Systematic evaluation of markers used for the identification of human induced pluripotent stem cells.

Autor:	Bharathan SP; Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India.; Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India., Manian KV; Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India.; Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India., Aalam SM; Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India., Palani D; Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India., Deshpande PA; Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India., Pratheesh MD; Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India., Srivastava A; Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India.; Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India., Velayudhan SR; Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India rvshaji@cmcvellore.ac.in.; Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India.
Jazyk:	angličtina
Zdroj:	Biology open [Biol Open] 2017 Jan 15; Vol. 6 (1), pp. 100-108. Date of Electronic Publication: 2017 Jan 15.
DOI:	10.1242/bio.022111
Abstrakt:	Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4 + and TRA-1-60 + cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs). Competing Interests: The authors declare no competing or financial interests. (© 2017. Published by The Company of Biologists Ltd.)
Databáze:	MEDLINE
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