The inaugural ‘Symposium for the Next Generation of Stem Cell Research’ (SY-Stem) was held on February 22-24 at the Vienna BioCenter in Austria. The meeting focused on having young researchers as speakers, and the program was of an impressively high quality. Here, we summarise key findings from this meeting, which brought together emerging leaders to discuss various topics, including pluripotency, organoids, endogenous regeneration, transcriptional regulation, clinical applications and emerging technologies.
In Vienna, the home of Mozart and the historical capital of one of the world's great empires, 292 researchers from 26 different countries met to participate in the first ever SY-Stem meeting. The meeting revolved around the topic ‘Advances in Stem Cell Biology’ (depicted in the meeting poster through a reworking of the famous Klimt painting Danaë, Fig. 1), and was organised and hosted by researchers from the Institute of Molecular Biotechnology (IMBA) and Institute of Molecular Pathology (IMP) – Elly Tanaka, Juergen Knoblich, Ulrich Elling, Sasha Mendjan and Chukwuma Agu – in the impressive and vibrant research facilities of the Vienna BioCenter. The vision for the meeting was to create an environment for the ‘free exchange of ideas and results’, and the speaker program was therefore deliberately composed of primarily young talented researchers (of whom 15 out of 31 were female), who were encouraged to share their unpublished data on the podium. Reflecting the theme of the meeting, the coffee breaks were vibrant, not only with young researchers planning new collaborations across borders, but also with small children and babies who were welcomed to come along with their parents to the meeting. We congratulate the organisers for putting together a gender-balanced 3-day program of impressively high-quality stem cell biology, and we look forward to this meeting hopefully being a recurring event. For those who missed it, this Meeting Review summarises the main findings presented.
Several talks at the meeting focussed on cell state transitions in stem cell cultures, and on the mechanisms driving or blocking these transitions. Joerg Betschinger (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) opened the meeting with a powerful demonstration of how functional genomic screens can reveal novel biological mechanisms that control stem cell differentiation. In contrast to the factors that sustain self-renewal of embryonic stem cells (ESCs), the machinery that regulates exit from pluripotency is not well defined. Building on a recent large-scale siRNA screen that identified the bHLH transcription factor Tfe3 as a gate-keeper of the exit from pluripotency (Betschinger et al., 2013), Betschinger screened for factors that regulate differentiation and Tfe3 subcellular localisation in mouse ESCs (mESCs). This identified components of the lysosomal amino acid sensing machinery (folliculin and lamtor), and Betschinger showed that a component of this machinery can phosphorylate and inactivate Tfe3 in mESCs. Mutations that render Tfe3 non-phosphorylatable block mESC differentiation and cause a human developmental syndrome. These findings reveal a previously unappreciated requirement for lysosomal signaling in developmental progression and suggest that ESC differentiation is controlled by a check point that senses the metabolic environment prior to exiting pluripotency.
Jacob Hanna (Weizmann Institute, Israel) presented ongoing efforts to identify epigenetic regulators that are important for control of naïve versus primed pluripotency in mESCs. He reminded us that naïve pluripotency is a unique state that, in contrast to primed pluripotency, largely tolerates the loss of epigenetic repression and is characterized by a conspicuous absence of the nucleosome remodeling complex NuRD under certain growth conditions. Hanna has previously shown that partial depletion of the NuRD complex component Mbd3, when combined with the Yamanaka factors, can increase reprogramming efficiency into naïve state to >95%, thereby making the reprogramming process deterministic (Rais et al., 2013). At this meeting, Hanna underpinned this finding by showing that depletion of another NuRD component, Gatad2a, showed a similar phenotype in relieving a roadblock in the reprogramming process and making the route towards pluripotency deterministic (Mor et al., 2018preprint).
Naïve pluripotency was also the focus of Hannah Stuart's talk (José Silva's group, Cambridge Stem Cell Institute, UK), who presented evidence that state transitions can occur through multiple routes. Using single-cell RNA sequencing (scRNAseq), Stuart showed how the reprogramming of primed epiSCs to naïve ESCs followed distinct transcriptional trajectories depending on the inducing transgene, even though the final naïve cell product appeared identical. Interestingly, these different routes exhibited different requirements for both signalling and genetic factors. This provided evidence that cellular identity can be conceptualised as a multidimensional attractor state, with distinct routes of approach in terms of both transcriptional trajectories and the underlying functional attributes.
Organoids and tissue engineering
Having set the scene of pluripotent network regulation, the meeting moved on to cover a diverse range of stem cell topics, with several focussing on three-dimensional (3D) culture of stem cells. Starting with the earliest morphogenetic events orchestrated during development, Magdalena Zernicka-Goetz (University of Cambridge, UK) delivered a breathtaking keynote presentation on the newest advances in engineering stem cell-derived ‘synthetic embryos’ by co-culturing mouse trophoblast stem cells (TS) and epiblast ESCs (EPI) in 3D (Harrison et al., 2017). Despite the absence of anterior visceral endoderm (AVE), a spontaneous break in symmetry is observed in about half of these synthetic embryos, with formation of mesoderm and primordial germ cells (PGCs) on only one side of the tissue. In Vienna, Zernicka-Goetz showed how her team was able to refine the model further by coating the TS/EPI synthetic embryos with a layer of extraembryonic visceral endoderm cells to achieve embryo structures that proceeded all the way to gastrulation. Remarkably, these triple cell-type synthetic embryos could generate both the anterior and posterior primitive streak, AVE and endothelial-to-mesenchymal transition. This model, which circumvents the need for animal-derived embryos, may prove highly valuable in enabling large-scale studies of blastocyst development and gastrulation.
A symphony of subsequent talks during the meeting demonstrated the power of organoid systems for modelling various developing tissues in normal and diseased state. Focussing on the pancreas, Anne Grapin-Botton (University of Copenhagen, Denmark) revealed that pancreatic organoids (Greggio et al., 2013) could not be formed from single mouse pancreatic progenitors, but required a minimum of four starting cells. Interestingly, organoid formation appeared to depend on paracrine interaction between Notch ligand and its receptor on neighbouring cells, as organoids only grew where there was at least one Hes+ and one Hes− cell in the starting population. Grapin-Botton also showed progress in generating human pancreatic organoids from hESC-derived PDX1+ pancreatic progenitors. Unlike the equivalent mouse ESC-derived organoids, the human organoids did not display spontaneous endocrine differentiation, but could be patterned by exogenous factors to produce either endocrine or ductal organoids separately.
On the hepatic side, Barbara Treutlein (Max Planck Institute, Leipzig, Germany) presented recently published work on characterising liver organoids as a model for studying liver bud development (Camp et al., 2017). These organoids were generated from hPSC-derived hepatic progenitors mixed with human endothelial cells (HUVECs) and mesenchymal stem cells, and scRNAseq showed that the transcriptome of the organoid hepatocytes resembled foetal, rather than adult, liver cells and that they were more similar to primary liver cells than to hepatocytes grown in 2D. The organoids (but not equivalent 2D cultures) displayed an angiogenic signature, which could possibly be a response to the hypoxia-related gene expression profile observed. Innovative means of analysing the transcriptomic dataset for receptor-ligand partners on different cell types revealed that hepatocytes in the organoids were more likely to interact with endothelial cells and stromal cells rather than with other hepatocytes, thereby presenting a new way of using transcriptomics to study organogenesis in complex systems.
Moving to the gut, Joep Beumer (Hans Clevers' group, Hubrecht Institute, The Netherlands) showed how intestinal organoids can be used to study the rare population of enteroendocrine cells (EECs), which constitute less than 1% of the epithelial population in the intestine. Beumer found that EECs located in crypts versus villi expressed different sets of hormones, where they reside in different BMP niches. Stimulation of BMP signalling in the organoids induced hormone switching in some subtypes of EECs, allowing them to produce villus-associated hormones such as secretin, thereby revealing some degree of endocrine plasticity in the EEC population. He also showed that EECs do not necessarily follow the same crypt-to-villus translocation observed for enterocytes, indicating that EECs are replenished in situ.
Giorgia Quadrato (Paola Arlotta's group, Harvard University, Boston, MA, USA; shortly starting her own group at the University of Southern California, Los Angeles, USA) summarised her impressive work using hPSC-derived neural organoids and large-scale scRNAseq for studying brain development and neural subtype specification (Quadrato et al., 2017). Quadrato went on to show that organoids generated from hPSCs heterozygous for a mutation in CHD8, a strong risk factor for autism and macrocephaly, grew much larger than organoids from control cells, thereby mimicking the human disease phenotype. Analysis of over 60,0000 single cells by RNAseq further revealed reproducible patterns of genotype-specific differential gene expression between the control and CHD8 mutant in both progenitor and neuronal subtypes.
Extending the theme of 3D tissue reconstruction, Agnete Kirkeby (University of Copenhagen, Denmark) presented an alternative to neural organoid models involving a microfluidic system for producing controlled morphogenic gradients in vitro. By exposing differentiating hPSCs to a gradient of WNT signaling, the cells in this microfluidic system produced a coherent neural tissue comprising forebrain-, midbrain- and hindbrain-patterned cells located in a spatially ordered manner. The system therefore mimics in a human context the WNT-dependent rostrocaudal patterning of the early neural tube, which has been shown to take place in model organisms . Kirkeby further showed how the system could be used to assess region-specific responses of neural cells to genes and growth factors.
Transcriptional regulation during differentiation and reprogramming
Precise regulation of transcription, and concomitant changes in chromatin architecture, are crucial for the acquisition of gene expression programs that govern cell identity. Chromatin remodelling is a defining feature of fertilisation, which generates the zygote – the ultimate totipotent stem cell. Kikuë Tachibana (Institute of Molecular Biotechnology, Vienna, Austria) provided an elegant view of chromatin reorganisation during the oocyte-to-zygote transition in mice, using a recently developed method for single cell chromosome conformation capture (Flyamer et al., 2017). Interestingly, global chromatin organisation of zygote nuclei is fundamentally different from that of other interphase cells. Moreover, Tachibana's work demonstrates a key role for cohesin-dependent loop extrusion in organising the genome during the oocyte-to-zygote transition (Gassler et al., 2017).
Epigenetic mechanisms, such as DNA methylation dynamics, are not only a defining feature of the earliest stages of organismal development but also a crucial hallmark of tissue specification. Judith Kraiczy (Matthias Zilbauer's group, School of Clinical Medicine, University of Cambridge, UK) demonstrated the importance of analysing epigenetic marks to determine the regional identity of human organoids derived from intestinal stem cells. Analysis of human biopsy-derived paediatric intestinal organoids showed that they retained gut segment-specific DNA methylation marks even after long-term culture. Similarly, organoids derived from individuals with Crohn's disease retained disease-specific alterations in methylation patterns. In contrast, foetal-derived organoids underwent drastic methylation changes in vitro, suggesting an in vitro maturation that could be used to model epithelial development.
One of the most striking examples of plasticity is the reprogramming of cellular identity by transcription factors. Marisa Karow (Ludwig Maximilians University, Munich, Germany) showed heterogeneity in the ability of human brain pericytes to reprogram into induced neurons. Interestingly, neuronal reprogramming of pericytes with Ascl1 and Sox2 involved the activation of certain ‘switch genes’, which displayed a peak in expression around the time of cell fate decision. These ‘switch genes’ were enriched for components of the BMP and NOTCH pathways, and modulation of BMP and NOTCH signalling had a profound effect on reprogramming efficiency.
A major highlight of the meeting was the stunning keynote presentation from Marius Wernig (Stanford University, CA, USA), providing evidence for a carefully orchestrated synergy between transcription factors during neuronal reprogramming. Contrary to what seems logical for reprogramming factors, Wernig showed that MYT1L – one of the factors required for reprogramming to neuronal fate - does not act as a transcriptional activator, but instead as a repressor. MYT1L repression targets a multitude of non-neuronal genes, reflecting the fact that MYT1L is a neuronal-specific transcription factor. Accordingly, a construct that fused the DNA-binding domain of MYT1L with a transcriptional activator domain could not induce neuronal reprogramming (Mall et al., 2017). Wernig further presented evidence of a surprisingly high degree of overlap in the DNA-binding sites of ASCL1 and MYOD, despite the fact that ASCL1 directs neural fates and MYOD directs muscle fates. This finding could explain why a fraction of cells expressing ASCL1 could go towards the muscle, rather than neural, lineage. ASCL1 displayed higher binding affinity to neural loci and MYOD to muscle loci, but substituting small domains of the MYOD protein with the corresponding domains from ASCL1 could convert MYOD into a neuronal reprogramming factor. Moreover, upon combining wild-type MYOD with MYT1L to repress all non-neuronal fates, MYOD now induced reprogramming into neurons instead of muscle. The work of Wernig therefore beautifully illustrates the important synergy between transcriptional activators and repressors in inducing efficient and lineage-specific reprogramming.
Regeneration and homeostasis
The meeting also provided a platform to discuss the remarkable regenerative feats of a menagerie of model organisms, including axolotls, newts, zebrafish and mice. Prayag Murawala (Elly Tanaka's group, Research Institute of Molecular Pathology, Vienna, Austria) revisited several long-standing questions about the source of blastemal cells in axolotl limb regeneration using sophisticated genetic lineage tracing and single cell sequencing technologies. Lineage reconstruction from scRNAseq data suggests that pre-existing blastemal precursors probably do not exist. Moreover, cells from the mature limb appear to de-differentiate and enter a transitional state, during which there is a loss of cellular heterogeneity, before initiating the regeneration program. These elegant technologies are set to transform our molecular understanding of tissue regeneration in the axolotl.
Nadia Mercader Huber (University of Berne, Switzerland) provided insights into the role of cardiac progenitor populations in heart regeneration by developing new tools for genetic lineage tracing in zebrafish (Sánchez-Iranzo et al., 2018). Remarkably, ablation of tbx5a-derived cardiomyocytes (the first heart-field population) in the embryo was compensated for by expansion of a tbx5a-negative population, suggesting that second heart-field progenitors can substitute for first heart-field progenitors during embryonic heart development. Moreover, lineage tracing of tbx5a-positive cardiomyocytes following cryoinjury in zebrafish suggests that trabecular cardiomyocytes can switch their fate and differentiate into cortical myocardium during adult heart regeneration. These findings indicate a high degree of cardiomyocyte cell fate plasticity in zebrafish, which may contribute to the regenerative capacity of this species.
Also looking at cardiac tissue, Enzo Porrello (Murdoch Children's Research Institute, Melbourne, Australia) built on a recently developed human cardiac organoid screening platform (Mills et al., 2017) to provide new insights into signalling pathways governing cardiac regeneration. Through combinatorial screening of small molecules, novel drug interactions were revealed, which uncovered crosstalk between the MST1 and GSK3 pathways in proliferating cardiomyocytes. High-throughput quantitative proteomics and loss-of-function experiments revealed that the mevalonate pathway was required for cardiomyocyte proliferation. This study highlights the power of combining drug screening and high-throughput proteomics in cardiac organoids to identify novel pathways and drug targets for cardiac regeneration.
In contrast to axolotls, newts and zebrafish, most adult mammalian tissues have a much more restricted regenerative potential. One exception is skeletal muscle, which harbours a resident stem cell population that can mediate efficient regeneration following injury. Tom H. Cheung (Hong Kong University of Science and Technology, China) highlighted the potential importance of RNA processing in the regulation of stem cell quiescence. Transcriptional profiling of highly purified muscle stem cell populations revealed a surprising prevalence of intron-retaining transcripts in quiescent versus activated stem cell populations. Bioinformatic analysis of publicly available datasets suggests that intron retention might be a general feature of a variety of quiescent stem cell populations, not only specific to muscle. Cheung speculated that the accumulation of pre-mRNA transcripts in quiescent stem cells could reflect a ‘poised’ state that is primed for rapid activation following muscle injury.
In a beautiful demonstration of the power of in vivo imaging, Sven Falk (Magdalena Götz's group, Hemholtz Center Munich, Germany) used two-photon microscopy to study in real-time the progeny of dividing radial glial cells (RGCs) in the lateral ganglionic eminence (LGE). With this technique, Falk could show that the quiescent neural stem cells (NSCs) in the subependymal zone of adult mice are derived from RGCs in the LGE at an early time point during development. He further showed that when genetically manipulating the mitotic spindle assembly to induce random orientation of the cleavage plane in these RGCs, the cells now generated new RGC daughter cells only infrequently, and instead gave rise to more differentiating progeny.
To relate the many basic science findings to the clinic, the meeting also included a number of clinically-relevant talks. On the disease modelling side, Patricia Garcez (University of Rio de Janeiro, Brazil) discussed her pioneering efforts using human neural organoids infected with Zika virus to demonstrate a causative link between the South American Zika virus epidemic and the high simultaneous incidence of microcephaly in newborns (Garcez et al., 2016). Curiously, Garcez revealed that the African strain of Zika virus caused much more severe impairment of neural organoid growth than the Brazilian strain, indicating that the microcephaly epidemic observed in South America may be linked to strain-dependent differences in crossing the placenta or that the African Zika virus (where infection has not been associated with microcephaly) may cause undetected early embryonic lethality. As an additional mechanism of pathogenesis, Garcez presented evidence that the virus can also impair blood vessel formation in the developing mouse brain.
Going into stem cell-based drug screening platforms, Jerome Chal (Coyne Scientific, Atlanta, GA, USA) presented an update on recent commercial applications of stem cell technologies, building on protocols that he developed for directed differentiation of skeletal myocytes from hPSCs (Chal et al., 2015). These protocols are now being used by Anagenesis Biotechnologies, a company he co-founded with Olivier Pourquié, to identify compounds that enhance myogenic differentiation. Chal recently joined Coyne Scientific, where he is now using a genetically diverse cohort of hPSC-derived cardiomyocytes as a potential drug toxicity assay, including identification of compounds with potential arrhythmogenic side-effects.
In the same realm, Meritxell Huch (The Gurdon Institute, Cambridge, UK) built on her groundbreaking work on the derivation of liver organoids (Huch et al., 2013, 2015) to unveil the power of this platform for biological discovery and drug screening. For example, Huch has recently developed methods for growing human liver tumour organoids, which closely recapitulate the tumour of origin and can be used to screen for known and novel drug sensitivities. This approach was recently used to identify an ERK inhibitor as a potential therapeutic target for primary liver cancer (Broutier et al., 2017).
On the cell therapy side, Frank Edenhofer (University of Innsbruck, Austria) presented an alternative regenerative function of transplanted NSCs in offering immune-modulatory properties in neuroinflammatory conditions (Peruzzotti-Jametti et al., 2018). When transplanting induced NSCs (iNSCs) into a mouse model of multiple sclerosis, the cells dampened macrophage-induced inflammation by converting activated macrophages to resting state and inducing PGE2 release. This anti-inflammatory activity was dependent on scavenging of extracellular succinate through the succinate receptor Sucnr1 on the iNSCs, thereby opening the potential for an autologous transplantation in humans employing patient-specific iNSCs. Future research will show whether such immune modulatory effects might also be obtained with other types of transplanted cells or by drug treatment.
Moving from cell replacement in the brain to cell replacement in the pancreas, Barbara Ludwig (University of Dresden, Germany) showed how transplantation of whole islets derived from dissociated pancreas tissue can efficiently stabilise blood glucose levels of patients and prevent severe hypoglycaemic events. For individuals undergoing pancreatic resection, auto-transplantation of isolated islets is furthermore a successful strategy in preventing surgery-induced diabetes. Ludwig's own group is currently working on developing a cassette for protection of transplanted islet cells from immune rejection to enable allogeneic transplantation in patients without the need for immunosuppressive treatment. Ludwig projected that future sources of islet cells for transplantation might even involve xenografts from pigs or, eventually, human islets derived from chimeric humanised pigs.
Fotios Sampaziotis (University of Cambridge, UK) gave a stunning presentation on the generation of cholangiocyte organoids, including potential clinical applications of bioengineered bile ducts. Recent efforts have focussed on the potential applications of cholangiocyte organoids to treat common bile duct disorders such as biliary atresia. Protocols have been developed for the isolation of primary cholangiocytes from human bile ducts, which can be expanded in vitro, thus allowing for scalable production of cholangiocytes for tissue engineering. After seeding cholangiocytes onto a biodegradable scaffold, cells self-organise to form a bioengineered tissue with biliary characteristics. Remarkably, the bioengineered bile duct could repair the gallbladder wall and biliary epithelium following implantation in vivo in a mouse model (Sampaziotis et al., 2017).
In addition to these exciting biological discoveries and translational applications of stem cells, the meeting also witnessed the emergence of a number of innovative technologies. Gabsang Lee (Johns Hopkins University, Baltimore, MD, USA) presented unpublished data on an optogenetic system that could be used to control FGFR activation to maintain proliferating pluripotent hESCs in the absence of exogenous FGF2. The optogentically modified hESCs could be maintained in culture with only once-weekly media change by applying blue-light stimulation of the opto-FGFR cells for 1 min every 2 h. Moreover, the resulting cells showed less heterogeneity in gene expression profiles compared with conventionally cultured hESCs in the presence of FGF2.
A common thread of the meeting was the use of single cell sequencing technologies to provide new insights into cellular heterogeneity during reprogramming, organoid differentiation, endogenous regeneration and development. Charles Chan (Stanford University, CA, USA) described a new approach to dissect the HSC niche with unprecedented resolution using an elaborate in vivo system based on transplantation of membrane dye-labelled HSCs from a wild-type donor into recipient mice. The cellular composition of the homing niche for the transplanted HSCs was subsequently deconstructed by purifying dye-labelled aggregates of cells from the bone marrow and dissecting the composition of these aggregates (i.e. niches) through index sorting and scRNAseq.
One limitation of current single-cell sequencing approaches is the high cost of sequencing large numbers of cells. Fatma Uzbas (Micha Drukker's group, Helmholtz Zentrum Munchen, Munich, Germany) described a new method termed barcode assembly for targeted sequencing (BART-seq), which enables analysis of thousands of samples using a small panel of known genes, with high coverage. She suggested that BART-seq could offer a relatively cheap solution for high-throughput analysis of bulk and single-cell samples (e.g. for drug screening applications or analysis of stem cell differentiation).
Adding to a growing repertoire of available tools for stem cell researchers, Malkiel Cohen (Rudolf Jaenisch's group, Rudolf Whitehead Institute, Cambridge, MA, USA) summarised recent advances in the generation of mouse-human chimaeras. Previous work from the group showed that human neural crest cells can contribute chimerically to melanocyte formation in the mouse (Cohen et al., 2016). Cohen reported that transplantation of cells modified to overexpress the neuroblastoma-associated oncogenes MYCN and ALKF1174L resulted in chimeric mice that developed neuroblastomas with phenotypic resemblance to tumours from patients. He speculated that the human tumour cells in the chimeric mouse model make use of an immune evasion strategy to avoid xenograft rejection.
Much like the classical compositions of Mozart, the inaugural SY-Stem meeting in Vienna was lively and invigorating. By bringing together emerging leaders in stem cell research, the organisers succeeded in creating a high-quality, open and collegial meeting that lays the foundations for a vibrant and dynamic stem cell community in the future (Fig. 2). The meeting covered enormous territory over 3 days, spanning a diverse range of topics from the nature of primed versus naïve pluripotency to transcriptional mechanisms governing endogenous regeneration and cellular reprogramming, as well as clinical applications of stem cell and organoid technologies. The rapid pace of development in these exciting fields promises a bright future for regenerative medicine, although with every discovery new questions arise that warrant further investigation. Some of these discoveries, such as the generation of human organs in a dish, go to the very heart of what makes us human and raise important biological and ethical questions for the scientific community and society. There is no doubt that these questions will fuel this vibrant field for many years to come and ensure that the next SY-Stem meeting is as stimulating as the first.
We thank the IMP/IMBA Graphics Department for permission to use the SY-Stem meeting poster illustration in this publication and the IMBA/pov.at for the photograph of meeting attendees.
E.R.P. is supported by the National Health and Medical Research Council of Australia, the Heart Foundation of Australia and Stem Cells Australia. The Murdoch Children's Research Institute is supported by the Victorian Government's Operational Infrastructure Support Program. A.K. is supported by funding from the Novo Nordisk Foundation (NNF17CC0027852).
The authors declare no competing or financial interests.