Unlocking the potential of SY-stem cells Free

Fantastic previous meetings of the SY-Stem Symposium set a high bar of expectations for the ‘Symposium focusing on the next generation of stem cell researchers’. Yet again, the scientific organizing team composed of Elly Tanaka (Research Institute of Molecular Pathology; IMP), Jürgen Knoblich (Institute of Molecular Biotechnology; IMBA), Christa Buecker (Max Perutz labs), Sofia Grade (IMBA), Joanna Jachowicz (IMBA) and Bon-Kyoung Koo (IBS Center for Genome Engineering) more than fulfilled their goal to set up a diverse program and create an atmosphere in which scientific exchange happened naturally (Fig. 1). Lea-Louisa Klement provided indispensable support to the entire organizing team, alongside PhD students Daria Filipczak (Foisner lab at Max Perutz labs) and Theresa Sommer (Rivron lab at IMBA), ensuring the event's success for the 230 participants (Fig. 2). Further promoting the idea of fostering scientific exchange, the symposium provided plenty of networking opportunities, for example during two lively poster sessions, and lunch arrangements facilitating discussions among attendees or with the speakers and journal editors. This was further topped by evening activities, such as a pub quiz, providing lively entertainment while disclosing scientific and historical knowledge about Vienna.

In line with the spirit of the SY-Stem symposium series, before the start of the scientific talks, a mentorship session led by Daria Filipczak included guest speakers from various scientific fields and career paths, such as Claudia Gerri (MPI of Molecular and Cell Biology and Genetics, Dresden), Josh Bagley (CSO, a:head AG, Vienna), Ruben van Helden (senior scientist, HeartBeat.bio AG, Vienna) and Manuel Llado Santaeularia (scientific communicator, IMBA and GMI). The speakers shared their career trajectories in an interactive session. Regardless of their current position, all emphasized the importance of showing resilience in scientific pursuits and the importance of seeking mentorship beyond one's immediate circle.

Keynote speakers Hiro Nakauchi and Magdalena Götz delivered enlightening talks on diverse topics in stem cell research, emphasizing the importance of understanding basic stem cell biology to carry forward their cutting-edge approaches in regenerative medicine. Unique to this particular meeting, the SY-Stem also spotlit early career scientists and young PIs, breaking new ground in their research fields, together creating a multi-faceted stem cell-centered event.

Studies of cellular and molecular mechanisms underlying early miscarriage and congenital pathologies are restricted by ethical and technical limitations. These might be overcome by advanced three-dimensional in vitro engineering approaches, exploiting the self-organizing properties of stem cells and recapitulating key spatiotemporal events of early human embryonic development. Employing these new model systems, it has been discovered in recent years that metabolism exerts functions beyond the energetic needs of a cell, including cell fate decisions, at different developmental stages and across different cell lineages.

Starting this session, Jan Żylicz (University of Copenhagen, Denmark) proposed transcriptional, epigenetic and metabolic changes acting in concert in an epi-metabolic coupling to navigate specific cell state changes. Although previous work in mice had shed light on glucose-regulating trophectoderm specification (the first lineage decision within blastomeres), Żylicz relies on human naive embryonic stem cells (ESCs) and their transition towards trophoblast stem cells (TSCs) and blastoids. Here, he identified metabolic rewiring as a bottleneck during the induction of GATA3⁺ TSC-like cells. He presented metabolic engineering as a potent approach promoting not only human ESC-to-TSC induction competence, but also blastoid development, polarization and expansion, all modulated via global epigenetic changes.

The importance of metabolic pathways for cell fate decisions was further promoted by Kristina Stapornwongkul, a postdoctoral fellow from Vikas Trivedi's Group (EMBL Barcelona, Spain). Using mouse and human ESC-based in vitro models for gastrulation, her work unveiled glycolysis inhibition to increase ectodermal cell fates at the expense of mesodermal and endodermal lineages. Here, glycolysis acts as an upstream activator of Nodal and Wnt signaling, enabling direct metabolic control of germ layer proportions through exogenous glucose levels. Further rescue experiments with signaling agonists demonstrated that the effect of glycolysis on signaling can be decoupled from its effect on growth.

However, metabolism is not only instructive in cell fate decisions between different germ layers, it is also dynamic within cell lineages, as introduced by Berna Sozen (Yale School of Medicine, New Haven, CT, USA). Using in vivo and ex vivo live imaging of gastrulating mouse embryos, in vitro stem cell and embryo-derived tissue explant models, Sozen described an instructive role of glucose metabolism navigating mouse gastrulation in time and space (Cao et al., 2023 preprint). Whereas the epithelial-to-mesenchymal transition uses the hexosamine biosynthetic pathway to metabolize glucose, newly formed mesoderm requires glycolysis. This transition is coordinated with fibroblast growth factor (FGF) activity and serves a functional role in supporting fundamental aspects of gastrulation (Cao et al., 2023 preprint). Sozen also introduced a new model system for studying early embryonic development, spanning up to the post-implantation stage, called human extra-embryoids. Derived from human pluripotent stem cells, this model accurately recapitulates the Carnegie stages 4-7. It captures the spontaneous differentiation and, most importantly, the tissue interaction between embryonic epiblast-like and extra-embryonic hypoblast-like lineages (Pedroza et al., 2023).

Importantly, nutritional regulation of cell fate decision-making and proper embryonic development is dependent on nutrient availability regulated by the fetal-placental interface. Claudia Gerri's lab (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany) is investigating how cell lineages are established, and how environmental cues are interpreted and incorporated to build complex architectures of the placenta. To recreate its development in a dish, Gerri is exploiting early embryos and placental organoids, investigating how the uterine microenvironment shapes the placental specification in various species. Her studies will help unravel the drivers of placental diversity and shed light on the molecular changes shaping organ morphogenesis.

Taken together, the speakers of this session centered on early aspects of embryogenesis, enlightened how embryos adapt to changes in their environment and opened up new non-invasive approaches to advance in vitro fertilization techniques and understand the origins of life.

A developmental step beyond, research focusing on organogenesis addresses cell fate decisions culminating in the formation of organs. How cells control gene expression programs to establish the complex vertebrate body plan is studied by Vicki Metzis' lab (Imperial College London, UK). During the epiblast precursor stage, Wnt induction leads to Cdx2 upregulation, a crucial step in the establishment of anterior-posterior identity. The onset and duration of Cdx2 expression are controlled by several nonredundant cis-regulatory elements (CRE), the dynamics of which Metzis characterized during directed differentiation of genome-engineered mouse ESCs towards spinal cord and mesodermal cells. She unveiled that the activity of distinct CREs is required for the onset, maintenance and termination of Cdx2 expression during the development of spinal cord and paraxial mesoderm. When switching a single motif within a silencer to a motif normally restricted to an enhancer in close proximity, Cdx2 is dysregulated. The findings propose dual-function enhancer-silencer CREs as a mechanism to provide cells with context-specific responses to extrinsic cues during posterior body formation (Amblard et al., 2024 preprint).

However, the integration of intrinsic cues is equally important for proper embryonic development. Gastruloids hold self-organizing potential and facilitate the investigation of early embryonic patterning. Alexandre Mayran, postdoctoral fellow at Denis Duboule's lab (École Polytechnique Fédérale de Lausanne, Switzerland) proposed a cell-specific switch from E- to N-cadherin underlying this remarkable property (Mayran et al., 2023 preprint). Snai1 represses both E-cadherin and a pluripotency-associated signature, thus allowing a proper switch to N-cadherin. N-cadherin hampers the morphogenic capability of gastruloids, installing a molecular and cellular mechanism integrating the exit from pluripotency and the pace of cell differentiation.

This balance between patterning and growth is relevant throughout embryonic development, resulting in the emergence of proper proportions of cell populations. Marek van Oostrom, a PhD student from Katharina Sonnen's lab (Hubrecht Institute, Utrecht, Netherlands), discovered a bidirectional feedback coupling between the segmentation clock and cell cycle progression during somitogenesis in mouse embryonic explants. Although the presomitic mesoderm cell population is maintained by proliferation, waves of oscillatory gene expression run from posterior to anterior, initiating new somite formation by differentiation. Thereby, Oostrom gave valuable insights on how proliferation and differentiation are coordinated with one another, ensuring proper embryonic development and body plan formation.

Once the body plan is established, a series of complex morphogenetic events is required for the formation of functional organs. Eyal Karzbrun from the Weizmann Institute of Science (Israel) unraveled a new chip-based model to recreate organ morphogenesis, specifically neural tube folding, using human ESCs in vitro. This process is driven by two mechanisms: apical contraction of neural ectoderm and extracellular matrix-based basal adhesion mediated by non-neural ectoderm. By proposing a mechanical model recreating neural-tube morphology, Karzbrun stressed the relevance of tissue mechanics to study organ morphogenesis.

Even though recapitulation of 3D tissues in vitro is a major field of research, certain types of analyses require a 2D morphology as a model. Xavier Trepat (Institute for Bioengineering of Catalonia, Barcelona, Spain) explained the intricate interplay between physical forces and cellular dynamics in open-lumen mouse intestinal organoids and tumoroids using traction force microscopy to map three-dimensional forces. These maps depict the stress distribution defining mechanical and functional compartments, crucial for crypt folding through apical constriction (‘bending’) and collective cell migration along a tensile gradient (‘buckling’) driven by mitotic pressure. These processes are facilitated by compartment-specific accumulation of actomyosin and subsequent differential cell surface tension, as explained by a 3D vertex model. However, the role of mechanics on tissue dynamics extends beyond physiology, as alterations in mechanical properties are observed in patient-derived colorectal cancer organoids helping to understand pathological processes.

As highlighted by Stefan Jahnel, postdoctoral fellow in Sasha Mendjan's lab (IMBA, Vienna, Austria), mechanical cues influence multiple aspects of early heart development, which can be replicated in human cardiac organoids (cardioids). These cardioids form fluid-filled cavities, which are filled with hyaluronic acid (HA), resembling the early developing heart. The formation of these cavities is inhibited when HA synthesis is genetically disrupted. Moreover, degradation of HA has an immediate effect on cardiomyocyte contractility, which can be rescued by oil injection restoring the internal pressure.

Despite immense progression in modeling certain aspects of development in vitro, understanding complex interactions with extra-embryonic cells requires in vivo characterization. During mouse development, extra-embryonic cells not only contribute to the gut endoderm but also closely match the transcriptional identity of embryonic gut cells. Julia Batki, postdoctoral fellow from Alexander Meissner's lab (Max Planck Institute for Molecular Genetics, Berlin, Germany), took on the challenge to lineage-trace extra-embryonic cells in vivo and assess their fate during murine gut development. Batki discovered that gut cells of extra-embryonic origin retain a non-canonical epigenetic landscape, undergo p53-dependent programmed cell death and their remnants are cleared by neighboring embryonic gut cells via non-professional phagocytosis by midgestation. These data on eliminating transient cell types show a selective cell clearance mechanism during mammalian organogenesis.

The evolution of human brain complexity encompasses multiple research directions, including how transposable elements (TEs) store a large portion of genetic information specific to primates and humans. However, only recently have technical advances allowed the obstacles posed by the TE highly repetitive, abundant and polymorphic nature to be surpassed by using individual-matched RNA-sequencing and long-read genome data for accurate in-depth analyses. Johan Jakobsson (Lund University, Sweden) identified the dynamic activity of long interspersed nuclear element 1 (L1) retrotransposon promoters in the developing and the adult human brain, inducing not only its own expression but also possibly acting as alternative promoters for their host genes. Interestingly, Jakobsson highlighted how an L1 insertion may have contributed to the evolution of the human brain, but also provided a putative link between L1s and the genetics of neuropsychiatric disorders.

Although it is well established that neurons, including the nuclear genomic DNA, can persist throughout life, the life span of non-coding nuclear RNAs has not yet been determined in adult tissues. Tomohisa Toda (German Center for Neurodegenerative Diseases, Dresden, Germany, and Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany) discovered that some nuclear RNAs, crucial for proper chromatin architecture and transcription regulation, do not turn over for years in the adult mouse brain (Zocher et al., 2024). Thereby, Toda proposes a novel concept of RNA metabolism and cell type-specific turnover rates, exploring possible functional roles for nuclear RNA in long-term epigenetic regulation and genome integrity.

Sex chromosome dosage compensation in female mammalian cells is achieved by X-chromosome inactivation (XCI). However, some X-linked genes can variably escape XCI, and clusters of these ‘escapees’ are often organized in topologically associating domain (TAD)-like structures on the inactive X-chromosome. How escapees evade Xist-mediated silencing, thereby overruling the repressive epigenetic signatures typical of XCI, is an active field of research. Agnese Loda, postdoctoral fellow from Edith Heard's lab (European Molecular Biology Laboratory, Heidelberg, Germany) highlighted the role of Xist RNA as a crucial regulator of escapees in differentiated cells and potentially throughout life (Hauth et al., 2024 preprint). By forcing enhanced Xist expression both in vitro, in cultured neural progenitor cells, and in vivo, during mouse embryonic development, Loda and colleagues found that Xist RNA has the capacity to modulate the expression levels of escapees and to erase TAD-like structures on the inactive X-chromosome in a SPEN-dependent manner. These findings suggest that XCI initiation can occur well beyond early embryogenesis and can result in progressive and irreversible silencing of the large majority of escapees on the inactive X.

X-chromosomal inactivation occurs in placental animals, yet is not comparable across species. However, studying the function of X-linked genes is particularly relevant for the field of neurodevelopmental diseases, which are oftentimes caused by mutations in X-chromosomal genes and characterized by a profound sexual dimorphism, with males being more often and more severely affected than females. In humans, the study of XCI is challenged by the limitations to selected in vitro model systems or postmortem tissues disregarding developmental trajectories. Marisa Karow (Friedrich-Alexander-Universität Erlangen-Nürnberg) and her colleagues used the clonal nature of human induced pluripotent stem cells (hiPSCs) to study allelic usage of X-chromosomal genes from a defined point-of-origin. They describe a dynamic, locus- and cell lineage-specific reactivation of selected X-chromosomal genes, re-expression of which directly impacts on a neurodevelopmental disease phenotype, as modeled in brain organoids (Käseberg et al., 2023 preprint).

Furthermore, the potential of brain organoids to explore the complexity of the different human brain regions was highlighted by In-Hyun Park (Yale Stem Cell Center, New Haven, USA). His work demonstrated the modeling of diverse nuclei identities within the diencephalon. By generating diencephalic thalamic organoids (ThOs) through varying BMP and FGF signaling, Park successfully produced both ventral and dorsal regions. These ThOs provide a valuable platform to study neurodevelopmental disorders affecting the diencephalic regions in greater depth.

Sakurako Nagumo Wong (IMBA, Vienna, Austria) provided an impressive example of the power of brain organoids to study disease etiology. She described the characterization of patient-derived organoids recapitulating tuberous sclerosis complex. An expansion of caudal late interneuron progenitor (CLIP) cells was found to be the starting point of the two main features of the patient's brain: the subependymal nodules (SENs) and focal dysplastic regions (cortical tubers). In addition, an imbalance caused by an overproduction of inhibitory interneurons along with alterations in calcium bursts or high-frequency oscillations in organoids carrying TSC2 mutations is consistent with the hypothesis of hyperexcitability and susceptibility to epileptic seizures.

Magdalena Götz (Chair of Physiological Genomics at Ludwig-Maximilians University and Director of the Institute for Stem Cell Research at Helmholtz Center Munich, Germany) explored novel mechanisms of neurogenesis and neural repair, focusing on organelle heterogeneity. Notably, nuclear Trnp1 and centrosomal Akna were identified as key regulators in neural stem cell regulation, with Trnp1 promoting self-renewal (Stahl et al., 2013; Esgleas et al., 2020) and Akna driving delamination and differentiation (Camargo Ortega et al., 2019). Using spatial proteome analysis of the centrosomes in hiPSC-derived neural stem cells and neurons, the lab uncovered cell type-specific centrosome protein composition, as a mechanistic basis of how ubiquitous expressed proteins have brain-specific phenotypes when mutated (O'Neill et al., 2022). In addition, Götz discussed mitochondrial heterogeneity and its role in direct neuronal reprogramming (Russo et al., 2021), including insights from murine models of traumatic brain injury that underscore the therapeutic relevance of astroglial response and their molecular regulators in cerebral pathologies.

In the field of stem cells, regeneration – exemplified by bone fracture healing – is a prominent topic. With 5-10% of larger bone fractures resulting in ‘non-union’ (i.e. incomplete healing), novel therapeutic strategies supporting bone bridging are needed. To discover such approaches, bone critical size defects (CSD) in axolotls are employed as a model for failed blastema formation and limb regeneration. Anastasia Polikarpova, postdoctoral fellow in Elly Tanaka's lab, proposed gene regulatory network rewiring by inducing early wound response signaling mimicking wound epidermis and fibroblast interaction as sufficient to induce a blastema-like phenotype, thereby possibly enabling bone bridging after CSD in axolotls.

Another bridging technique to regenerate neurons was presented by Francesca Merighi, PhD student in Vittoria Raffa's and Marco Onorati's lab (University of Pisa, Italy) addressing the challenge of spinal cord injuries. Merighi highlighted the absence of effective treatments while exploring innovative therapeutic approaches. These include the use of human spinal cord-derived neuroepithelial stem (NES) cells to promote spinal cord repair through the formation of relay circuits (Dell'Anno et al., 2018). Merighi showed the application of paramagnetic nanoparticles (MNPs) and magnetic fields to induce neuronal stretch-growth, stimulating axonal outgrowth mechanically (De Vincentiis et al., 2020; Falconieri et al., 2023). The illustrated technique was applied on MNP-labeled spinal cord-NES cell-derived neurons in vitro and engrafted into spinal cord tissue in an ex vivo model of spinal cord organotypic slices (De Vincentiis et al., 2023), facilitating stretch growth and integration. Using cortico-spinal assembloids and organotypic spinal cord cultures, her research aims to model human cortico-spinal tracts and validate the efficacy of stem cell-based approaches combined with mechanical stimulation for spinal cord injury treatment.

Challenging the conventional understanding of the mechanisms driving neurodegenerative diseases, Mina Gouti (Max Delbrück Center for Molecular Medicine, Berlin, Germany) discussed the crucial role of dysfunction in both muscles and the nervous system in diseases, such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Gouti used hiPSC-derived axial stem cells to generate 3D neuromuscular organoids, representing functional neuromuscular junctions and central pattern generator-like neuronal circuits (Faustino Martins et al., 2020). Furthermore, Gouti introduced a self-organizing 2D neuromuscular junction model (Urzi et al., 2023), which paves the way for high-throughput drug screening. The integration of the 2D and 3D models could significantly advance our understanding of neuromuscular disease mechanisms and support the discovery of new therapeutic strategies.

Opposite to neurodegeneration is the field of rejuvenation and tissue remodeling. Addressing this topic was Daniel Del Toro (University of Barcelona, Spain), who investigated the consequences of overexpressing the Yamanaka factors (cMyc, Klf4, Oct4 and Sox2) on the brain. By inducing the Yamanaka factors in the mouse brain during both embryonic development and adult stages, Del Toro observed significant effects on neurogenesis and behavior (Shen et al., 2023 preprint). During embryonic induction of Yamanaka factors, the cell identity of progenitors and neurons was perturbed, but transient and low-level expression was tolerated, leading to expanded neurogenesis and an increased number of upper cortical neurons (25%). Adult brain induction of Yamanaka factors also showed improvements in a neurodegenerative mouse model. Del Toro highlighted the use of the i4F system for controlled Yamanaka factor induction, resulting in increased cortical expansion and enhanced motor and social behavior in adult mice. The talk highlighted global interest in reversing aging and the potential of transient reprogramming, showing promise in expanding the neocortex and protecting against neurodegeneration.

Ultimately, with the goal of tackling the continuous and major demand for human organ transplantation, Hiro Nakauchi (Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, USA, and Tokyo Medical and Dental University, Tokyo, Japan) explored the generation of rejection-free, xeno-created organs for transplantation, focusing on the role of the stem cell niche. He introduced the concept of an ‘organ niche’ to generate human ESC-derived organs in vivo and shared the success of generating a mouse-sized rat pancreas in mice, marking the first demonstration of interspecies organ generation (Kobayashi et al., 2010). Challenges included overcoming xenogeneic barriers and coordinating development in interspecies chimeras. Nakauchi's work offers hope for patients awaiting transplants through innovative approaches such as in utero transplantation of keratinocytes, ultimately aiming at creating chimeric pigs with transplantable human skin.

To date, due to the lack of experimental platforms and technical limitations, durable inheritance mechanisms of epigenetic marks remain elusive. To better evaluate epigenetic memory, Minhee Park (Korea Advanced Institute of Science and Technology, Daejeon, South Korea) generated a minimal epigenetic system in human cells (in Ahmad Khalol's lab, Boston University) using a bottom-up synthetic biology approach. The combination of synthetic initiator, readout and read-write modules proved sufficient for spreading an artificial DNA modification and regulation of distant genes. Subsequently, Park aimed to employ super-resolution 3D chromatin imaging techniques to explore the relationship between epigenetic memory and chromatin organization at the single-nucleus level.

A sequencing-based approach to analyze and visualize the 3D genome organization on a single-cell level was presented by Joanna Jachowicz (IMBA, Vienna, Austria). She developed ‘single-cell split-pool recognition of interactions by tag extension’ (scSPRITE), a tool to tag DNA fragments and their spatial arrangements measuring multidirectional intra- and interchromosomal interactions within the same nucleus (Arrastia et al., 2022). Its application will be particularly valuable for heterogeneous populations including organoids and complex tissues, amongst others. Further proving its functional versatility, simultaneous capture of the subcellular localization of RNA (RD SPRITE) proposed a permissive model for transcription during early developmental stages with genes remaining in the inactive B-chromatin compartments, despite being actively transcribed.

Neural fate acquisition is governed by epigenomic trajectories controlling target gene expression and the activity of regulatory elements. Fides Zenk (École Polytechnique Fédérale de Lausanne, Switzerland) generated a genome-wide single-cell atlas of histone modification changes during human brain organoid development, integrating datasets from scCUT&Tag and single-cell RNA-sequencing (scRNA-seq). This allowed deciphering of a time-dependent course of decision-making across multiple developmental time points and brain regions, identifying region-specific modes of fate acquisition and epigenetic priming of neuronal genes. Underpinning the versatility of this approach, these findings were supported by retrieving transcriptome and chromatin accessibility data from the same cells of primary human developing brain tissue.

Despite the crucial role of the interplay between epigenetics and transcription during embryonic development, our understanding of it remains limited. Peter Zeller, postdoc in Alexander van Oudenaarden's lab (Hubrecht Institute-KNAW, Netherlands), introduced T-ChIC (Transcriptome+Chromatin ImmunoCleavage), a method enabling the simultaneous acquisition of full-length transcripts and histone mark positions from single cells. Studying mouse gastruloids enabled Zeller to monitor the growth of all three germ layers from stem cells, revealing complex chromatin regulation via T-ChIC, which tracks the local positions of H3K27me3 (repressed regions) and H3K4me3 (active promoters). The findings suggest an initial loss of repressive H3K27me3 and de-repression of bivalent marked genes (H3K27me3+H3K4me3 positive), indicating a general mechanism for ectodermal trajectory, following the induction of other germ layers at specific times.

Nike Walther, postdoc in Robert Tjian's and Xavier Darzacq's lab (University of California, Berkeley, CA, USA) introduced an automated live-cell single-molecule imaging and tracking (SMT) technique to monitor transcription factors at the single-cell level, enabling the correlation of single-molecule dynamics with cellular profiles and phenotype changes at the tissue level. By focusing on the expression of the transcription factor Sox9, Walther investigated the molecular dynamics associated with differentiation and morphological changes in intestinal organoids, revealing that Sox9 overexpression led to a proliferative cell state transition displaying regenerative features and hallmarks of cancer.

Kirstin Meyer, postdoc in Orion Weiner's lab (University of California San Francisco, CA, USA) discussed how the transcriptional regulator YAP drives developmental decisions, such as pluripotency and differentiation via transmitting extracellular signals (Meyer et al., 2023). Using optogenetics and live-imaging techniques, Meyer delved into the role of YAP in controlling stem cell fate and proliferation in the context of pluripotency, revealing how the dynamic YAP concentration can drive information transmission for developmental gene activation.

Taken together, SY-Stem 2024 provided two and a half exciting days fueled with inspiring talks, scientific exchange and networking opportunities. The meeting leaves behind the anticipation for the next edition in 2025.