Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2

Polanco, Juan Carlos, Wang, Bei, Zhou, Qi, Chy, Hun, O'Brien, Carmel and Laslett, Andrew L. (2013) Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2. Journal of Visualized Experiments, 82: . doi:10.3791/50856

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Author Polanco, Juan Carlos
Wang, Bei
Zhou, Qi
Chy, Hun
O'Brien, Carmel
Laslett, Andrew L.
Title Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2
Journal name Journal of Visualized Experiments   Check publisher's open access policy
ISSN 1940-087X
Publication date 2013-12-06
Sub-type Article (original research)
DOI 10.3791/50856
Open Access Status File (Publisher version)
Issue 82
Total pages 8
Place of publication Cambridge, MA, United States
Publisher Journal of Visualized Experiments
Language eng
Formatted abstract
Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30Hi-GCTM-2Hi hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30Neg-GCTM-2Neg). In a further study, pluripotent stem cell-free samples of differentiated TG30Neg-GCTM-2Neg cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30Hi-GCTM-2Hi hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.
Keyword Antibodies
Cell surface antigens
Differentiation
FACS
Human embryonic stem cells (hESC)
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Queensland Brain Institute Publications
 
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