Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

<div><p>Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.</p> </div

Histological Analysis of Teratomas formed after Transplantation of RPE Cells Spiked with Undifferentiated HES-1 Cells.

<p>(<b>A–B</b>): H&E staining of a section of a teratoma and an RPE graft generated after transplantation of 5×10⁵ HES-1-derived RPE cells spiked with 1×10⁴ undifferentiated HES-1 cells. (<b>C</b>): Efficiency of teratoma formation after transplantation of 5×10⁵ HES-1-derived RPE cells spiked with decreasing numbers of undifferentiated HES-1 cells.</p

Analysis of HES-1-derived RPE Cell Grafts.

<p>Following transplantation of 5×10⁵ HES-derived differentiated RPE cells, the transplanted cells remained as small RPE-grafts at the site of injection. (<b>A</b>): The grafts could be visualized because RPE cells are pigmented. The arrow indicates the pigmented graft. (<b>B</b>): H&E staining of a section of a RPE-graft revealed pigmented cells. (<b>C–D</b>): Immunofluorescence staining of a section of the graft for markers specific for RPE cells, Bestrophin (C, red), and RPE65 (D, red), together with DAPI (blue) showed that the majority of cells within the graft expressed these markers.</p

Proposed protocol for a standardized teratoma assay of pluripotency.

<p>The scheme describes our recommendations for the performance and analysis of a standardized teratoma assay of pluripotency.</p

Histological Analysis of Teratomas Formed after Transplantation of undifferentiated HES-1 Cells.

<p>Images of sections of teratomas, formed after s.c. transplantation of undifferentiated hESCs with their feeders and Matrigel into NOD/SCID mice. (<b>A–C</b>): A teratoma formed from transplantation of 5×10⁵ hESCs. (A) A lower magnification (× 4) of a section of a teratoma showing derivatives of all three germ layers. (B–C) A higher magnification (× 20) of regions of (A) showing ectodermal, and endodermal structures (B) and mesodermal and endodermal structures (C). (<b>D–G</b>): A teratoma formed from transplantation of 100 hESCs. (D) A lower magnification (× 1.25) of a section of a teratoma showing derivatives of all three germ layers (E–G) a higher magnification (× 20) of regions of (D) showing ectodermal structures (E), mesodermal structures (F) and endodermal structures (G).</p

Kinetics of Teratoma Formation after Transplantation of Various Specific Numbers of Undifferentiated HES-1 Cells.

<p>Specific numbers of undifferentiated HES-1 cells were mixed with MMC-treated foreskin fibroblasts (to a total of 1×10⁶ cells) and Matrigel, and transplanted s.c. into NOD/SCID mice. The transplanted animals were monitored weekly for the appearance of tumors, and for the progression of tumor size. The endpoint of the experiments was when the tumors reached a size of ≥ 1 cm³ or 30 weeks after transplantation. (<b>A</b>): Efficiencies of teratoma tumor formation after transplantation of decreasing numbers of undifferentiated HES-1 cells. The trendline is depicted in dotted line. (<b>B</b>): A Kaplan-Meier plot showing the percentage of surviving mice transplanted with decreasing numbers of hESCs, at various time-points (1 W–30 W) during the transplantation experiments. The mice were sacrificed when the tumors reached a volume of ≥ 1 cm³. (<b>C</b>): The average time interval between transplantation and the detection of tumors. (<b>D</b>): The average volume of the teratomas at the time of animal sacrifice. All data relate to animals that developed teratomas. Data presented as mean ± SEM.</p