Thomas O'Leary

Tracking the progression of the human inner cell mass during embryonic stem cell derivation

The different pluripotent states of mouse embryonic stem cells (ESCs) in vitro have been shown to correspond to stages of mouse embryonic development. For human cells, little is known about the events that precede the generation of ESCs or whether they correlate with in vivo developmental stages. Here we investigate the cellular and molecular changes that occur during the transition from the human inner cell mass (ICM) to ESCs in vitro. We demonstrate that human ESCs originate from a post-ICM intermediate (PICMI), a transient epiblast-like structure that has undergone X-inactivation in female cells and is both necessary and sufficient for ESC derivation. The PICMI is the result of progressive and defined ICM organization in vitro and has a distinct state of cell signaling. The PICMI can be cryopreserved without compromising ESC derivation capacity. As a closer progenitor of ESCs than the ICM, the PICMI provides insight into the pluripotent state of human stem cells.

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

The combination of inhibitors of FGF/MEK/Erk and GSK3β signaling increases the number of OCT3/4- and NANOG-positive cells in the human inner cell mass, but does not improve stem cell derivation.

In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3β pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3β pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3β pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3β significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3β signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

Treatment of human embryos with the TGFβ inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation

STUDY QUESTION Is there an effect of the TGFβ inhibitor SB431542 (SB) on the epiblast compartment of human blastocysts, and does it affect subsequent human embryonic stem cell (hESC) derivation? SUMMARY ANSWER SB increases the mean number of NANOG-positive cells in the inner cell mass (ICM), and allows for subsequent hESC derivation. WHAT IS KNOWN ALREADY It is known that inhibition of TGFβ by SB has a positive effect on mouse ESC self-renewal, while active TGFβ signalling is needed for self-renewal of primed ESC. STUDY DESIGN, SIZE, DURATION From December 2011 until March 2012, 263 donated spare embryos were used from patients who had undergone IVF/ICSI in our centre. PARTICIPANTS/MATERIALS, SETTING, METHODS Donated human embryos were cultured in the presence of SB or Activin A, and immunocytochemistry was performed on Day 6 blastocysts for NANOG and GATA6. Moreover, blastocysts were used for the derivation of hESC, with or without exposure to SB. MAIN RESULTS AND THE ROLE OF CHANCE Immunocytochemistry revealed a significantly higher number of NANOG-positive ICM cells in the SB group compared with the control (12.0 ± 5.9 versus 6.1 ± 4.7), while no difference was observed in the Activin A group compared with other groups (6.7 ± 3.7). The number of GATA6-positive ICM cells did not differ between the SB, Activin A and control group (8.8 ± 4.3, 8.0 ± 4.6 and 7.2 ± 4.0, respectively). Blocking TGFβ signalling did not prevent subsequent hESC line derivation. LIMITATIONS, REASONS FOR CAUTION The number of human blastocysts available for this study was too low to reveal if the observed increase in NANOG-positive epiblast cells after exposure to SB affected the efficiency of hESC derivation (12.5% compared with 16.7%). WIDER IMPLICATIONS OF THE FINDINGS This work can contribute to the derivation of naive hESC lines in the future. STUDY FUNDING/COMPETING INTEREST(S) M.V.d.J. is holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT, grant number SB093128), Belgium. G.D. and this research are supported by the Research Foundation Flanders (FWO), grant number FWO-3G062910) and a Concerted Research Actions funding from BOF (Bijzonder Onderzoeksfonds University Ghent, grant number BOF GOA 01G01112). S.M.C.d. S.L. is supported by the Netherlands Organization of Scientific Research (NWO) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). P.D.S. is holder of a fundamental clinical research mandate by the FWO. We would like to thank Ferring Company (Aalst, Belgium) for financial support of this study. The authors do not have any competing interests to declare. TRIAL REGISTRATION NUMBER Not applicable.

Derivation of human embryonic stem cells using a post–inner cell mass intermediate

Little is known about the true developmental origin of human embryonic stem cells (hESCs) or the events that initiate their generation. Recently, we have shown that hESCs originate from a post–inner cell mass (ICM) intermediate (PICMI), a unique transient epiblast-like structure that is different from both its ICM progenitor and its subsequent hESC fate. As a closer progenitor of hESCs than the ICM, the PICMI could be used to provide further insight into the human pluripotent state. Here we provide a detailed (7-d) protocol for the culture of the human preimplantation embryos in order to derive the PICMI. Subsequent identification and cryopreservation of the PICMI are described, in addition to hESC derivation. The initial hESC outgrowth is visible within 2–7 d after PICMI plating. By using the protocol provided, we observed PICMI formation in 21.3% of plated blastocysts with good-quality ICMs. Of the PICMIs used for hESC derivation, 80.6% showed hESC outgrowth after further culture.

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

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

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 (naïve) to human (primed), it has recently been shown in mice that a similar process of self-organization at the transition from ICM to (naïve) 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 naïve (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 naïve 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.

Inhibition of Transforming Growth Factor β Signaling Promotes Epiblast Formation in Mouse Embryos

Early lineage segregation in preimplantation embryos and maintenance of pluripotency in embryonic stem cells (ESCs) are both regulated by specific signaling pathways. Small molecules have been shown to modulate these signaling pathways. We examined the influence of several small molecules and growth factors on second-lineage segregation of the inner cell mass toward hypoblast and epiblast lineage during mouse embryonic preimplantation development. We found that the second-lineage segregation is influenced by activation or inhibition of the transforming growth factor (TGF)β pathway. Inhibition of the TGFβ pathway from the two-cell, four-cell, and morula stages onward up to the blastocyst stage significantly increased the epiblast cell proliferation. The epiblast formed in the embryos in which TGFβ signaling was inhibited was fully functional as demonstrated by the potential of these epiblast cells to give rise to pluripotent ESCs. Conversely, activating the TGFβ pathway reduced epiblast formation. Inhibition of the glycogen synthase kinase (GSK)3 pathway and activation of bone morphogenetic protein 4 signaling reduced the formation of both epiblast and hypoblast cells. Activation of the protein kinase A pathway and of the Janus kinase/signal transducer and activator of transcription 3 pathway did not influence the second-lineage segregation in mouse embryos. The simultaneous inhibition of three pathways--TGFβ, GSK3β, and the fibroblast growth factor (FGF)/extracellular signal-regulated kinases (Erk)--significantly enhanced the proliferation of epiblast cells than that caused by inhibition of either TGFβ pathway alone or by combined inhibition of the GSK3β and FGF/Erk pathways only.