In March 2009, the fifth International Meeting of the Stem Cell Network North Rhine Westphalia took place in Aachen, Germany. Numerous fascinating presentations about reprogramming, stem cells, and therapeutic devices were given. A number of excellent speakers from all over the world were invited to present their work. Over 20 high-profile presentations were given on 2 days under the five different topics: reprogramming, mechanisms regulating stem cells, stem cell differentiation, cancer stem cells, and therapeutic devices. Young researchers had opportunity to present their work in over 120 posters.
The meeting started with the main topic: reprogramming. Austin J. Cooney, Baylor College of Medicine, Houston, USA, gave an exiting presentation about alternative pathways to maintain pluripotency. His work is focused on the regulation of Oct4 by nuclear receptors, specifically LRH-1, which regulates directly Oct4 expression and the role of canonical wnt signalling relating to β-catenin, which potentiates reprogramming (Gu et al., 2005). His group showed that ES cells deficient in LRH-1 and β-catenin lose their pluripotency faster than wild-type ES cells. The group established a model for generation of β-catenin −/− ES cells. In a second model, the effect of LRH-1 was analyzed and the effect of BIO, a GSK3β inhibitor, was evaluated. The results showed that wnt3a induces LRH-1 in a β-catenin-dependent manner, because β-catenin binds directly to TCF elements in the LRH-1 promotor (Lluis and Cosma, 2009; Mullen et al., 2007).
Theodore Rasmussen, University of Connecticut, USA, talked about direct reprogramming of somatic cells: from ES cell fusion to iPS. Rasmussen and his working group chose the method of somatic cell nuclear transfer (SCNT) to reprogram differentiated somatic cells to a more pluripotent state. He pointed out that iPS technology is highly promising because it can yield immunocompatible cells by relatively simple, noncontroversial means. Rasmussen's group used the superb cell biology allowed by SCNT to study rapid and dynamic chromatin remodeling events such as histone replacement, which occur within hours after nuclear transfer with kinetics similar to those of preimplantation development (Chang et al., 2005). FMR reprogramming takes longer than SCNT, but is faster than iPS. FMR is well suited for genetic analyses, because polymorphic differences between the somatic and ES cell fusion partners can be used to trace reprogrammed gene expression (Ambrosi et al., 2007). In addition, genes can be manipulated in ES cells prior to fusion to evaluate their importance for reprogramming. Together, SCNT and FMR offer unique advantages for the investigation of reprogramming mechanisms.
The presentation of Sir John B. Gurdon, Cambridge, UK, a pioneer in this field, was focused on nuclear reprogramming by nuclear transfer. John Gurdon and his group try to identify the components of eggs that cause the nuclear reprogramming. One method is to transfer multiple nuclei into the germinal vesicle of growing eggs, the oocytes. These result in lots of mammalian somatic cell nuclei that can be induced to start expressing genes for pluripotency such as Oct4, Nanog, and Sox2. The researchers detect that the following steps are involved in reprogramming: the chromatin becomes highly decondensed, differentiation marks are removed, the provision of transcription factors does not appear to be necessary in reprogramming, and linker histones of the H1 class are exchanged in transplanted nuclei (Gurdon and Melton, 2008). The presentation of Huck-Hui Ng, Genome Institute of Singapore, was focused on the major role of transcriptional regulation in specifying the unique properties of embryonic stem cells. They used chromatin immunoprecipitation coupled to high-throughput sequencing technology to map the binding sites for 13 different transcription factors (Oct4, Sox2, Nanog, Smad1, Stat3, Esrrb, Tcfcp2l1, Klf4, c-Myc, n-Myc, Zfx, E2f1, and Ctcf ) in mouse ES cells (Chen et al., 2008). The data revealed hot spots for transcription factor binding. These hot spots occur in two major clusters, namely, the Oct4-centric clusters and the Myc-centric clusters. Dr. Ng also presented data on a new reprogramming factor, Esrrb (Feng et al., 2009). Esrrb is a nuclear orphan receptor that works in conjunction with Oct4 and Sox2 to reprogram mouse fibroblasts to induced pluripotent stem cells. This study demonstrates that one of the core reprogramming factor Klf4, can be substituted by Esrrb.
Three outstanding presentations on the mechanisms regulating the stem cell state were given in the second session. First, Ian Cambers, University of Edinburgh, UK, presented his group's recent studies on the role of Oct4 and Nanog in controlling pluripotent cell identity. A combination of biophysical studies and ES cell assays show that Nanog functions as a dimmer (Mullin et al., 2008). Constitutive expression of Nanog allows autonomous ES cell self-renewal. However, the Chambers lab have shown that Nanog fluctuates in ES cells with downregulation of Nanog predisposing but not committing cells toward differentiation. Targeted Nanog null ES cells remain competent to colonize the three embryonic germ layers. However, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge, a defect rescued by repair of the mutant allele. Together with the requirement of Nanog for specification of pluripotent cells in the preimplantation embryo, these results correlate a Nanog requirement with changes in the epigenetic status of the X-chromosome. The effect of deletions of Nanog and Oct4 on regulation of Xist, the inactive X-specific transcript responsible for X-chromosome inactivation, were therefore investigated (Navarro et al., 2008). Loss of Nanog causes partial Xist derepression despite continued Oct4 and Sox2 binding. In contrast, Oct4 elimination abrogates Nanog and Sox2 binding to Xist chromatin resulting in robust Xist expression. Together, these results indicate that the level of Nanog directly correlates with the efficiency of undifferentiated ES cell colony formation, and that Nanog therefore functions as a differentiation rheostat.
Niall Dillon, Imperial College London, UK, and his team focused on the fact that the ES cells have the ability to activate many different gene expression programs as the cells commit to specialized lineages. They provide evidence that the ES cell transcription factors Sox2 and Foxd3 facilitate this process by priming genes for expression at later stages of differentiation. Binding of Sox2 epigenetically marks the gene by generating a localized peak of histone H3 dimethylation at the enhancer in ES cells, whereas binding of Foxd3 suppresses transcription of the intergenic sequences between the gene where the enhancer is located. In pro- and pre-B cells where the genes are expressed, two related factors, Sox4 and FoxP1, bind to the enhancer, and we show that mutation of the DNA sequences that bind these factors abolishes the enhancer effect. The working group also identifies similar patterns of factor binding at the Pax5 and Blnk/Dntt loci, both of which encode key regulators of B cell development. Based on these findings, we propose a factor relay model where ES cell factors establish epigenetic marks at enhancers and promoters before being replaced by related factors at later stages of cell differentiation.
On the second day of the meeting, stem cell differentiation, cancer stem cells, and therapeutic devices were the main topics. The day started with a presentation about the generation of embryonic stem cell (ESC) like cells of germline stem cells (GSC) by Kinarm Ko, Münster, Germany. In their current study, they first demonstrate that mouse pluripotent stem cells, so-called germline-derived pluripotent stem (gPS) cells, can be converted from established adult unipotent GSCs by using a defined culturing procedure. To understand the underlying mechanism by which unipotent cells are converted into pluripotent cells, a robust conversion protocol is a prerequisite. By microscopic evaluation of fluorescent pluripotency markers during the time course of conversion the group of Dr. Ko noticed a critical window of culture time in which only certain subpopulations of cells in GSC colonies are converted into gPS cells. They also found that the conversion process requires a certain microenvironment, which apparently can be sustained by the developed conversion methods. The established protocol is reproducible in that they found that the gPS cell conversion rate was compatible for that of viral induction of somatic cells into iPS cells. The conversion method will allow for studying the underlying mechanisms by which unipotent GSCs are converted into gPS cells. Furthermore, insights from this study could uncover mechanisms that are involved in human disease concerning testicular germ cell cancer formation (Kim et al., 2009).
Andreas Trumpp, Heidelberg, Germany, gave a really stimulating presentation in the session about cancer stem cells and presented his work on “Waking up dormant stem cells” (Essers et al., 2009). Trumpp and his group characterized long-term dormant population within the most immature HESCs (Lin−Sca1+ckit+CD150+CD48−CD34−) using two types of label-retaining assays. Computational modeling suggests that dormant hematopoietic stem cells (d-HSCs) divide every 145 days or five times per lifetime. Although they form a silent reservoir of HSC during homeostasis, they are activated to self-renew in response to bone marrow injury or granulocyte-colony stimjulating factor (G-CSF) stimulation. Activated HSC can turn to dormancy after reestablishment of homeostasis. They are not entering stochastically the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress. One reason that cancer stem cells can escape antiproliferative chemotherapy is their relative dormancy. Trumpp and his group showed that treatment of mice with interferon-α leads to activation of dormant HSCs in vivo. HSCs lacking either the interferon-α/β-receptor, STAT1 or Sca1, are insensitive to INF-α stimulation, demonstrating that STAT1 and Sa-1 mediate IFN-α-induced HSC proliferation.
Christine Mummery, Leiden Medical Center, The Netherlands, gave a presentation about differentiation of human embryonic stem cells into cardiomyocytes. Microarray analysis had shown that the major known cardiac genes are upregulated but that novel genes are also expressed. HESC were marked by GFP and cells could be traced following transplantation into the mouse heart. Results showed short-term functional benefit after 4 weeks but no long-term benefit after 12 weeks. Short-term benefits result from neoangiogenesis around the infarct border resulting from the presence of cardiomyocytes. The results suggest that early benefits are derived from amelioration of ischemic damage by the neovasculature. More immediate applications of HESC-derived cardiomyocytes that have also been investigated were presented by Christine Mummery. Results presented showed a series of cardiac and noncardiac drugs in clinical use highly predictive dose responses on the electrical activity of the HESC-derived cardiomyocytes. By the effects of a QT interval by microelectrode array the drugs could be catagorized in groups as high risk or low risk. The results correlated well with clinical observations (Mummery 2007; Mummery et al., 2003, van Laake et al., 2007).
In conclusion, it was an outstanding meeting with forward-looking presentations. The participants had the great opportunity to get an excellent overview on diverse topics in reprogramming and stem cell research. The meeting was very well-organized, and gave a nice framework for inspiring conversations.
