Periodic Reporting for period 4 - CELLNAIVETY (Deciphering the Molecular Foundations and Functional Competence of Alternative Human Naïve Pluripotent Stem Cells)
Berichtszeitraum: 2022-05-01 bis 2022-10-31
. An important goal of stem cell therapy is to create “customized” cells that are genetically identical to the patient, which upon transplantation can restore damaged tissues. Such cells can be obtained by in vitro direct reprogramming of somatic cells into embryonic stem (ES)-like cells, termed induced pluripotent stem cells (iPSC). This approach also opens possibilities for modelling human diseases in vitro. However, major hurdles remain that restrain fulfilling conventional human iPSC/ESC potential, as they reside in an advanced primed pluripotent state. Such hurdles include limited differentiation capacity and functional variability. Further, in vitro iPSC based research platforms are simplistic and iPSC based “humanized” chimeric mouse models may be of great benefit.
In this period we have published and established the following on Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs. In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.
In this period we have published and established the following on Post-gastrulation synthetic embryos generated ex utero from mouse naive ESCs. In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.
1) High-Resolution Dissection of the Trajectory Underlying Successful Somatic Reprogramming toward Naïve Ground-State Pluripotency
The ability to reprogram somatic cells into iPSCs with the four Yamanaka factors has provoked immense scientific interest over the past decade. Different studies sought to define inhibitors and facilitators of this process, as well as map the molecular changes accompanying cells undergoing reprogramming. However, our group decided to take a different approach to this challenge, focusing on characterizing the molecular synthesis processes underlying the successful conversion of somatic cells into iPSCs while simultaneously attempting to overlook the “noise” from populations that fail to complete the process. This gap in understanding results from the inefficiency of conventional reprogramming methods and the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. The near-deterministic reprogramming strategies recently developed by our group (Rais et al. Nature 2013) and others open the way to the dissection of molecular events accompanying a synchronized and non-saltatory progression toward iPSCs, thereby providing an opportunity to reveal the critical molecular details needed to decipher reprogramming dynamics without the need for cell sorting or repeated passaging, which greatly bias results.
2) Different conditions have been devised to isolate MEK/ERK signalling independent human naïve pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better correspond to pre-implantation developmental stages. While the naïve conditions described thus far endow human PSCs with different extents of naivety features, isolating human pluripotent cells that retain characteristics of ground state pluripotency while maintaining differentiation potential and genetic integrity, remains a major challenge. We engineer reporter systems that allow functional screening for conditions that can endow both the molecular and functional features expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NFκB, WNT/ßCATENIN and PKC signalling pathways is essential for enabling expansion of teratoma competent fully naïve human PSCs in defined or xeno-free conditions. Divergent signalling and transcriptional requirements for maintaining naïve pluripotency were found between mouse and human. Finally, we establish alternative naïve conditions in which MEK/ERK inhibition is substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human naïve PSCs with diminished risk for loss of imprinting and deleterious global DNA hypomethylation. Our findings set a framework for the signalling foundations of human naïve pluripotency and may advance its utilization in future translational applications.
3) SUMOylation of linker histone H1 drives chromatin condensation and restriction of embryonic cell fate identity. The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.
The ability to reprogram somatic cells into iPSCs with the four Yamanaka factors has provoked immense scientific interest over the past decade. Different studies sought to define inhibitors and facilitators of this process, as well as map the molecular changes accompanying cells undergoing reprogramming. However, our group decided to take a different approach to this challenge, focusing on characterizing the molecular synthesis processes underlying the successful conversion of somatic cells into iPSCs while simultaneously attempting to overlook the “noise” from populations that fail to complete the process. This gap in understanding results from the inefficiency of conventional reprogramming methods and the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. The near-deterministic reprogramming strategies recently developed by our group (Rais et al. Nature 2013) and others open the way to the dissection of molecular events accompanying a synchronized and non-saltatory progression toward iPSCs, thereby providing an opportunity to reveal the critical molecular details needed to decipher reprogramming dynamics without the need for cell sorting or repeated passaging, which greatly bias results.
2) Different conditions have been devised to isolate MEK/ERK signalling independent human naïve pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better correspond to pre-implantation developmental stages. While the naïve conditions described thus far endow human PSCs with different extents of naivety features, isolating human pluripotent cells that retain characteristics of ground state pluripotency while maintaining differentiation potential and genetic integrity, remains a major challenge. We engineer reporter systems that allow functional screening for conditions that can endow both the molecular and functional features expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NFκB, WNT/ßCATENIN and PKC signalling pathways is essential for enabling expansion of teratoma competent fully naïve human PSCs in defined or xeno-free conditions. Divergent signalling and transcriptional requirements for maintaining naïve pluripotency were found between mouse and human. Finally, we establish alternative naïve conditions in which MEK/ERK inhibition is substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human naïve PSCs with diminished risk for loss of imprinting and deleterious global DNA hypomethylation. Our findings set a framework for the signalling foundations of human naïve pluripotency and may advance its utilization in future translational applications.
3) SUMOylation of linker histone H1 drives chromatin condensation and restriction of embryonic cell fate identity. The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.
The overall novelty of our proposal (Objectives 1-4) lies in the fact that this is project will be the first comprehensive study aiming at employing multi-approach dissection of a new pluripotent state recently detected in human PSC cultures, and seeks to biologically characterise these cells and unveil pathways that will underlie and facilitate their robust growth and adequate differentiation. The latter will lead to the design of strategies that will accelerate the safe medical application of human naïve PSCs and accelerate their use in disease specific research and basic and applied stem cell research.