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

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

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

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

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

Prediction analysis of networks and transcription factors regulating <i>IKBKAP</i> and <i>IKBKAP</i> co-regulated functional candidate genes.

<p>“String” analysis of protein networks showing the potential interaction between the majority of the <i>IKBKAP</i> co-regulated functional candidate genes. (B) Prediction of transcription factors (TF) related to <i>IKBKAP</i> and co-regulated functional candidate genes. (C) Shows relative quantification levels represented as mean ± s.d. of TF gene candidates taken from the total cDNA microarray analysis showing difference between WT and FD-hESC derived PNS neurons. * P<0.05 **P<0.01, Non statistical significance (NS).</p

GO analysis of differential gene expression between hESC-derived PNS neurons and embryo brains of WT and FD origins.

<p>The major enriched GO analysis groups showing processes (A) and gene functions (B) from the two in vivo and in vitro cDNA microarray chip data. The enrichment groups are arranged by number of downregulated and upregulated genes detected in each group. Only genes above 2-fold change are included. FDR q-value (adjusted p-values of result significance), Enrichment = (number intersection genes / input genes) / (total number of genes of specific GO term / background genes).</p

Characterization of IKAP localization in PNS WT and FD hESC-derived cultured neurons.

<p>Immunofluorescence confocal microscopy analysis was performed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.g003" target="_blank">Fig 3</a>. IKAP together with peripherin and Rab3a expression are shown within hESC derived neurons in WT (A-F) and FD (G-L) genetic backgrounds. Images B and H show the expression and localization of IKAP in WT and FD derived-neurons respectively. C and I, D and J show the expression of peripherin and Rab3a respectively. IKAP and Rab3a co-localization levels are shown in WT (F) and FD (L). Quantitative analysis of the mean intensity of IKAP and RAB3a colocalization levels in WT and FD axons is shown in M. 3D stacks of sequential confocal images were de-convolved using Huygens (scientific volume imaging-SVI) software and the analyzed for colocalization between the signals was performed using Imaris (Bitplan). Bar sizes are indicated in representative images. Mouse anti-hIKAP (BD Biosciences) was used in these experiments.</p

Comparative transcriptome analysis between human 12 weeks fetal WT and FD brains to WT and FD hESC derived neurons.

<p>Prior to cDNA microarray chip analysis mRNA and protein extracts from the brain samples were used to validate the FD phenotype of the FD splicing mutation in the <i>IKBKAP</i> gene at the transcriptional and translational levels by: (A) RT-PCR analysis of the expression of <i>IKBKAP</i> in human 12 weeks fetal WT and FD brains showing WT (upper lane) and FD (mis-spliced, lower lane) mRNA isoforms in the FD brain only. (B) Western blot analysis of IKAP protein levels in WT and FD brains. Note the almost complete absence of IKAP in the FD brain. β-Actin served as loading control. Comparative transcriptome analysis between data obtained from both cDNA microarray chips of WT and FD fetal brains and hESC-derived PNS neurons was performed by cross-referencing genes which their expression levels differ significantly (>2 fold) between FD and WT. (C) Venn Diagram representation of a wide genome transcriptome analysis of common genes that are differentially expressed in FD-hESC-derived PNS neurons (in green) and in two other known FD stem cell neural-derived models (FD fibroblasts derived iPSC (in yellow) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.ref023" target="_blank">23</a>] and FD-hOE-MSC (in blue) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.ref024" target="_blank">24</a>] and in FD Fetal brain (in red). Cross-referencing genes with their expression levels difference of >2 fold change for hESC p<0.05 or >1.5fold change p<0.05 for iPSC and hOE-MSC between WT and FD were considered for analysis. The number at the crossection between the diagrams represents the number of genes that are shared by these multiple analyses. For the list of genes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138807#pone.0138807.s009" target="_blank">S3 Table</a>. (D) Gene sets were divided into downregulated and upregulated genes in both hESC-derived PNS neurons and Fetal brain biological systems. Venn diagram represents the results from this analysis showing in blue, the number of genes that differ in WT and FD in the fetal <i>in vivo</i> and in yellow, the number of genes that differ in WT and FD hESC derived neurons <i>in vitro</i>. The crossection between the diagrams represent the number of genes that are shared by both analyses. Upper diagrams represent the downregulated genes while the lower ones represent the upregulated genes.</p