According to the results, and are highly sensitive and robust markers for detecting residual undifferentiated cells in human iPSC-derived cell products differentiated into each of the three germ cell lineages. a highly sensitive manner using qPCR. In addition, such markers could be used to detect residual undifferentiated cells from various differentiated cells, including hepatic cells and pancreatic cells for the endodermal lineage, endothelial cells and mesenchymal cells for the mesodermal lineage, and neural cells for the ectodermal lineage. Our method facilitates robust validation and could enhance the safety of the cell products through the exclusion of undifferentiated iPSC. could be used to detect residual undifferentiated cells in iPSC-derived differentiated retinal pigment epithelial (RPE) cells21, which was already applied to patients. Such methods are often optimized for specific differentiation protocols and are not always applicable to the other lineages. Therefore, it is critical to develop more versatile methods to facilitate the detection of residual undifferentiated cells in differentiated cells. Here, we report a method for detecting undifferentiated cells amongst iPSC-derived cells in all three germ layers. Results is not suitable for detecting undifferentiated iPSC in hepatic differentiation expression Alvespimycin was examined to validate the potential application of in the detection of residual undifferentiated cells during iPSC differentiation toward hepatic lineage cells. While expression was high in hepatic endoderm (HE), it remained unaltered in the immature hepatocyte (IH) stage (Figs.?1a and S1). We considered two possible explanations for Alvespimycin the observation. One is that is expressed in hepatic lineage cells and; therefore, is not suitable for the detection of undifferentiated iPSC in hepatic lineage cells. The other potential explanation is is actually the undifferentiated iPSC marker and there were undifferentiated iPSCs in the differentiated cells in the present study. Alvespimycin To explore the possibility of the above cases, we evaluated gene expression in the developing mouse liver and observed that hepatic cells expressed some amounts of mouse during liver development (Fig.?1b). This result suggests that express during hepatocyte differentiation and might not suitable to detect undifferentiated cells in differentiated, but immature hepatic progenitors. Open in a separate window Figure 1 is not suitable for detecting undifferentiated iPSC during hepatic differentiation. (a) Human expression during hepatic differentiation from iPSC. DE, definitive endoderm; HE, hepatic endoderm; IH, immature hepatocyte; MH, mature hepatocyte. The relative?expression levels were normalized by the amount of 18S rRNA in each sample.?(b) Mouse expression in hepatic cells during liver development. For samples from embryonic day 9.5 (E9.5) to post natal day 3 (P3) total RNA was isolated from nonhematopoietic (CD45???TER119?) cells. For 8 week (8w) old sample, hepatic cell fraction was isolated by centrifugation. Subsequently, we evaluated whether undifferentiated iPSCs were present in the differentiated cells in the present study. We utilized re-seeding method, by which we re-seeded differentiated cells and cultivated them for ~1 week in iPSC maintenance state to derive undifferentiated cell colonies to facilitate the direct observation of the contamination with undifferentiated cells in the culture22. To validate this re-seeding method, we spiked-in (mixed) undifferentiated iPSC to the differentiated cells and detected at least 0.0025% of spiked-in undifferentiated cells in our condition (data not shown). Notably, the method is robust and the more cells are seeded in culture, the more the detection limit can be lowered, although it requires at least 1 week to grow undifferentiated cell colonies. No undifferentiated cell colonies were detected from HE cells when cells were seeded at densities of 8 104 cells/cm2 and 1.6 105 cells in three independent experiments. The results indicate that is not suitable for detecting undifferentiated iPSC in hepatic differentiation (Fig.?1 and see below). Identification of a marker gene for residual undifferentiated iPSC We have previously reported the use of single-cell RNA sequencing (scRNAseq) for the reconstruction of hepatocyte-like lineage development from pluripotency under two-dimensional culture14. We explored our scRNAseq data, and we selected genes consistent with following criteria: (1) Specific expression in Rabbit Polyclonal to GAK the iPSC stage to exclude genes expressed during directed hepatic differentiation, (2) high expression in iPSC to facilitate high-level and sensitive detection even at low levels of undifferentiated Alvespimycin iPSC contamination, and (3) considerable difference in expression level between iPSC and target cells i.e., hepatic endoderm (HE) cells. Twelve genes were selected as illustrated in Fig.?2a which expressed highly, specifically, and abundantly in iPSC. Marker gene expression was confirmed using quantitative reverse transcription-polymerase chain reaction (qPCR), and were.