title: Systematic evaluation of markers used for the identification of human induced pluripotent stem cells.
abstract: 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).
authors.affiliation: 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.
authors.forename: Sumitha Prameela
authors.initials: SP
authors.lastname: Bharathan
authors.affiliation: 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.
authors.forename: Kannan Vrindavan
authors.initials: KV
authors.lastname: Manian
authors.affiliation: Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India.
authors.forename: Syed Mohammed Musheer
authors.initials: SM
authors.lastname: Aalam
authors.affiliation: Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India.
authors.forename: Dhavapriya
authors.initials: D
authors.lastname: Palani
authors.affiliation: Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India.
authors.forename: Prashant Ajit
authors.initials: PA
authors.lastname: Deshpande
authors.affiliation: Centre for Stem Cell Research (Unit of InStem, Bengaluru), Christian Medical College Campus, Vellore, Tamil Nadu, India.
authors.forename: Mankuzhy Damodaran
authors.initials: MD
authors.lastname: Pratheesh
authors.affiliation: 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.
authors.forename: Alok
authors.initials: A
authors.lastname: Srivastava
authors.affiliation: 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.
authors.forename: Shaji Ramachandran
authors.identifier: http://orcid.org/0000-0002-3228-6758
authors.initials: SR
authors.lastname: Velayudhan
keywords: Colony identification; Human induced pluripotent stem cells; Pluripotency; Reprogramming; Retroviral silencing; hiPSC morphology