Bridging the gap from basic epigenetic research to medical

The understanding of neural fate acquisition during embryogenesis is exciting in its own right. In addition to that however, from a medical point of view, the knowledge of the basic molecular developmental mechanisms is essential, because the same signalling factors and transcriptional regulators are also expressed in endogenous niches of neural stem cells in the adult brain.

Human embryonic stem cells (hESCs) can efficiently differentiate into functional neurons and glia with a mechanism akin to in vivo development (Hu et al, 2009; Li et al, 2005; Perrier et al, 2004; Roy et al, 2006; Yang et al, 2008). Therefore, the

developmental program that specifies neural fate in the embryo is important to programme neural stem cells to repopulate damaged tissue. However, due to ethical concerns, the opportunities surrounding hESCs is under discussion. In addition, a potential immune rejection due to their allograft character must not be ignored. According to studies in mice and with human somatic skin cells, which were reprogrammed by a set of core pluripotent transcription factors induced pluripotent stem cells (iPSCs), which are truly pluripotent (Boland et al, 2009; Okita et al, 2007; Park et al, 2008b; Takahashi et al, 2007; Yu et al, 2007; Zhao et al, 2009). Interestingly, neural differentiation of human iPSCs was shown to also follow developmental principles. In addition, human iPSCs have been shown to convert to neuro-epithelial cells following the same differentiation program as hESCs and could express neural marker genes. Their neural identity was determined by the expression of Pax6 and Sox1. The neural differentiation process was improved by regulating FGF and BMP signalling (Hu et al, 2010). This finding is very interesting in relation to this work, as I demonstrate the influence of CHD4 dependent chromatin remodelling during neural induction and neural cell differentiation that is regulated by FGF and BMP signalling processes. In addition, I could demonstrate that CHD4 chromatin remodelling regulates the expression of neural marker genes, including Pax6 and Sox2. This resembles the results achieved in hESCs. Consequently, epigenetic regulation plays a fundamental role during cellular (re-)programming processes and could thus provide opportunities to enhance cellular differentiation protocols for medical applications. Most recent evidence that epigenetic mechanisms, especially ATPase dependent chromatin remodelling, plays a role in the generation of iPSCs through somatic cell reprogramming is provided by Hans Schöler’s group (Singhal et al, 2010). They demonstrated that the ATP-dependent BAF chromatin remodelling complex significantly increases the reprogramming efficiency when used together with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. In addition to that, it was shown that gene suppression by pluripotency factors in ESCs is associated with the recruitment of repressive chromatin remodelling complexes, such as CHD4/NuRD (Kaji et al, 2006). A further example that underlines the role of chromatin remodelling during cell (re-)programming was provided by our laboratory, as we demonstrated that the chromatin remodelling factor CHD4 plays a crucial role for the boundary formation between neuroectoderm and mesoderm. We showed that this is specifically controlled by the Nodal input via Sip1 for the Xbra transcription (Linder et al, 2007). Most recent data provides evidence that Nodal signalling acts through Sip1 to regulate the cell-fate decision between neuroectoderm and mesendoderm in human pluripotent stem cells (hPSCs) (Chng et al, 2010).

Thus, it is possible that signalling information for the neuroectoderm/mesendoderm cell-fate decision in hPSCs by Sip1 is also regulated by CHD4 chromatin remodelling. As I demonstrate in this work that CHD4 and Sip1 could shift the ectoderm/neuroectoderm boundary for the benefit of neuroectoderm, my data can help to understand how stem cells could be programmed from neural commitment to neural differentiation, and reprogrammed back to a higher potent state by epigenetic chromatin remodelling.

A further step in cellular (re-)programming is provided by the recent results of Marius Wernig’s group, which efficiently converted mouse embryonic and postnatal fibroblasts into functional neurons in vitro, referred to as induced neuronal cells (iN) (Vierbuchen et al, 2010). The induced cellular phenotypes are defined and reinforced by lineage-specific transcription factors, leading to cell-type-specific gene expression patterns. These patterns are further stabilized by epigenetic modifications that allow faithful transmission of cell-type-specific gene expression patterns over the lifetime of an organism (Bernstein et al, 2007; Jenuwein & Allis, 2001). Emphasising the crucial role of epigenetic gene regulation in their study, Wernig argues that changes in transcriptional activity result in a genome-wide adjustments of repressive and active epigenetic features such as DNA methylation, histone modifications and changes of chromatin remodelling complexes, which further stabilize the new transcriptional network (Jaenisch & Young, 2008; Zhou & Melton, 2008). A further possible notion that can be considered is that certain subpopulations of cells are ‘primed’ to respond to inducing factors, depending on their pre-existing transcriptional or epigenetic states (Yamanaka, 2009). Interestingly, the principle of reprogramming a differentiated cell into a different cell type by changing epigenetic patterns in combination with specific transcription factors has already been described very early in Xenopus. It was shown that fibroblasts could be converted to stable myoblasts by 5-azacitidine-treatment and transfection with the muscle specific transcription factor MoyD (Davis et al, 1987). Incorporation of 5-azacitidine into DNA inhibits methyltransferases, therefore leads to demethylation and de-repression of methylated gene loci. This data demonstrate that one type of differentiated cell can directly be converted into another, notably; this approach does not always require a stem-cell intermediate stage.

During the late 1950s, Briggs and King established the technique of somatic cell nuclear transfer (SCNT), or “cloning”. This technique demonstrated the developmental potential of isolated nuclei derived from late stage embryos and tadpoles by transplanting them into enucleated oocytes (Briggs & King, 1952; King & Briggs, 1955). Together with work by Gurdon (Gurdon, 1962; Gurdon et al, 1975),

this illustrated that differentiated amphibian cells preserve the genetic information that is necessary to develop into cloned frogs. The conclusion that can be drawn from these early findings is that the genome undergoes reversible epigenetic, rather than irreversible genetic changes during cellular differentiation. This implies that these epigenetic changes have to be reversed, when the process of cell differentiation wants to be reprogrammed to generate iPSCs. Interestingly, this epigenetic contribution to cell reprogramming is emphasised by most recent data that describes an “epigenetic memory” in iPSCs by transcription factor-based reprogramming, compared to reprogramming by SCNT (Kim et al, 2010). They describe that iPSCs, derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin. This “epigenetic memory” of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than iPSCs. Consequently, nuclear transfer was suggested to be more effective at establishing pluripotency than factor-based reprogramming, which can leave an “epigenetic memory” of the tissue of origin (Kim et al, 2010). That memory could influence applications in disease modelling or treatment. Nevertheless, in addition to serve for basic studies in development and epigenetic reprogramming, iPSCs have therapeutic potential for two fundamental concepts: First for custom-tailored or personalized cell therapy, and second for so-called “disease modelling”. For cell therapy, the advantages are obvious, as therapy by organ transplantation is complicated, limited and require life long immunosuppression. Here, iPSCs from patients could provide the solution as they could be differentiated into the desired cell type that is already genetically matched with the patient. An additional approach could be the repairing of disease causing-mutations by homologous recombination. Promising data was provided by Jaenisch and colleagues in a mouse model for sickle cell anaemia (Hanna et al, 2007). They reprogrammed mouse skin cells into iPSCs, fixed the disease-causing mutation and differentiated the repaired cells into healthy blood- forming progenitors. The progenitor cells were transplanted into the anaemic mice, where they formed healthy red blood cells, and cured the disease. This method was also applied to correct haemophilia A in mice (Xu et al, 2009). The principle could be applied to any human disease with a known mutation that can be treated with cell transplantation.

The second approach is referred to as “disease modelling”, which means that iPSCs, derived from patient skin cells could be differentiated in vitro into the diseased cell

type, thereby recapitulating the disease in vitro. This model of the disease could help to identify novel drugs to treat the disease. Several laboratories have isolated iPSCs from patients suffering from amyotrophic lateral sclerosis (ALS) (Dimos et al, 2008), Huntington’s disease, Pakinson’s disease (Soldner et al, 2009), juvenile diabetes, muscular dystrophy, Fanconi anaemia (Raya et al, 2009), Down syndrome, immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome, Gaucher disease type III, Duchenne and Becker muscular dystrophy and others (Park et al, 2008a). In addition, in vitro therapeutic approaches have been reported for spinal muscular atrophy (Ebert et al, 2009), familial dysautonomia (Lee et al, 2009) and the LEONARD syndrome (Carvajal-Vergara et al, 2010). For review about the history, the mechanisms and the applications of induced pluripotency please see (Stadtfeld & Hochedlinger, 2010).

The possibility to reverse the cellular differentiation process by a few transcription factors, combined with epigenetic mechanisms that enable and stabilize stage specific gene expression profiles also influenced our view back on normal and disease development. Comparable to reprogrammed iPSCs, the mechanisms that stabilize and regulate gene expression profiles are often reversed in cancer cells, which show characteristics of stem cells and de-differentiation (Stadtfeld & Hochedlinger, 2010). In contrast, induced neural differentiation could be achieved from human embryonic carcinoma stem cells. Interestingly, neural differentiation was accompanied by significant changes in the acetylation and methylation patterns of histone H3, and expression level of the histone variant H2A.Z. The epigenetic changes occurred on the regulatory regions of Oct4, Nanog, Nestin, and Pax6 (Shahhoseini et al, 2010).

Specific signalling pathways that are mutated cancer cells are also associated with iPSCs formation. This illustrates similarities of tumourgenesis and cellular re- programming and emphasises the role of epigenetic gene regulation in both healthy and diseased cells. The understanding of cancer development in recent years has identified epigenetic abnormalities as a common factor in tumourigenesis. One epigenetic factor is the dysregulation of histone deacetylases (HDACs) in both haematological and solid tumours. Research over the past decade consequently led to the development of HDAC inhibitors (HDACI) as anticancer agents. For a recent review about HDAC inhibitors and cancer therapy please see (Atadja, 2010).

In relation to my work, data suggests epigenetic regulation by ATPase dependent chromatin remodelling to be specifically crucial in cellular differentiation, especially for neural tissue. As an example, Chd1-deficient embryonic stem cells have been shown to be no longer pluripotent, because they are incapable to give rise to

primitive endoderm with a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. These results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells and for somatic cell reprogramming to the pluripotent state (Gaspar-Maia et al, 2009).

A recent study revealed an evolutionarily conserved role for CHD7, which was identified to orchestrate neural crest gene expression programs, which provides insights into the synergistic control of distal elements by chromatin remodelers and illuminated the patho-embryology of the CHARGE syndrome. The CHARGE syndrome is a sporadic, autosomal dominant disorder characterized by malformations of the craniofacial structures, peripheral nervous system, ears, eyes, and heart. This observation is suggested to represent a broader function for CHD7 in the regulation of cell motility (Bajpai et al, 2010).

A further interesting syndrome that links epigenetic regulation in neural development with disease formation is the Mowat-Wilson syndrome (MWS). MWS is caused by Sip1 mutations. It was shown that aberrant Sip1 protein is unable to recruit NuRD/CHD4. This defective NuRD/CHD4 recruitment due to mutant human Sip1 can be a MWS-causing mechanism (Verstappen et al, 2008). MWS is characterized by a number of health defects including delayed growth and motor development, congenital heart disease, genitourinary anomalies and absence of the corpus callosum, mental retardation, and Hirschsprung’s disease. Hirschsprung’s disease arises when ganglion nerve cells in the gut fail to develop and mature correctly. Considering these human developmental defects, MWS could consequently be understood as a clinical representation of a disorder in the epigenetic regulatory mechanism by CHD4/Sip1, which I describe in this work.

During the last years, these examples demonstrated that epigenetic regulation of cell differentiation revealed promising opportunities in medical applications. The study of epigenetic mechanism for cell (re-)programming and cellular differentiation in developmental model organism are crucial steps to understand how cells can be modulated for the benefit of patients. The knowledge of the basic mechanism of epigenetic regulation during cellular differentiation and (re-)programming has been acquired by basic research in model organisms like Xenopus.

Concluding, in reference to the introduction, epigenetic cell modification has a high potential to serve as useful tools for medical applications. Epigenetically enhanced (re-)programming of iPSCs could overcome the problems of hESCs to study diseased cells for pathological studies, drug screening, and regenerative medical approaches.