Highly Sensitive In Vitro Methods for Detection of Residual Undifferentiated Cells in Retinal Pigment Epithelial Cells Derived from Human iPS Cells

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research

Characterization of in vivo tumorigenicity tests using severe immunodeficient NOD/Shi-scid IL2Rγnull mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products

The contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic cells is one of the major concerns in the manufacturing and quality control of hCTPs. However, no quantitative method for detecting the tumorigenic cellular impurities is currently standardized. NOD/Shi-scid IL2Rγnull (NOG) mice have shown high xeno-engraftment potential compared with other well-known immunodeficient strains, e.g. nude mice. Hypothesizing that tumorigenicity test using NOG mice could be a sensitive and quantitative method to detect a small amount of tumorigenic cells in hCTPs, we examined tumor formation after subcutaneous transplantation of HeLa cells, as a model of tumorigenic cells, in NOG mice and nude mice. Sixteen weeks after inoculation, the 50% tumor-producing dose (TPD50) values of HeLa cells were stable at 1.3 × 104 and 4.0 × 105 cells in NOG and nude mice, respectively, indicating a 30-fold higher sensitivity of NOG mice compared to that of nude mice. Transplanting HeLa cells embedded with Matrigel in NOG mice further decreased the TPD50 value to 7.9 × 10 cells, leading to a 5000-fold higher sensitivity, compared with that of nude mice. Additionally, when HeLa cells were mixed with 106 or 107 human mesenchymal stem cells as well as Matrigel, the TPD50 values in NOG mice were comparable to those of HeLa cells alone with Matrigel. These results suggest that the in vivo tumorigenicity test using NOG mice with Matrigel is a highly sensitive and quantitative method to detect a trace amount of tumorigenic cellular impurities in human somatic cells, which can be useful in the quality assessment of hCTPs

Tumorigenicity-associated characteristics of human iPS cell lines.

Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61-77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used

Comparison of the tumorigenicity-associated assays.

*<p>Not based on the calculation found in Reference #21 because the background signal from the negative controls (primary RPE cells) was not detectable.</p

Detection of undifferentiated hiPSCs by qRT-PCR assay.

<p>(A) The relative mRNA expressions in primary RPE cells of Lin28, Oct-3/4, Sox2, Nanog, Rex1, Klf4, and c-Myc were determined by qRT-PCR analysis. (B–D) qRT-PCR analysis of hiPSCs spiked into primary RPE cells and five lots of primary RPE cells. Single-cell hiPSCs (1%, 2.5×10³ cells; 0.1%, 2.5×10² cells; 0.01%, 25 cells) were spiked into 2.5×10⁵ primary RPE cells, and total RNA was isolated from the mixed cells. The mRNA levels of Nanog (B), Oct3/4 (C) and Lin28 (D) are shown as a relative expression. Limit of detection was calculated as the mean plus 3.3 fold the standard deviation of the measurement of the five lots of primary RPE cells. (E) Lin28 expression of hiPSCs differentiating into RPE and purified hiPSC-derived RPE cells (passage 3 and 4). All values are expressed as mRNA levels relative to those in undifferentiated hiPSCs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037342#s2" target="_blank">Results</a> are means ± standard deviation (n = 3).</p

Detection of undifferentiated hiPSCs by flow cytometry assay.

<p>(A) Flow cytometry analysis of hiPSCs (blue) and primary RPE cells (red). Cells were fixed, permiabilized and stained with anti-TRA-1-60, anti-TRA-1-81, anti-Sox2, anti-Oct3/4 and anti-Nanog antibodies labeled with fluorophore. (B) Five lots of primary RPE cells were analyzed by flow cytometry with anti-TRA-1-60 antibody. (C) HiPSCs (0.1%, 2.5×10² cells; 0.01%, 25 cells) were spiked into primary RPE cells (2.5×10⁵ cells) and analyzed by flow cytometry with anti-TRA-1-60 antibody. (D) Flow cytometry analysis of hiPSC-derived RPE cells was performed with anti-TRA-1-60 antibody. Ten thousand cells (A) and 1×10⁵ cells (B–D) were used for one assay of flow cytometry analysis. The numbers indicate the quantity of cells contained in the gate.</p

Soft agar colony formation assay of hiPSCs and teratocarcinoma PA-1 cells.

<p>(A) Phase-contrast images of hiPSCs, primary RPE cells, hiPSC-derived RPE cells and PA-1cells spiked into primary RPE cells (1%) cultured in soft agar medium for 30 days. Arrows indicate the colonies of PA-1 cells. (B) PA-1 cells (1%, 100 cells; 0.5%, 50 cells; 0.25%, 25 cells; 0%, 0 cells) were spiked into 1.0×10⁴ primary RPE cells and grown in soft agar for 10, 20 and 30 days. Cell growth was quantified using a CytoSelect kit. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037342#s2" target="_blank">Results</a> were expressed as a relative fold change of the value of blank well. Statistical significance was determined using two-way ANOVA and Bonferroni's post-hoc test (*<i>P</i><0.05 compared with the 0% control). (C) HiPSC-derived RPE cells, three lots of primary RPE cells and PA-1 cells spiked into primary RPE cells were grown in soft agar for 30 days. Quantification of the results is described in (B). Limit of detection was calculated as the mean plus 3.3 fold the standard deviation of the measurement of the three lots of primary RPE cells. Error bars represent the standard deviation of the measurements (n = 3).</p