Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells

Abstract

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by enforced expression of transcription factors. Using serial live imaging of human fibroblasts undergoing reprogramming, we identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters and differentiation into teratomas, we determined that only one colony type represents true iPS cells, whereas the others represent reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B and REX1 can be used to distinguish the fully reprogrammed state, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT and NANOG are insufficient as markers. We also show that reprogramming using chemically defined medium favors formation of fully reprogrammed over partially reprogrammed colonies. Our data define molecular markers of the fully reprogrammed state and highlight the need for rigorous characterization and standardization of putative iPS cells.

Two-Phase Analysis of Molecular Pathways Underlying Induced Pluripotent Stem Cell Induction

“…We replaced Tgfbr1 and Tgfb2 with Smad7, as Smad7 can block TGF-b signaling, which mimics the effects of downregulating Tgfbr1 and Tgfb2 [37,40]. An immunostaining assay for Rex-1, a specific pluripotency stem cell marker [41], was used to screen for iPS-like cells. We introduced all five genes (Smad7, Nanog, Nr6a1, Zfp371, and Zfp459) into the OKM cells by retroviral transduction (OKM-5F), which generated a much higher number of Rex-1-positive colonies compared with MEFs directly transduced with the four reprogramming factors (OSKM) or OKM cells transduced with Sox2 (OKM-S; Fig.…”

Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

“…There are reports that suggest a fluctuating expression of Nanog in murine ESCs [34]. In contrast, other groups confirm bona fide human iPS cells by qRT-PCR for NANOG [18,25] and this correlation held true in downstream experiments such as teratoma formation in immunodeficient mice [18]. We also have chosen Nanog as a marker to identify truly reprogrammed murine iPS cells in situ.…”

“…However, in most cases, a definite verification of gene expression is impossible on living cells. The detection of endogenous pluripotency markers such as OCT4, SOX2, NANOG, GDF3, and REX1 requires fixation of the cells prior to staining [16][17][18] or is accomplished only after lysis by an RT-PCR analysis [17,18]. With such a manipulation, the cells are lost for further culture.…”

“…Therefore, detection of pluripotency markers on live cells is highly desirable. This is feasible and routinely performed for cell membrane-based antigens such as TRA-1-60 or SSEA4 [18] although the antibodies might nevertheless perturb the cells [19] and these "early pluripotency markers" do not allow discrimination of terminally stable reprogrammed iPS cells. Also for alkaline phosphatase (AP), which is another early pluripotency marker mostly assessed on fixed cells [16], a fluorescent reporter dye ("AP Live Stain") has been lately developed for live screening [20].…”

Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm

“…Mouse intermediate reprogrammed cells can be labelled by stagespecific embryonic antigen (SSEA)-1 that is recognized as a specific marker of mouse embryonic stem cells (ESCs)/iPSCs [5][6][7] . We demonstrated that human cells positive ( þ ) for tumorrelated antigen (TRA)-1-60 8,9 induced by OSKM are intermediate reprogrammed cells to being iPSCs 10 . Behaviour of TRA-1-60 ( þ ) intermediate reprogrammed cells suggested that maturation, but not the initiation step, is a bottleneck of cell fate conversion towards human iPSCs.…”