Simple differentiation method using FBS identifies DUSP6 as a marker for fine-tuning of FGF-ERK signaling activity in human pluripotent stem cells.

Assessment of differentiation potential is a basic requirement to obtain qualified human pluripotent stem cells (hPSCs). Here, we report a simple differentiation method using fetal bovine serum (FBS) to estimate differentiation potential and propensity of hPSCs. PluriTest using RNA-sequencing showed that cells differentiated after treatment with 5% FBS. Expression patterns of three germ layer markers revealed that cells cultured in Knockout Serum Replacement-containing medium (KSR) with mouse feeder cells had higher differentiation potential than cells cultured in a chemically defined medium (E8) with recombinant matrix proteins, especially into the mesoderm and endoderm lineages. Analysis of differentially expressed genes between KSR and E8 identified DUSP6 as a marker for where cells had been cultured. Expression of DUSP6 correlated with FGF-ERK signaling activity. Fine-tuning of FGF-ERK signaling activity to a range that can shut down DUSP6 transcription but sustain NANOG transcription partially increased the differentiation potential. Our data suggest that differentiation with 5% FBS is good to estimate differentiation potential and propensity at the early stage, and that DUSP6 is an excellent marker to monitor ERK signaling activity.

Nuclear receptor gene alteration in human induced pluripotent stem cells with hepatic differentiation propensity.

AIM: Human induced pluripotent stem (hiPS) cells are an alternative cell source of regenerative medicine for liver disease. Because variations in hepatic differentiation efficacy among hiPS cells exist, it is important to select a hiPS cell line with hepatic differentiation propensity. In addition, nuclear receptors (NR) regulate essential biological processes including differentiation and development. In this study, we identified the hiPS cell line with hepatic differentiation propensity and examined expression levels of 48 NR during this process. METHODS: We screened 28 hiPS cell lines, which are established from various tissues of healthy persons with various reprogramming methods, using a three-step differentiation method, and examined expression levels of 48 NR by quantitative real-time polymerase chain reaction during the differentiation process in the selected cells. RESULTS: hiPS-RIKEN-2B and hiPS-RIKEN-2F cells have hepatic differentiation propensity. Differentiation propensity towards endoderm was affected by donor origin but not by reprogramming methods or cell type of origins. Expression levels of NR were closely associated with those of hepatic differentiation markers. Furthermore, expression patterns of NR were categorized as five patterns. In particular, seven NR such as chicken ovalbumin upstream promoter transcription factor 1, retinoic acid receptor α, peroxisome proliferator-activated receptor-γ, progesterone receptor, photoreceptor cell-specific nuclear receptor, tailless homolog orphan receptor and glucocorticoid receptor were identified as the genes of which expression gradually goes up with differentiation. CONCLUSION: These findings will be useful for not only elucidating mechanisms of hepatic differentiation of hiPS cells but also cell-based therapy for liver diseases.

Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Variation of DNA methylation on the IRX1/2 genes is responsible for the neural differentiation propensity in human induced pluripotent stem cells.

Introduction: Human induced pluripotent stem cells (hiPSCs) are useful tools for reproducing neural development in vitro. However, each hiPSC line has a different ability to differentiate into specific lineages, known as differentiation propensity, resulting in reduced reproducibility and increased time and funding requirements for research. To overcome this issue, we searched for predictive signatures of neural differentiation propensity of hiPSCs focusing on DNA methylation, which is the main modulator of cellular properties. Methods: We obtained 32 hiPSC lines and their comprehensive DNA methylation data using the Infinium MethylationEPIC BeadChip. To assess the neural differentiation efficiency of these hiPSCs, we measured the percentage of neural stem cells on day 7 of induction. Using the DNA methylation data of undifferentiated hiPSCs and their measured differentiation efficiency into neural stem cells as the set of data, and HSIC Lasso, a machine learning-based nonlinear feature selection method, we attempted to identify neural differentiation-associated differentially methylated sites. Results: Epigenome-wide unsupervised clustering cannot distinguish hiPSCs with varying differentiation efficiencies. In contrast, HSIC Lasso identified 62 CpG sites that could explain the neural differentiation efficiency of hiPSCs. Features selected by HSIC Lasso were particularly enriched within 3 Mbp of chromosome 5, harboring IRX1, IRX2, and C5orf38 genes. Within this region, DNA methylation rates were correlated with neural differentiation efficiency and were negatively correlated with gene expression of the IRX1/2 genes, particularly in female hiPSCs. In addition, forced expression of the IRX1/2 impaired the neural differentiation ability of hiPSCs in both sexes. Conclusion: We for the first time showed that the DNA methylation state of the IRX1/2 genes of hiPSCs is a predictive biomarker of their potential for neural differentiation. The predictive markers for neural differentiation efficiency identified in this study may be useful for the selection of suitable undifferentiated hiPSCs prior to differentiation induction.

Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells.

BACKGROUND: Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. OBJECTIVE AND RATIONALE: Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. SEARCH METHODS: Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. OUTCOMES: Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. WIDER IMPLICATIONS: As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.