Our long-range goals are to understand epigenetic regulation of gene expression in mammalian development and disease. An important question is to understand the different epigenetic conformations that distinguish differentiated cell states and to define strategies to transdifferentiate one differentiated cell type into another. Embryonic stem cells are of major significance because they have the potential to generate any cell type in the body and, therefore, are of great interest for regenerative medicine. A major focus of our work is to understand the molecular mechanisms that allow the reprogramming of somatic cells to an embryonic pluripotent state and to use the potential of patient specific pluripotent cells to study complex human diseases.
In vitro reprogramming of somatic cells to a pluripotent state
The recent success in reprogramming somatic cells to pluripotent Induced Pluripotent Stem (iPS) cells by defined factors has opened exciting possibilities not only for the investigation of complex human diseases in the Petri dish but also for the ultimate application in transplantation therapy. A major focus of our work is (i) to study the molecular mechanisms of somatic reprogramming and to devise efficient approaches for the reprogramming of mouse and human somatic cells; (ii) to derive patient specific iPS cells for the generation of tissue culture models of major human diseases; and (iii) to establish efficient gene editing approaches in human cells.
(i) Molecular mechanisms: The study of induced pluripotency is complicated by the need for infection with high titer retroviral vectors resulting in genetically heterogeneous cell populations. We have generated genetically homogeneous “secondary” mouse and human somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. This system facilitates the characterization of reprogramming and provides a unique platform for genetic or chemical screens to enhance reprogramming or replace individual factors. For example, the secondary system has allowed us to describe the role of stochastic epigenetic events and of cell proliferation during the reprogramming process. Single cell expression analysis was used to define an initial stochastic followed by a deterministic phase in the reprogramming process.
(ii) Patient specific iPS cells: Most current methods for reprogramming human somatic cells preclude consideration for cell replacement therapies since they rely on the delivery of the four reprogramming factors by retroviral transduction, which carries the risk of tumor formation. Also, a concern is that the low level of provirus expression that is consistently detected in the iPS cells may affect other biological characteristics such as differentiation potential. An important issue of the field is to generate vector free iPS cells and our most recent reprogramming protocol uses RNA instead of DNA to induce iPS cells. A major goal is to establish in vitro differentiation systems that allow us to study the pathogenesis of neurodegenerative diseases such as Parkinson’s, Alzheimer disease or ALS in the Petri dish and to eventually isolate small molecules that could be used for therapy.
(iii) Genetic manipulation of human ES and iPS cells: In contrast to mouse ES cells, homologous recombination in human pluripotent cells is extremely inefficient impeding progress in using the potential of human ES and iPS cells for disease research. We have used Zinc Finger Nucleases (ZFN) and TALENs (Transcription Activator Effector Like Nucleases) to engineer genes. Most recently we have adapted the CRISPR/Cas9 gene editing technology to efficiently generate human ES and iPS cells as well as mice carrying multiple mutations. These novel approaches are efficient in introducing specific alterations into cellular genes including deletions, insertions and point mutations. It is likely that these approaches will vastly expand our ability to establish in vitro model system to study complex human disease in the Petri dish by generating genetically defined disease specific and matched control iPS or ES cells.
iPS cell technology for the study of human disease
(i) Rett syndrome: Rett syndrome (RTT) is a postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like syndromes that manifests in girls during early childhood. The disease is caused by mutation of the X-linked DNA binding protein MECP2 (Methyl CpG-binding Protein 2) and represents the second most common cause of intellectual disability in females (males are more severely affected and die early). Previously we have established a transgenic mouse strain carrying a mutation in the Mecp2 gene. The phenotype of the Mecp2 mutant mice strikingly resembles the patient phenotype and has been used to establish therapies to treat progression of symptoms in mice. Based on these results in mice, clinical trials have been initiated yielding promising results.
To study Rett syndrome in the human system we have used TALEN mediated gene editing to generate isogenic pairs of human mutant and control ES and patient derived iPS cells that differ exclusively at the disease causing mutation. Using this well-defined in vitro system we found that MECP2 mutant ES or iPS cells differentiated into mature neurons display key molecular and cellular characteristics of the disorder including a smaller cell size, decreased spine complexity and significantly reduced electric activity. Importantly, the cellular phenotype of the cultured mutant neurons was reversible by small molecules providing an attractive therapeutic strategy.
(ii) Parkinson Disease: Parkinson’s disease (PD) is the second most common chronic progressive neurodegenerative disease with a prevalence of more than 1% in the population over the age of 60, thus constituting a major global health problem of the aging population. The overall goal of our work is to establish a genetically defined experimental in vitro system for studying the molecular and biological mechanisms of sporadic PD in the dish and to identify novel genes that are causally involved in the pathogenesis of Parkinson’s. We are using 2 approaches.
(i) Mutations in some genes such as SNCA coding for synuclein cause dominant early onset disease. We used the novel gene targeting technology described above to either “correct” the SNCA-A53T mutation in patient-derived hiPSCs or to “knock-in” the same mutation in a human embryonic stem cell (hESC) line, resulting in two pairs of isogenic stem cells that differ exclusively at the SNCA. Neurons differentiated from these cells have been used to define cellular phenotypes of the disease and to screen for small molecules that could be used for therapy.
(ii) Genome-wide association studies (GWAS) have pointed to more than 50 genomic loci, typically associated with enhancers, that contribute to the risk of sporadic PD, which the most common form of the disease. However, these studies are mostly descriptive and little functional and molecular understanding has been gained into which and how these genetic variants contribute to the increased risk for PD. Using CRISPR/Cas-mediated gene editing we are exchanging genomic candidate regions to generate isogenic pairs of cells that differ exclusively at multiple disease associated loci. Our aim is to gain molecular understanding of the complex interactions between multiple genetic risk alleles and to identify key genes of which the expression level affects the PD specific cellular phenotype.
(iii) Adrenoleukodystrophy (ALD – Lorenzo’s oil disease): ALD is a lethal de-myelinating disease caused by the mutation of an X-linked fatty acid transporter gene. The mutation causes progressive demyelination, likely caused by oligodendrocyte dysfunction and neuroinflammation. Using patient derived iPS cells we are establishing culture systems that allow to investigate the interaction of mutant neurons with glia to setup a screenable in vitro system, in which drugs or genetic modifiers can be tested for therapeutic intgervention.
Neural Crest related diseases
Neural crest (NC) cells arise from the neuro-ectoderm at neurulation and migrate over long distances to contribute to multiple cell types and tissues throughout the body. We focus on NC based diseases because mutations in neural crest cells are the cause of a wide variety of diseases such as the lethal developmental disorder Familial Dysautonomia or cancers such as Neurofibromatosis and melanoma. However, study of neural crest derived diseases by iPS technology is complicated as these disorders originate in embryogenesis because an in vitro approach using patient specific iPS cells cannot recapitulate the developmental aspects of a disease. Also, conventional study of human diseases such as NC derived cancer involves the transplantation of human tumor cells into immune-suppressed animals, an approach that does not allow studying early stages of tumor formation and its late stage manifestation in the microenvironment of a normal animal.
The objective of this project is introduction of neural crest cells, which have been differentiated from patient-derived iPS cells, into mouse embryos with the goal to generate mouse-human NC chimeras. Such an experimental system would enable studying the initiation of neural crest diseases during embryogenesis and its progression and manifestation over the lifetime of the animal using patient derived cells. Establishing robust procedures that allow to incorporate human cells into tissues of the developing mouse would provide a novel approach not only for investigating the pathogenesis of a disorder but would also offer a relevant animal model to assess the effect of candidate drugs on the relevant human cells for treatment of the disease.
Tracing dynamic changes of DNA methylation in real time and at single cell resolution
Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. We have established a Reporter of Genomic Methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. Insertion of the RGM reporter into the proximity of CpG islands associated with gene promoters, allows faithful reporting on gain and loss of DNA methylation. Importantly, RGM allows reporting on endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Also, the reporter activity faithfully reflects the methylation state of the two alleles of imprinted genes (one being expressed and non-methylated and the other one methylated and silent). Loci-specific DNA methylation changes, and its correlation with transcription was visualized during cell state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single cell resolution.
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