluripotent stem cell (hPSC)‐derived cardiomyocytes (CMs) for cardiac regenerative therapy is one of the most active and complex fields to achieve this alternative to heart transplantation and requires the integration of medicine, science, and engineering. Mortality in patients with heart failure remains high worldwide. Although heart transplantation is the sole strategy for treating severe heart failure, the number of donors is limited. Therefore, hPSC‐derived CM (hPSC‐CM) transplantation is expected to replace heart transplantation. To achieve this goal, for basic research, various issues should be considered, including how to induce hPSC proliferation efficiently for cardiac differentiation, induce hPSC‐CMs, eliminate residual undifferentiated hPSCs and non‐CMs, and assess for the presence of residual undifferentiated hPSCs in vitro and in vivo. In this review, we discuss the current stage of resolving these issues and future directions for realizing hPSC‐based cardiac regenerative therapy.

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Section: In Vitro Tumorigenicity Tests For Hpsc ...mentioning

confidence: 99%

Section: In Vitro Tumorigenicity Tests For Hpsc ...mentioning

confidence: 99%

Section: In Vitro Tumorigenicity Tests For Hpsc‐derived Productsmentioning

confidence: 99%

Section: In Vitro Tumorigenicity Tests For Hpsc‐derived Productsmentioning

confidence: 99%

See 2 more Smart Citations

Scalable manufacturing of clinical‐grade differentiated cardiomyocytes derived from human‐induced pluripotent stem cells for regenerative therapy

et al. 2022

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“…For instance, Lin-28 Homolog A (Lin28A), although it is widely clinics for detecting undifferentiated cells in retinal pig-ment epithelial (RPE) cells derived from hiPSCs, similar efficacies in hepatic differentiation could not be replicated [11]. Also, despite OCT4 and TDGF1 being useful detection markers, they are mainly specific to hiPSC-derived neural stem cells [15]. Thus, an important limitation in current research is that the markers identified/developed are specific to certain cell types and cannot be used in other cell lineages.…”

Section: Introductionmentioning

confidence: 99%

Universal Markers for hiPSCs Residue Detection

Shi¹,

Feng²,

Wang³

et al. 2022

Front. Biosci. (Landmark Ed)

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Background: Residual undifferentiated induced pluripotent stem cells (iPSCs) detection is essential for both Embryonic Stem Cells (ESCs) and iPSCs application in final cell therapy products. However, specific differentiated cells require specific genes for residual detection; identifying the suitable marker is costly and time-consuming. Thus, a universal marker for iPSCs residue detection for all three germline cells would greatly benefit PSC-derived cellular therapies. Methods: Next-generation sequencing (NGS) was performed on total RNAs isolated from the iPSC cell lines and embryonic stem cells (H9), the top 30 expressed genes were selected as candidates. By analysis expression fold change comparing iPSC cells to the differentiated cells, seven genes were highly expressed in iPSCs but showed minimal background expression in differentiated cells. Tissue expression pattern of the candidate genes were explored in the Genotype-Tissue Expression (GTEx) project database, candidate genes were narrowed down to two genes. Spike-in experiments were performed to determine the detection limit and correlation with the number of iPSCs and gene expression by ddPCR. Results: By next-generation sequencing (NGS), we identified two marker genes (ESRG and ZSCAN10) suitable for universal undifferentiated iPSC detection. Both ESRG and ZSCAN10 are highly expressed in iPSCs. ZSCAN10 is slightly expressed in the testis, pituitary, and cerebellum; ESRG is highly expressed in the vagina and scarcely expressed in the other tissues. Furthermore, the ddPCR method with a probe and primers for ESRG and ZSCAN10 detected a trace of undifferentiated hiPSCs to a spiked level of 0.0001%. Conclusions: These results suggest that targeting ESRG/ZSCAN10 transcripts is highly sensitive, quantitative, and could be broadly applied to quality control of almost all iPSC-derived cell therapy products.

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Highly Sensitive Detection of Human Pluripotent Stem Cells by Loop-Mediated Isothermal Amplification

Yasui

Matsui

Sekine

et al. 2022

Stem Cell Rev and Rep

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For safe regenerative medicines, contaminated or remaining tumorigenic undifferentiated cells in cell-derived products must be rigorously assessed through sensitive assays. Although in vitro nucleic acid tests offer particularly sensitive tumorigenicity-associated assays, the human pluripotent stem cell (hPSC) detectability is partly constrained by the small input amount of RNA per test. To overcome this limitation, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays that are highly gene specific and robust against interfering materials. LAMP could readily assay microgram order of input sample per test and detected an equivalent model of 0.00002% hiPSC contamination in a simple one-pot reaction. For the evaluation of cell-derived total RNA, RT-LAMP detected spiked-in hPSCs among hPSC-derived trilineage cells utilizing multiple pluripotency RNAs. We also developed multiplex RT-LAMP assays and further applied for in situ cell imaging, achieving specific co-staining of pluripotency proteins and RNAs. Our attempts uncovered the utility of RT-LAMP approaches for tumorigenicity-associated assays, supporting practical applications of regenerative medicine. Graphical Abstract

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Detection of small numbers of iPSCs in different heterogeneous cell mixtures with highly sensitive droplet digital PCR

Cited by 9 publications

References 28 publications

Scalable manufacturing of clinical‐grade differentiated cardiomyocytes derived from human‐induced pluripotent stem cells for regenerative therapy

Scalable manufacturing of clinical‐grade differentiated cardiomyocytes derived from human‐induced pluripotent stem cells for regenerative therapy

Universal Markers for hiPSCs Residue Detection

Highly Sensitive Detection of Human Pluripotent Stem Cells by Loop-Mediated Isothermal Amplification

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