In May 2017, the 9th International Meeting of the Stem Cell Network North Rhine Westphalia (see www.congress.stemcells.nrw.de) took place in Münster, Germany and was hosted by Martin Götte and Hans Schöler. This year's scientific program comprised numerous exciting presentations and discussions with a focus on “Stem Cell Differentiation,” “Stem Cells in Regenerative Medicine,” and “Disease Modeling.” Other studies dealt with three-dimensional (3D) organoids, induced pluripotent stem cells (iPSCs), cancer stem cells, and the role of epigenetics. 600 participants, presenting a new record of over 160 posters, joined the congress providing an excellent platform for interdisciplinary discussion and new collaborations. This meeting report emphasizes the most interesting presentations from a broad spectrum of challenging approaches in stem cell research.
Frank Grosveld (Erasmus University Rotterdam, Rotterdam, Netherlands) introduced 3D structures of the genomes in hematopoietic development. Enhancers regulate gene expression through interactions with the promoter region of their target genes and can act over megabase distances. The genome of mammalian cells consists of a large series of folded domains known as topologically associating domains (TADs). These TADs are generated through the binding of the transcription factor (TF) CTCF and cohesin, a ring-like protein complex, resulting in an overall structural organization of the genome as a series of rosettes connected by linker chromatin. Within the TADs' chromatin, the enhancer-promoter interactions take place to form “functional” loops within “structural” TAD loops, a process that appears to occur in transcriptional foci known as transcription factories. Using the developing and differentiating hematopoietic system as the biological model, different aspects of the 3D dynamics of the genome have been studied, including the activation of new enhancers during the process of differentiation. In an embryonic stem cell differentiation system, both the GATA1/TAL1/LDB1 TF complex and the co-TF EVI1 are essential to differentiate between hematopoietic and cardiac lineages. In later stages, the appearance of the co-TF RUNX1 leads to the use of novel enhancers and the formation of stem cells to be followed by the co-TF KLF1, to lead to erythroid differentiation. In summary, Frank Grosveld highlighted how TFs physically cooperate to regulate transcription in the hematopoietic system.
Gerd Blobel (Children's Hospital of Philadelphia, Philadelphia, PA) described work relating to transcriptional enhancers and how their gene specificity is established. He provided an overview of enhancer function and stressed that the majority of enhancers form looped contacts with the genes they regulate. He summarized previous work from his laboratory showing that enhancer-promoter contacts are very dynamic and can be perturbed for scientific or therapeutic purposes. Even though enhancers control tissue-specific gene expression they are generally promiscuous and can activate any reporter gene to which they are coupled. Moreover, chromosomal translocations that inappropriately pair enhancers with proto-oncogenes can trigger their activation. Therefore, a key question in the field is how enhancers are normally prevented from activating the wrong genes. In agreement with Frank Grosveld, he supported the widely held concept indicating that chromosomes are physically and functionally partitioned into TADs and sub-TADs, within which most enhancer-promoter interactions occur. Frequently, the boundaries between these structures are demarcated by binding sites for the architectural TF CTCF. He then described a recent discovery in his laboratory that two so-called BET proteins, Brd2 and to a lesser extent Brd3, associate with CTCF and contribute to the formation of CTCF boundary function. BET proteins are normally recruited to enhancers and promoters through their ability to bind to acetylated TFs and histones. Recently developed pharmacologic BET inhibitors have entered clinical trials for a variety of malignancies and have raised the interest in how BET proteins function mechanistically. The Blobel laboratory found that CTCF recruits Brd2 to a large fraction of its sites. Using a variety of approaches, including genome editing, single molecule messenger RNA (mRNA)-fluorescence in situ hybridization (FISH), and high-throughput chromosome conformation capture (Hi-C), it was found that Brd2 contributes to the formation of chromatin boundaries and prevents enhancers from activating genes inappropriately. Pharmacologic BET inhibitors thus influence gene expression not only locally but likely also through perturbation of chromatin boundaries.
Juan Vaquerizas (Max-Planck-Institute for Molecular Medicine, Münster, Germany) described his work on Drosophila, where he investigated chromatin conformation in tightly staged, hand-sorted embryos at different developmental time points. Three-dimensional chromatin organization maps for these embryos revealed that chromatin structure is significantly remodeled at hundreds of loci during embryo development. Furthermore, he demonstrated that specifically expressed loci serve as nucleation sites for early TAD boundaries. Of note, pharmacological inhibition of RNA polymerase II does not preclude the formation of TADs, suggesting that transcription is not necessary for the establishment of such domains. However, reduction of RNA polymerase II activity results in a decrease of insulation between topological domains. Furthermore, his laboratory identified DNA motifs enriched at TAD boundaries and demonstrated that the pioneer TF Zelda is necessary to establish insulation at early stage boundaries. These results have important implications for the understanding of how the 3D structure of the genome is established in the early embryo and the mechanisms that trigger this organization.
Jason Spence (University of Michigan, Ann Arbor, MI) presented on the complex architecture of the lung, which is established through a process called branching morphogenesis. Specialized epithelial progenitor cells, called “bud-tip progenitors,” undergo repeated rounds of bifurcations to set up the stereotyped network of airways and alveoli in the adult (Dye et al., 2016b; Miller and Spence, 2017). Human pluripotent stem cells (hPSCs) have been used to model some aspects of the complex process of lung development and have been directed to differentiate into complex 3D lung organoids (Dye et al., 2015), which possess airway structures and a limited number of alveolar cell types. These lung organoids can be transplanted into immunocompromised mice, where they mature and resemble the adult human airway (Dye et al., 2016a). However, despite these advances, there has been limited evidence to show that hPSCs can be differentiated into a bud-tip progenitor-like cell. Novel work from the Spence laboratory was aimed at understanding the biochemical cues that act to maintain bud-tip progenitor cells cultured in vitro when explanted from the native lung and attempts to induce this population of cells in hPSC-derived lung organoid cultures.
During the past decade, the study of human neurodegenerative diseases has benefited from the advent of human patient-specific iPSC-based models. However, at the same time, such models have revealed stark limitations in the reflection of the cellular age of their origin, as the reprogramming process reliably erases the aging memory from old cells (Lapasset et al., 2011; Miller et al., 2013). In this context, Jerome Mertens (Salk Institute for Biological Studies, La Jolla, CA) described how it might be possible to factor in age-related cellular phenotypes into human neuronal disease model systems. Unlike iPSC reprogramming, the direct conversion of fibroblast into induced neurons (iNs) bypasses the embryo-like state and directly yields human neurons that display stark transcriptomic signatures depending on the fibroblast donors' ages (Mertens et al., 2016). Thus, iNs possess similarities with postmortem cortical samples from old individuals. Based on these data, the nuclear pore complex-associated transport receptor RanBP17 emerged as a potential master regulator of cell aging. Its age-dependent loss is associated with nuclear transport and compartmentalization defects in old human cells (Mertens et al., 2015). Furthermore, iNs from old donors show age-related mitochondrial deficits that might prove important for the development of human neuronal models for neurodegenerative disorders.
Zena Werb's presentation (University of California, San Francisco, CA) focused on understanding mammary development, cancer, metastasis, and the microenvironment in the age of single cell biology. In that context, single cell RNA sequencing is a powerful strategy to identify cell types and states in a given population of cells. The Werb laboratory discovered previously unrealized expression patterns of known epithelial markers, including potential mammary stem cells in normal human breast epithelium using single-cell transcriptomic analysis. After they discovered about five luminal and five to seven basal subpopulations, they identified and validated new markers for basal and luminal subpopulations—including cell surface receptors for prospective cell isolation and functional interrogation. Using the Monocle algorithm, they developed new insights into differentiation pathways for breast epithelium. This single-cell analysis of normal breast epithelium has given insights into breast cancer. Using patient-derived xenografts, Werb proposed that they will gain insight into how metastasis is regulated both at the level of the tumor cell and the tumor microenvironment. They discovered that primary tumors contain rare (1.4%) basal-/stem-like cells. Once the tumor cells reach the blood, a subpopulation (17%) of circulating tumor cells are stem like. Metastatic sited low-burden or indolent metastatic cells possess a basal-/stem-like expression signature. In contrast, high-burden metastatic cells are more heterogeneous, and differentiated. Low-burden metastatic cells are tumorigenic and can produce differentiated cancer cells. These data give new insights into the processes of metastasis (Lawson et al., 2015).
A highlight of this year's meeting was a comprehensive talk given by Robert Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA), who is known for his outstanding contributions to cancer research. His laboratory focuses on the molecular mechanisms that control carcinoma progression and metastasis. The process of epithelial-mesenchymal transition (EMT) is active in a variety of carcinomas and confers on the constituent carcinoma cells a variety of traits associated with high-grade malignancy. This includes motility, invasiveness, and elevated resistance to apoptosis, a heightened refractoriness to chemotherapy, and an ability to disseminate to found metastatic colonies. In addition, it is clear that tumor-initiating cells, also termed cancer stem cells, are generated upon activating a previously latent EMT program. Indeed, it is plausible that the formation of metastatic colonies requires the tumor-initiating powers of disseminated carcinoma cells to initiate the colonies; in addition, once initiated, to achieve robust outgrowth of metastatic colonies, the founding stem cells must generate large numbers of more differentiated descendants, doing so by reversal of the EMT program, termed the MET (mesenchymal-epithelial transition).
As of late, the Weinberg laboratory has been examining the responses of the innate and adaptive immune systems to more mesenchymal and epithelial cells within a carcinoma using a mouse model of mammary adenocarcinoma pathogenesis. They have found that the more mesenchymal-like carcinoma cells are relatively resistant to elimination by the immune system, in part, through their ability to recruit regulatory T-cells, to recruit immunosuppressive M2-type macrophages, to downregulate major histocompatibility complex (MHC) Class I expression on the cell surface, and to induce functional exhaustion in the T-cells that succeed in entering into tumors; in addition, the mesenchymal carcinoma cells are more resistant to anti-CTLA4 checkpoint immunotherapy, in contrast to the more epithelial cells, which are very sensitive to this therapy. Implanting in mice an admixture of one part mesenchymal cells to nine parts epithelial carcinoma cells yields tumors that are relatively resistant to anti-CTLA4 therapy. This indicates that minority subpopulations of more mesenchymal carcinoma cells can confer resistance on the tumor as a whole.
Previously, Paul S. Frenette (Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine of Albert Einstein College of Medicine, Bronx, NY) described that nerves from the sympathetic nervous system (SNS) regulate the hematopoietic stem cell migration from the bone marrow (Katayama et al., 2006; Méndez-Ferrer et al., 2008). The idea was that similar mechanisms might be involved in the migration of cancer-initiating cells in tumors. They have tested this hypothesis in prostate cancer models using orthotopic xenografts and transgenic mice. It became clear that distinct functions of the two branches of the autonomic nervous system in which adrenergic signals from the SNS were critical for tumor initiation, whereas cholinergic signals from the parasympathetic nervous system played a prominent role in tumor cell invasion and metastasis (Magnon et al., 2013). Recently, they have investigated the target cell of adrenergic nerves in the prostate and its anticancer mechanisms. Loss of beta-adrenergic receptor (Adrb) signaling in the prostate stroma inhibited tumor growth by altering glucose metabolism in endothelial cells and turning off the angiogenic switch. These results thus suggest that targeting the cross-talk between nerves and endothelial metabolism may serve as a novel anticancer therapeutic strategy.
In addition to presentations showing the latest findings in stem cell research, ethical and legal aspects of stem cell research were discussed. Dieter Birnbacher (Heinrich-Heine University, Düsseldorf, Germany) provided an ethical view for prospects of human germline modification by CRISPR-Cas9. He pointed out that gene editing holds the promise of revolutionizing many fields in which human interventions have hitherto proved to be insufficient to meet major global challenges like nutrition and environmental protection. It is controversial, however, how far this method might also be applied to the human germline with a view to preventing the transference of grave genetic diseases to offspring. While there is a near consensus that gene editing, at the present stage of science, should not be applied clinically, it is unclear whether this extends also to clinically oriented research. Dieter Birnbacher argued that, among the arguments against interventions in the human germline, the only one that is sufficiently strong to be practically relevant is the uncertainty whether the challenge imposed by off-target effects with potentially fatal health consequences can be met. Since nearly all objectives of human germline gene editing can be attained by preimplantation genetic diagnosis (except for the rare case of parents homozygous with respect to the relevant genes), there are good grounds to orient clinically oriented research to the improvement of this alternative method, which has proved to be without substantial risks and poses no ethical problems over and above those posed by gene editing.
Ralf Müller-Terpitz (University of Mannheim, Mannheim, Germany) addressed the concept of embryo under German and European law. Contrary to what is often pretended in legal discussions, he came to the conclusion that the concept of embryo is a congruent one, both in national (Embryo Protection Act, Stem Cell Act) and in European Union (EU) law (Biopatent Directive 1998, Tissue Directive 2004). Therefore, the legal situation in Germany and Europe does currently not face a legal contradiction with regard to the definitions of human embryos in national and in EU law. Although in national law, the concept of embryo is rather old as it goes back to the year 1990, there exists a surprising uncertainty regarding the scope of the definition of a human embryo. This uncertainty is caused by the unclear and not-defined notion of the “individual,” which serves as a vanishing point of the national definitions of the term “embryo.” According to this vanishing point, only a human entity that is capable to develop to such an “individual” is regarded as being a protected embryo in the sense of the national laws. Therefore, this term is crucial for the question whether research with human embryos or cells derived from such embryos is admissible or inadmissible. Hence, a clarification of this term by the national legislator would be desirable, although this appears to be rather unlikely. This legal opinion, including some practical guidelines for researches working with stem cells derived from human embryos, will also be published in the upcoming Yearbook of Science and Ethics.
In summary, the 9th International Meeting of the Stem Cell Network North Rhine Westphalia in Münster, Germany, provided a broad spectrum of outstanding studies in modern stem cell research fields. Similar to meetings of this series in the previous years (Fleischmann and Horn, 2009; Görgens and Horn, 2015; Radtke and Horn, 2011, 2013), it was very well organized and offered excellent opportunities for inspiring discussions. Students and well-established researchers in their fields enjoyed a stimulating atmosphere to engage in conversations with their peers and the industry partners to initiate new collaborations.
