The regenerative potential of somatic stem cells declines with age. Our subproject within the FWF-funded SFB F78 (“Neuro Stem Modulation”; Coordinator: Dr. Jürgen Knoblich) aims at deciphering the transcriptional network of neural stem cell (NSC) decline and gain of quiescence to understand regenerative processes in the adult brain. Since experimental access to young vs. old human brain tissue is limited, we employ reprogramming technology as a proxy to analyze physiologic neural regeneration. We use iN- and iNSC-type direct conversion from isogenic sources to gain human NSCs of distinct age. We assess reprogramming trajectories during direct conversion of human fibroblasts into induced NSCs by serial combinatorial cell-indexing. This will enable for parallel capture of clonal history and cell identity to reconstruct multilevel lineage trees as a proxy for regeneration pathways. Moreover, we will perform a CRISPR/Cas-based perturbation screen to study the function of human-specific instructors of neurodevelopment.
Induced neural stem cells (iNSCs) carry a strong potential for clinical application. Recently, we demonstrated that iNSCs can ameliorate pathophysiology of multiple sclerosis in a relevant mouse model. Within a cooperative project with the Paracelsus Medical University, Salzburg funded by the wings for life foundation we assess the therapeutic capacity of iNSC to cure traumatic spinal cord injury (SCI). We will apply a combination of anti-inflammatory intervention at the acute phase with transplantation of iNSCs during the chronic phase to induce and maintain a neuroregenerative environment. Stem cells of the nervous system do represent an ideal cellular source for cell replacement; however, the access to appropriate cells from adult patients is limited. By employing patient-specific iNSCs we will make use of a novel, safe and autologous cellular source for transplantation in the chronic phase. Our previous studies have shown that iNSCs can be generated much faster and are safer than iPSC-derived neural cells. However, iNSCs can be cryopreserved, proliferate virtually indefinitely and are able to mature into neuronal and glial cells.
Establishing an authentic human disease model of the lysosomal storage disease (LSD) Mucopolysaccharidosis IIIB (MPS IIIB), often referred to as Sanfilippo Syndrome, is the goal of this project. Using cellular reprogramming of patient-derived somatic cells, we aim to better understand the cellular dysfunction as well as to develop novel therapeutic approaches for MPS IIIB. In detail, we will investigate the contribution of lysosomal pathway dysfunction to neurodegeneration employing models of the brain and eye using patient-derived reprogrammed cells. By applying specific differentiation protocols, we will generate and analyze patient-specific adherent neuronal networks, retinal cells as well as cortical organoids. By the means of state-of-the-art analytic methods, we will explore to which extent the disease is recapitulated in a petri dish and consequently enhance the understanding of the molecular hallmarks and disease mechanisms of MPS IIIB. The collaboration with Nicole Muschol from UKE, Hamburg, Germany enables us to assess samples from MPS IIIB patients enrolled in a clinical study. Thus, allowing a direct patient-specific analysis and eventually the prediction of therapy efficacy. We anticipate major advances in MPS IIIB disease understanding in particular and rare neurological diseases in general to facilitate clinical translation to novel therapeutic cures.
Voltage-gated calcium channels (VGCCs) play important roles in physiological functions of many human cells and are particularly important in the central nervous system. VGCCs are key modulators of early neural development and essential for proper functionality of the adult human brain. Consequently, mutations within VGCCs contribute to a range of neurological disorders such as Schizophrenia, Epilepsy and Autism-Spectrum-Disorders (ASD). Previous electrophysiological studies in mouse models indicate that particular mutations affect the gating properties of Cav channels, permitting a gain-of-channel-function. We assess Cav1.3 mutations in iPSC-derived disease-relevant human neurons. For that, we use both patient-specific iPSCs and perform genome engineering by CRISPR/Cas9 in wild-type iPSC and iNSC, respectively. For functional validation we subject mutated NSCs to in vitro differentiation protocols to obtain disease-relevant human cortical glutamatergic, midbrain dopaminergic and striatal GABAergic medium spiny neurons. Immunocytochemical stainings, qPCR analysis, electrophysiological recordings and calcium imaging will be employed to assess how the mutations interfere with early neurodevelopment and to which extent those pathophysiological alterations can be compensated by pharmacological intervention.
Ageing is a major risk factor for cardiovascular disease. At organismic level cardiovascular ageing is characterized by a gradual change of vascular structure and cardiac homeostenosis resulting in vascular and cardiac insufficiency. Within the FFG-funded COMET center VASCage we aim to get a further understanding of cardiovascular ageing at cellular and molecular level. Employing stem cells we will determine cardiovascular ageing profiles with high predictive power and try to identify putative molecular targets for therapeutic intervention. Current research is limited by the poor accessibility to functional human tissues. Human pluripotent stem cells (PSCs) offer a virtually unlimited access to functional human cells to model cardiovascular ageing. We generate hiPSCs from patients suffering from cardiomyopathy and stroke, respectively, employ robust in vitro differentiation into cardiovascular lineages and develop human 3D in vitro models for myocard and vascular structures. We will determine genome-wide ageing profiles by omics technologies defined by both physiological and synthetic, premature ageing.
Parkinson’s Disease (PD) is a neurodegenerative disease characterized by a progressive loss of striatal dopaminergic (DA) projecting neurons of the substantia nigra. The pathological hallmarks of PD are intracellular inclusions of proteins called Lewy bodies and Lewy neurites that are predominantly composed of misfolded and aggregated forms of the presynaptic protein alpha-Synuclein (a-Syn), encoded by the SNCA gene. This neuronal degeneration leads to marked decrease of dopamine levels in the striatum, which triggers a range of motor deficits characterising PD as a movement disorder such as tremor at rest, rigidity and bradykinesia. Recent studies indicate important roles of synuclein proteostasis, mitochondrial function, neuroinflammation and ageing in PD pathogenesis. To analyse these processes in relevant human cells we generate novel human iPSC-derived 3D model systems in vitro. Human models will be derived from genetic and idiopathic PD iPSC lines, subjected to premature ageing and a-Syn aggregation and pro-inflammatory cytokine production will be analysed.