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

Maintenance of <i>Xist</i> Imprinting Depends on Chromatin Condensation State and <i>Rnf12</i> Dosage in Mice

<div><p>In female mammals, activation of <i>Xist</i> (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal <i>Xist</i> is preferentially expressed whereas maternal <i>Xist</i> (Xm-<i>Xist</i>) is silenced. Unlike autosomal imprinted genes, <i>Xist</i> imprinting for Xm-<i>Xist</i> silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-<i>Xist</i> imprinting is poorly understood. Here, we revealed that Xm-<i>Xist</i> silencing depends on chromatin condensation states at the <i>Xist</i>/<i>Tsix</i> genomic region and on <i>Rnf12</i> expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-<i>Xist</i> derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-<i>Xist</i> derepression, Xm-<i>Xist</i> was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-<i>Xist</i> was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXp^Δ lethality, indicating that loss of Xm-<i>Xist</i> imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-<i>Xist</i>/<i>Tsix</i> genomic loci. In parthenogenetic embryos, <i>Rnf12</i> overdose caused Xm-<i>Xist</i> derepression via Xm-<i>Tsix</i> repression; physiological <i>Rnf12</i> levels were essential for Xm-<i>Xist</i> silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of <i>Rnf12</i> dosage were crucial for <i>Xist</i> imprint maintenance by silencing Xm-<i>Xist</i>.</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

Xm-<i>Xist</i> induces global X-linked gene silencing in XmXp^Δ embryos.

<p>(a) Experimental scheme to construct Kdm4b/TSA-XmXp^Δ embryos. (b) RNA/DNA-FISH analysis in Kdm4b/TSA- or Egfp/DMSO-XmXp^Δ embryos at the 4-cell stage. The graph shows quantification of RNA-FISH signals. The upper and lower graphs show the percentages of <i>Xist</i>-expressing and biallelic cells, respectively. n, the number of cells analysed. Scale bars show 20 μm. (c) RNA/DNA-FISH analysis in Kdm4b/TSA or control blastocysts. Each circle shows individual embryos. n, the number of embryos analysed. Scale bars show 20 μm. (d) Strand-specific RT-PCR analysis for <i>Xist</i> detection. Individual Kdm4b/TSA-XmXp^Δ blastocysts were used for the assay. (e and f) Hierarchical clustering analysis by RNA-Seq from individual blastocysts of WT-XmXp, Egfp/DMSO-XmXp^Δ, and Kdm4b/TSA-XmXp^Δ. The genes expressed in at least one sample (TMM > 10) were used for analysis. All genes expressed (e) and X-linked genes (f). (g) Heat map showing X-linked genes expression. Colours show expression levels; blue: low, black: middle, and yellow: high. The percentages are the genes with > 3-fold upregulated compared with WT.</p

Developmental competency of Kdm4b/TSA-XmXp^Δ embryos.

<p>(a) % of implantation sites at E19.5. n, the number of transferred embryos. (b) % of embryos developed to term at E19.5. n, the number of recovered embryos. (c) PCR analysis using cDNA and genomic DNA (gDNA) in rescued XmXp^Δ females. cDNA from the tails and placentas and gDNA from placentas were used for the assay, respectively. (d) The rescued females developed to adults with normal reproductive ability.</p

<i>Rnf12</i> over-dosage induces Xm-<i>Xist</i> derepression in XmXm, XmY and XmXp embryos.

<p>(a) Average <i>Rnf12</i> expression levels in single XmY, XmXp, and XmXm cells from oocytes to morula embryos. n, the number of cells analysed. The average expression levels of MII oocytes were set as one. (b) <i>Rnf12</i> expression profiles in individual cells. The average expression levels of XmXp embryos were set as one in each stage. (c) <i>Xist</i>/<i>Tsix</i> RNA-FISH analysis in Rnf12KD-XmXm and Rex1/Rnf12 double KD XmXm morulae. Representative images from scramble (siRNA control), Rnf12KD, and Rex1/Rnf12 double KD embryos. The graphs show quantification of FISH signal patterns. The P-values were calculated by the Fisher’s exact test. n, the number of cells analysed. Scale bars show 20 μm. (d) Experimental scheme of construction of RNF12 overexpressing fertilized embryos by <i>Rnf12</i> mRNA injection. <i>Egfp</i> mRNA was used for the control. (e and f) Immunofluorescence combined with RNA/DNA-FISH analysis of Rnf12 overexpressing XmY (Rnf12-XmY) and control XmY (Egfp-XmY) (e), Rnf12-XmXp, and Egfp-XmXp (f). Representative images from <i>Rnf12</i> or <i>Egfp</i> overexpressing embryos. The graph shows % of <i>Xist</i> cloud cells in XmY and XmXp embryos or of <i>Xist</i> biallelic cells. n, the number of cells expressing RNF12 or EGFP. Scale bars show 20 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

Proposed model.

<p>(a) Irreversible Xm-<i>Xist</i> imprinting. The transient alteration of histone modifications through loss of H3K9me3 and gain of histone acetylation induces stable Xm-<i>Xist</i> derepression and results in the rescue of lethality in XmXp^Δ without gene manipulation. The imprinting switch in XCI does not affect cellular integrity. (b) Maintenance and erasure of the Xm-<i>Xist</i> imprint. In early preimplantation phases, chromatin at Xm-<i>Xist</i>/<i>Tsix</i> regions is condensed by various epigenetic modifications. At late stage, Oct4 localizes to nucleus (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006375#sec010" target="_blank">discussion</a>) and serves as a chromatin opener at Xm-<i>Xist</i>/<i>Tsix</i> regions, creating transcriptional permissive states around <i>Xist</i>/<i>Tsix</i> regions. In XmXm embryos, <i>Rnf12</i> expression levels represent a double dose compared with those of XmY or XmXp embryos, leading to Xm-<i>Xist</i> activation by <i>Tsix</i> silencing, which depends on REX1 state. In XmY and XmXp embryos, on the other hand, physiological expression levels of <i>Rnf12</i> are essential for Xm-<i>Xist</i> silencing by Xm-<i>Tsix</i> activation.</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