Recent evidence suggests that hESCs combined with a transcriptomic technique have the potential to predict human relevant embryotoxicity. Within this context, we have developed in vitro methods based on hESCs, using transcriptomics to predict developmental toxicity of different courses of developmental toxicants. Specifically, we tested six histone deacetylase inhibitors and six mercury compounds [ 11 , 12 , 15 , 25 ]. Human teratogenic drugs, like thalidomide and VPA, also provide been tested using the UKK test system, covering earlier and late differentiation processes of hESCs. Their modified transcriptomic profiles determined the teratogenic mechanisms for these medications [ 13 , 14 ], resolving the in vivo teratogenic a result of these drugs. Furthermore, to quantify levels of developmental degree of toxicity, the indices D p and G we were established, using several differentially expressed genes induced by teratogenic compounds, such as thalidomide and VPA [ 10 ]. Because of the ethical concerns about the use of hESCs, hiPSCs were investigated as an alternative for individual relevant in vitro toxicity testing of potential developing toxicants. Here, we compared transcriptome responses of 2 different hiPSCs (foreskin and IMR90 hiPSCs) with hESCs, using the UKK test system, and VPA as a developing toxicant. We quantified the toxicity potential of VPA using both D p plus D i . According to the UKK test program, cells from all three cell lines were differentiated for 14 days in the presence and absence of VPA.
The PCA of the transcriptomes of day 0 and day 14 based on most microarray data showed significant differences between undifferentiated hESCs and hiPSCs (day 0) and their differentiated (14 days) cells. Interestingly, differences in the transcriptomes were pretty uniform for all three cell lines. The differentially portrayed genes on day 14, designated as developmental genetics, showed significant differences between the hESCs and both hiPSC lines, although the transcriptomes in undifferentiated hESCs, and each hiPSCs lines were very similar. A GO analysis of the upregulated genes at day 14 encompassed more than 30% associated with embryonic development-related biological processes (developmental GOs) in all 3 cell lines. Further analysis of these developmental genes within hESCs and hiPSCs revealed that approximately 50% of such similarities were attributed to upregulated developmental GOs, irrespective of whether hESCs or hiPSCs had been differentiated. Similarities were also noticed for KEGG pathways for all three cell lines. Many common GOs, such as “ anatomical system development”, “ nervous system development”, “ embryonic morphogenesis”, related to wanting development, were identified in the developmental genes.
A CellNet analysis revealed almost consistent ESC scores for all three undifferentiated cell lines upon day 0, and an uniform reduction of ESC ratings on day 14 of differentiation, along with an increase within cell/tissue type scores, such as fibroblast, lung, skin, kidney, heart and liver. A few developmental genes from all of three cell lines were from the ectoderm; mesoderm plus endoderm lineages, indicating a partial recapitulation of within vivo embryonic development at the transcriptomic level. Our CellNet analysis showed that hiPSCs and hESCs have comparable differentiation potential, suggesting that hiPSCs can recapitulate developing processes of differentiated hESCs.
VPA has teratogenic potential, inducing spina bifida with steady state concentrations of 0. 51 ± 0. 17 mM in humans. In this study, we all used a C max around two times above this level [ 26 , 27 ]. Exposure to VPA during the 14-day differentiation period resulted in deregulation of developing genes, with opposing induction, i. e., upregulated developing genes were downregulated, while downregulated developmental genes had been upregulated. Very few VPA deregulated genes were common for all three cell lines. We found that more downregulated genes belonged to embryonic development-related GOs than upregulated types. This clearly shows the inhibitory effects of VPA upon differentiation. The common VPA upregulated developmental genes in all 3 cell lines were associated with anatomical structure and anxious system development, whereas VPA downregulated developmental genes had been related to nervous system development, neurogenesis, and brain advancement. These results are consistent with our earlier published findings, showing that VPA repressed neural tube and dorsal forebrain developmental genes, and upregulated axonogenesis and ventral forebrain associated genes in differentiating hESCs [ 13 ].
However , we also noted variations between the three cell lines in genes associated with wanting development and regulated by VPA. Specifically, upregulated genetics associated with neural crest cell development were identified within differentiated H9 ESCs, whereas oligodendrocyte differentiation and bacteria cell development were identified in differentiated IMR90 plus foreskin hiPSCs, respectively. Downregulated genes associated with telencephalon growth were identified in differentiated H9 hESCs, whereas genetics involved in the metencephalon development and heart tube development had been identified in IMR90 and foreskin hiPSCs, respectively. Obviously, GOs identified in hESCs and hiPSCs do not allow the quantification of the toxic effect of developmental toxicants.
Given that a conclusion as to whether hiPSCs can replace hESCs for developmental toxicity testing depending on a GO analysis is not possible, we proposed the use of 2 indices: D p and D i based on VPA deregulated developmental genes. D p symbolizes the intersection of VPA-deregulated genes with developmental genetics and its value directly correlates with the developmental toxicity possible. D i represents the ratio of developmental genes among VPA deregulated overall genes; a high overrepresentation value means that VPA preferentially deregulates developmental genes. D p and M i actually values were estimated for different fold change values for the developmental genes (> two, > 5 and > 10). D p values demonstrated a linear increase for the same cell line with boosting fold change, but varied among cell lines. Oddly enough, the D i values were similar for all three cell outlines, for any given fold change value. The greatest increase in D i ideals occurred for a fold change from two to five. Generally there also was a moderate increase in D i from fivefold to tenfold change in developmental genes, indicating that a fivefold modify for developmental genes is most critical for the D i calculation. Therefore, this index has strong potential for prediction of developing toxicants.
Among the VPA-deregulated genetics common to all three cell lines, several developmental genetics were of particular interest for assessing in vivo observed teratogenic effects of VPA. In particular, we identified 2 upregulated developmental genes, which become downregulated by VPA ( DOK6 plus BCL2 ), plus two downregulated developmental genes that become upregulated simply by VPA (CLDN10 and PRKCB ).
Treatment with VPA while pregnant in women has resulted in teratogenic malformations in infants, including neural tube defects, microcephaly, ventricular septal problems, craniofacial abnormalities, ear abnormalities and urogenital abnormalities [ 28 ]. The gene Docking Protein 6 ( DOK6 ), a member of the DOK family, plays a role in Ret tyrosine kinase signalling, which stimulates neurite outgrowth (Crowder et al., 2004). In a computer mouse model, knockdown of Dok6 by specific RNAi resulted in decreased neurite outgrowth (Li et al., 2010). B-cell CLL lymphoma 2 ( BCL2 ) has been described as a key regulator of embryonic development. Despite the fact that Bcl2 knockout in mice is not lethal, it still exhibits different malformations during postnatal development, including growth retardation, smaller sized ears, atrophic thymus and spleen [ 29 , 30 ]. Bcl2 knockout rodents exhibited progressive degeneration of motor neurons of the face region [ 31 ]. Claudin 10 ( CLDN10) is a downregulated developmental gene that becomes upregulated by VPA. Gain of perform studies in chicken demonstrate that CLDN10 is crucial for normal center tube looping [ 32 ]. The Protein Kinase D Beta ( PRKCB ) is also upregulated by VPA, and recently, significant duplicate number variation has been found in human patients with ventricular septal defects [ 33 ]. In accordance with our findings, it is often established that VPA stimulates PRKCB in several cell types [ 34 , 35 ].