Inadvertent presence of pluripotent cells in monolayers derived from differentiated embryoid bodies

Abstract

The therapeutic use of embryonic stem (ES)-derived cells is restricted by a risk of teratoma formation. To test the hypothesis that some cells with pluripotency characteristics remain following the differentiation of embryoid bodies (EB) into monolayer cells, we transformed mouse ES cells using constructs comprised of the mTert promoter coupled to green fluorescent protein. In this manner, EBs could be identified as showing gradually diminishing expression of the fluorescent marker as a consequence of differentiation. After 2 weeks of incubation, however, small groups of fluorescent cells remained in the differentiated monolayer. When these cells were isolated, cultured and expanded under ES cell culture conditions, they recovered their ES cell morphology (herein denoted ES-EB). We found by immunocytochemistry, reverse transcription-PCR and bisulphite analysis that despite the fact that some of these ES-EB lost their capacity to express some pluripotency markers characteristic of ES cells and undergo the epigenetic modification (hypo-methylation) of some retrotransposons (RT), after several passages in ES media, the cell colonies recovered their capacity for both pluripotency marker expression and RT methylation. Furthermore, when assessed for their ability to form chimeras, most ES-EB lines were unable to do so, although they recovered this potential for chimera production after some passages in ES cell media. Our results highlight the need for specific screening of differentiated cells before their therapeutic use and indicate that under adequate culture conditions, cells that loose their potential for expressing key markers of pluripotency can recover this fundamental embryonic stem cell property.

Effect of Stem Cell Activation, Culture Media of Manipulated Embryos, and Site of Embryo Transfer in the Production of F0 Embryonic Stem Cell Mice1

“…ES cell lines were initially checked for pluripotency based on the expression of molecular markers specific for ES cells [17], and the ability to successfully produce chimeras with germline transmission. Karyotype analysis was realized as previously described [18]. Both cell lines were XY.…”

“Footprint-Free” Human Induced Pluripotent Stem Cell-Derived Astrocytes for In Vivo Cell-Based Therapy

“…Experimental allotransplantation of undifferentiated ESC and iPSC into the brain or other tissues results in teratoma formation [48][49][50][51][52]. This very serious risk has hindered the use of hiPSC-derived cells from clinical trials.…”

Most regions of mouse epididymis are able to phagocytose immature germ cells

“…The ESC line was initially checked for pluripotency based on the expression of molecular markers specific for ESCs and its ability to successfully produce chimeras showing germline transmission. Karyotype analysis was performed as described previously (Ramirez et al 2007).…”

“…Cellular clones were selected by geneticin resistance over 7 days and screened for GFP expression under fluorescence microscopy. DNA from the transformed ESCs was processed for PCR analysis to confirm transgene integration using standard protocols (Ramirez et al 2007). …”