Abstract
ABSTRACTLow 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).
Highlights
IntroductionHuman induced pluripotent stem cells (hiPSCs) resemble human embryonic stem cells (hESCs) at molecular and functional levels
Human induced pluripotent stem cells resemble human embryonic stem cells at molecular and functional levels
The expression pattern of fibroblast and pluripotent cell markers in reprogramming cells and emerging Human induced pluripotent stem cells (hiPSCs) colonies Several studies have been carried out to correlate expression patterns of individual surface markers with the transformation of cells during human somatic cell reprogramming, as a means to enrich the population of pluripotent stem cells in order to increase the efficiency of reprogramming and for studying the mechanisms of reprogramming (Abujarour et al, 2013; Chan et al, 2009; Hotta et al, 2009; Kahler et al, 2013; Tanaka et al, 2015)
Summary
Human induced pluripotent stem cells (hiPSCs) resemble human embryonic stem cells (hESCs) at molecular and functional levels. Combining positive and negative surface markers allows the selection and expansion of iPSCs with a relative reduction in the effort required to culture partially reprogrammed iPSC clones (Abujarour et al, 2013; Dick et al, 2011; Kahler et al, 2013; Quintanilla et al, 2014; Valamehr et al, 2012). Such enrichment methods generate heterogeneous colonies that lack clonal identity as they consist of cells originated from different donor cells. The ability of pluripotent stem cells to silence the transgenes expressed from retroviral vectors has been explored as a marker for identification and isolation of pluripotent clones
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
