Disease modeling using induced pluripotent stem cells (iPSC) and organoids for future drug development
Research area
Genetics, iPSC/organoid disease models, Molecular and Cell biology
Brief description
Neurodevelopmental and neurodegenerative disorders comprise a heterogeneous group of conditions for which treatment options are limited. Our group develop cell models of central nervous system disorders using induced pluripotent stem cells (iPSC). The long-term objective is to identify disease biomarkers that can be used for rescue-screens in search for candidate compounds and drug development.
Aim
The master projects (several offered) aim to characterize cell models of neurodevelopmental and neurodegenerative disorders in vitro using iPSC derived cells (neural stem cells, neurons, neural crest cells, organoids). The projects are focused on methodologies to identify cellular and molecular abnormalities associated with distinct disease phenotypes. Disease-associated abnormalities are evaluated for future rescue screening and drug development.
A specific master project will be selected according to the student’s interest within one of the projects below.
Background
A limiting factor for the analysis of disease mechanisms in disorders of the central nervous system has been access to appropriate model systems to recapitulate human brain pathophysiology. The introduction of induced pluripotent stem cell (iPSC) technology provides the possibility to overcome this limitation (Takahashi et al. Cell, 2007). iPSC has the ability to differentiate into mature neuronal cells that recapitulate functions in vivo. Our group has developed neural iPSC models of different central nervous system disorders with a known genetic cause (e.g. for Down syndrome, epilepsy, Dravet syndrome, etc). The iPSC models are derived from somatic cells obtained from affected individuals, or from genome edited (CRISPR/Cas9) wild-type cells, followed by differentiation into neuronal subtypes or 3D models (i.e. organoids). Various methods are used to identify disease specific cellular and molecular perturbations, such as imaging, high through-put RNA sequencing, mass spectrometry and different functional assays.
Concurrent projects
Down Syndrome (DS)
iPSC lines from DS patients and trisomy for chromosome 21 show dysregulated genes in various brain related models (e.g. organoids, neural stem cells, neurons, neural crest cells). We have recently established a protocol to model cranial neural crest cell (CNCC) development. Using scRNAseq we identified overexpression of the RUNX1 gene as a candidate for DS pathology.
In this project, we aim to further investigate the importance of RUNX1 (encoding a transcription factor) for CNCC development by introducing an inducible shRNA to knock-down expression of RUNX1 in DS derived cells.
Methods: IPSC culture. Generate stable iPSC lines, verify RUNX1 vector integration and downregulation upon doxycycline induction, CNCC differentiation, analyze selected genes by qRT/PCR and cell types by immunostaining. If time permits, we plan to evaluate CNCC migration and development in an organoid/ assembloid model system.
Incontinentia pigmenti – NEMO deficiency
NEMO (or IKBkG) deficiency causes Incontinentia Pigmenti (IP), characterized by abnormalities in neural crest lineage derivatives, such as different ectodermal and neuroectodermal tissues. We have generated an iPSC line depleted of NEMO (using CRISPR/Cas9) to model the disease. Following differentiation into the neural crest lineage analysed by scRNAseq, we recently identified dysregulated genes and cellular phenotypes (e.g. upregulation of ECM protein Decorin).
In the project, we aim to confirm our findings by overexpressing NEMO in our K.O. line (rescue) and by downregulation of NEMO in the control line by RNAi to mimic the IP phenotype.
Methods: IPSC culture. Rescue NEMO deficiency in iPSC K.O. line. Transfect and express a NEMO transgene, verify integration and overexpression. Mimic NEMO deficiency in W.T. cells after transgene expression of shRNA against NEMO mRNA. Transfection, crest cell differentiation, analysis of cells for expression of selected genes by qRT/PCR and by immunostaining. Generating crest cell spheres and characterize composition of ECM by immunostaining. If time permits, we plan to evaluate CNCC migration and development in an organoid/ assembloid model system.
Generation of fluorescent iPSC lines for improved cell line tracking and advanced disease modeling
Complex 3D models using organoids and/or assembloids are made up by different iPSC derived cell types. However, it is important to continuously track the progression of distinct cell line progenywhen different iPSC derivatives are combined in single structures. In this project, we aim to generate several fluorescently tagged iPSC lines that enable to track the origin of individual iPSC lines in complex disease models.
Methods: IPSC culture. CRISPR/Cas9 editing of iPSC lines, transfection, evaluation of editing, characterization of modified iPSC line(s). Evaluate CNCC migration and development in an organoid/ assembloid model system using fluorescently tagged iPSC lines.
Contact details
Jens Schuster, Docent; Jens.schuster@igp.uu.se
Niklas Dahl, Prof, Niklas.dahl@igp.uu.se
Research group Prof Niklas Dahl
Phone number (Jens) 076-8907223 (also text)