Influence of activin A supplementation during human embryonic stem cell derivation on germ cell differentiation potential

Human embryonic stem cells (hESCs) are more similar to primed mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro

Direct comparison of distinct naive pluripotent states in human embryonic stem cells

Until recently, human embryonic stem cells (hESCs) were shown to exist in a state of primed pluripotency, while mouse embryonic stem cells (mESCs) display a naive or primed pluripotent state. Here we show the rapid conversion of in-house-derived primed hESCs on mouse embryonic feeder layer (MEF) to a naive state within 5-6 days in naive conversion media (NCM-MEF), 6-10 days in naive human stem cell media (NHSM-MEF) and 14-20 days using the reverse-toggle protocol (RT-MEF). We further observe enhanced unbiased lineage-specific differentiation potential of naive hESCs converted in NCM-MEF, however, all naive hESCs fail to differentiate towards functional cell types. RNA-seq analysis reveals a divergent role of PI3K/AKT/mTORC signalling, specifically of the mTORC2 subunit, in the different naive hESCs. Overall, we demonstrate a direct evaluation of several naive culture conditions performed in the same laboratory, thereby contributing to an unbiased, more in-depth understanding of different naive hESCs

A causal role for TRESK loss of function in migraine mechanisms

The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target

Human embryonic stem cells : different shades of pluripotency and differentiation potential

In this thesis, we investigated the two fundamental characteristics of human embryonic stem cells (hESCs), being their pluripotency and differentiation potential. Firstly, we have shown that hESC derivation in the presence of Activin A (ActA) predisposes these ActA-derived hESC lines towards a primordial germ cell (PGC) fate unlike the hESCs derived in standard culture conditions. The undifferentiated ActA-derived hESC lines demonstrated an inherent increased expression of early PGC markers STELLA and cKIT. Following their differentiation for seven days in the presence of bone morphogenic protein 4 (BMP4), widely known to induce germ cell differentiation, the pre-meiotic marker VASA was significantly upregulated both at the transcript and protein level in comparison to the differentiated hESC lines derived in standard culture conditions. We also observed an active TGFβ/Activin signalling during the differentiation process as proven by the nuclear translocation of SMAD2/3 following phosphorylation. Next, we further investigated the role of ActA in inducing in vitro PGC fate upon its exogenous supplementation during hESC differentiation. Differentiation of ActAderived and standard hESC lines in the presence of both ActA and BMP4 significantly improved PGC gene expression compared to BMP4 alone. We also demonstrated the need to mimic in vivo environment for differentiation purposes as exposure of these seven-day old in vitro-derived PGCs to reproductive hormones resulted in the generation of pre-meiotic germ cells. Additionally, we suggest a synergistic developmental relationship between the endodermal and PGC lineages during in vitro differentiation of hESCs, which has not been reported earlier. As the biggest success in mouse for in vitro germ cell formation from stem cells was achieved when starting from naïve pluripotent stem cells, we attempted to convert existing primed hESCs into their naïve state. We were able to generate naïve hESCs using a novel naïve pluripotency inducing media, which also allowed the derivation of naïve mESCs. Hence, we suggest that alternative routes exist to induce naïve pluripotency in human

Use of pluripotent stem cells for reproductive medicine: are we there yet?

In recent years, pluripotent stem cells have demonstrated to be exciting tools to understand embryonic development, cell lineage specification, tissue generation and repair, and various other biological processes. In addition, the identification and isolation of germ line stem cells has given more insight into germ cell biology at the molecular level and into the underlying causes of infertility which was not possible earlier. The recent derivation of in vitro derived sperm and oocytes from pluripotent stem cells in the mouse model represents a major breakthrough in the field and substantiates the critical relevance of stem cells as a potential alternative resource for treating infertility. Although the past years have yielded compelling information in understanding germ cell development via in vitro stem cell assays, extended investigative research is necessary in order to derive fully functional artificial gametes' in a safe way for future therapeutic applications

The influence of patient and cohort parameters on the incidence and developmental potential of embryos with poor quality traits for use in human embryonic stem cell derivation

Human embryonic stem cells (hESCs) are most commonly derived from the inner cell mass (ICM) of blastocyst stage embryos. While the majority of hESC lines originate from good-quality embryos donated after cryogenic storage, poor-quality embryos (PQEs) not suitable for clinical use have also been shown to generate hESC. This provides a newfound function for embryos that would otherwise be discarded following IVF or ICSI. Owing to their lack of clinical importance, however, data on the poorest embryos in a cohort go largely unreported in the literature. It is therefore of interest to better understand the availability of PQEs from IVF/ICSI cycles and to determine their ability to develop into blastocysts with good-quality ICMs for use in hESC derivation. In this study, we investigate the influence of patient parameters and embryo cohort on PQE incidence, blastocyst development, ICM quality and successful hESC derivation from donated PQEs. PQEs from 736 patient cycles that did not meet our clinical criteria for transfer or cryopreservation were cultured until Day 6 of development and assessed for blastocyst formation and ICM quality. A subset of blastocysts with good-quality ICMs were then used for hESC derivation attempts. Anonymous patient data such as maternal age, embryo history and cohort parameters were then retrospectively compiled and analysed. PQEs made up 46.8 of two pronucleate embryos created from IVF/ICSI. Including embryos with abnormal fertilization, a mean of 3.6 2.8 embryos were donated per cycle with 32.6 developing to the blastocyst stage. Good-quality ICM were produced in 13.9 of PQEs cultured. Of good-quality ICM, 15.4 of those used in hESC derivation attempts resulted in a novel line. The PQEs that originated from older patients (37 year) or from cycles that did not result in pregnancy had significantly diminished blastocyst development and ICM quality. Maternal age was also shown to further influence the ability of good-quality ICMs to generate hESC. PQEs are an abundant source of embryos capable of developing to blastocysts with good-quality ICMs and subsequently generating novel hESC. We have shown that prognostic variables used to predict IVF/ICSI outcome can also help predict which PQEs have the best hESC developmental potential. Owing to the diversity of PQE origin, experiments designed to compare hESC derivation techniques or efficiency using PQEs should consider clinical IVF/ICSI parameters to establish groups with equal developmental competence. Additional investigation is needed to determine if these results are applicable to hESC derivation using good-quality embryos

Culture optimisation to direct differentiation of human embryonic stem cells into primordial germ cells

Stem cells & developmental biolog

The post-inner cell mass intermediate : implications for stem cell biology and assisted reproductive technology

BACKGROUND: Until recently, the temporal events that precede the generation of pluripotent embryonic stem cells (ESCs) and their equivalence with specific developmental stages in vivo was poorly understood. Our group has discovered the existence of a transient epiblast-like structure, coined the post-inner cell mass (ICM) intermediate or PICMI, that emerges before human ESC (hESCs) are established, which supports their primed nature (i.e. already showing some predispositions towards certain cell types) of pluripotency. METHODS: The PICMI results from the progressive epithelialization of the ICM and it expresses a mixture of early and late epiblast markers, as well as some primordial germ cell markers. The PICMI is a closer progenitor of hESCs than the ICM and it can be seen as the first proof of why all existing hESCs, until recently, display a primed state of pluripotency. RESULTS: Even though the pluripotent characteristics of ESCs differ from mouse (naive) to human (primed), it has recently been shown in mice that a similar process of self-organization at the transition from ICM to (naive) mouse ESCs (mESCs) transforms the amorphous ICM into a rosette of polarized epiblast cells, a mouse PICMI. The transient PICMI stage is therefore at the origin of both mESCs and hESCs. In addition, several groups have now reported the conversion from primed to the naive (mESCs-like) hESCs, broadening the pluripotency spectrum and opening new opportunities for the use of pluripotent stem cells. CONCLUSIONS: In this review, we discuss the recent discoveries of mouse and human transient states from ICM to ESCs and their relation towards the state of pluripotency in the eventual stem cells, being naive or primed. We will now further investigate how these intermediate and/or different pluripotent stages may impact the use of human stem cells in regenerative medicine and assisted reproductive technology