A Novel <i>In Vitro</i> Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

<div><p>Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple <i>in vitro</i> method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.</p></div

Characterization of 253G1 cells subcultured on laminin-521 in Essential 8 medium.

<p>(A) Expression levels of undifferentiated cell markers (<i>OCT3/4, NANOG, SOX2 and LIN28</i>) in 253G1 cells subcultured on laminin-521 in Essential 8 were determined using qRT-PCR. Relative mRNA expression levels are presented as ratios to the level of that in control cells on Matrigel. Results are the mean ± SD (n = 3). (B) <i>In vitro</i> differentiation analysis of 253G1 cells subcultured on laminin-521 in Essential 8 medium. Immunostaining of the markers for three germ layers are shown: endoderm (alpha-fetoprotein (AFP)), mesoderm (α-smooth muscle actin (SMA)) and ectoderm (βIII tubulin). Scale bars, 200 µm. (C) Expression levels of differentiated cell markers in embryoid bodies (EBs) derived from 253G1 cells: endoderm (<i>GATA6, SOX17</i>), mesoderm (<i>CDH5, FOXF1</i>), ectoderm (<i>SOX1, PAX6</i>). Relative mRNA expression levels are presented as ratios to the level of that in control cells (EBs at Day 10). Results are the mean ± SD (n = 3). (D-E) Teratomas derived from 253G1 cells cultured on laminin-521 in Essential 8 medium are shown. Hematoxylin and eosin staining showed the features of three germ layers: Ep, epithelium-like tissue (endoderm); Ca, cartilage (mesoderm); Ne, neural rosette-like tissue (ectoderm); P, pigmented neuroectodermal resembling meranocyte (ectoderm); Br, brain-like tissue (ectoderm). Scale bars, 200 µm.</p

Robust proliferation of 253G1 cells cultured on laminin-521 in Essential 8 medium.

<p>(A) Morphology of the 253G1 cells expanded on laminin-521 in Essential 8 or mTeSR1 medium after dissociation into single cells. Scale bars, 500 µm. (B) Quantification of the number of dissociated 253G1 cells expanded on laminin-521 or Matrigel in Essential 8 or mTeSR1 medium. Data are presented as the mean ± standard deviation (SD) of three independent experiments (**<i>P</i><0.01, two-way ANOVA followed by Bonferroni t-test as post-hoc test). LN521, laminin-521; MG, Matrigel.</p

Detection of residual undifferentiated cells contained in differentiating cell cultures.

<p>(A) Differentiation scheme of 253G1 cells into MSCs is shown. (B) Expression levels of undifferentiated cell markers (<i>OCT3/4, NANOG</i> and <i>LIN28</i>) in each cell culture were determined using qRT-PCR. Relative mRNA expression levels are presented as ratios to the level of that in 253G1 cells at Day 1. Results are the mean ± SD (n = 3). (C) Numbers of the forming colonies derived from residual undifferentiated cells in differentiating cell culture at Day 6 or Day 14 are shown. Experiments were carried out in duplicate. Data are present as raw data in each well (shown by plots) or the mean of well 1 and well 2 (shown by bar graphs). (D) Phase contrast images of forming colonies derived from residual undifferentiated cells are shown. Cells at Day 6 of differentiation (EBs) were dissociated into single cells by Accutase and cultured on laminin-521-coated wells in Essential 8 medium (5×10⁴/well). After 4 days of culture, small clusters emerged and then started to grow rapidly. Finally, they formed colonies that were positive for TRA-1-60 (shown by immunofluorescence staining, green). Arrowheads indicate a colony derived from same origin. Scale bars, 500 µm.</p

Rapid cell proliferation of hiPSCs plated at low cell density on laminin-521 in Essential 8 medium.

<p>(A-I) Quantification of the number of 253G1, 201B7 and 409B2 cells expanded on laminin-521 in Essential 8 or mTeSR1 medium. Cell numbers were counted every 24 h after plating at 3.2×10⁴ cells/cm² (A, D, G), 1.6×10⁴ cells/cm² (B, E, H) and 8.0×10³ cells/cm² (C, F, I), respectively. Data are presented as the mean ± standard deviation (SD) of three independent experiments (*<i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001, two-way repeated-measures ANOVA followed by a Bonferroni post-hoc test).</p

Detection of hiPSCs spiked into hMSCs on the culture system using laminin-521 and Essential 8 medium.

<p>(A) Morphologies of forming colonies derived from 409B2 cells spiked into hMSCs are shown (images in the left). 409B2 cells (1%, 1000 cells; 0.1%, 100 cells; 0.01%, 10 cells; 0%, 0 cells) were spiked into hMSCs (100,000 cells) and co-cultured on laminin-521-coated wells in 6-well plates in Essential 8 medium for 7 days. Expression of the undifferentiated marker, TRA-1-60, in these colonies was assessed using immunofluorescence staining (images in the right). Each experiment was carried out in duplicate. Scale bars, 500 µm. (B) Numbers of the colonies detected in each spiked sample in (A) are shown. Data are present as raw data in each well (shown by plots) or the mean of well 1 and well 2 (shown by bar graphs). (C) Morphology of a forming colony derived from 253G1 cells spiked into hMSCs at the ratio of 0.001% (6 hiPSCs to 600,000 hMSCs) is shown (images in the left). Mixture of those cells was co-cultured on a 100-mm cell culture dish coated with laminin-521 in Essential 8 medium for 9 days. Forming colony was stained with anti TRA-1-60 antibody (images in the right). Experiment was carried out in duplicate. Scale bars, 500 µm.</p

Detection of hiPSCs spiked into human neurons on the culture system using laminin-521 and Essential 8 medium.

<p>Morphologies of forming colonies derived from 253G1 cells spiked into human neurons are shown (images in the left). 253G1 cells (1%, 1000 cells; 0.1%, 100 cells; 0.01%, 10 cells; 0%, 0 cells) were spiked into human neurons (100,000 cells) and co-cultured on laminin-521-coated wells in 12-well plates in Essential 8 medium for 6 days. Forming colonies were stained with anti TRA-1-60 antibody (images in the right). HNs, human neurons. Scale bars, 500 µm.</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

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 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