Conference Agenda

Human Pluripotent Stem Cells as Tools for Modelling Neurodegeneration

Oliver Brüstle

University Bonn, Germany

Human pluripotent stem cells and their neural derivatives open exciting avenues for modelling neurological disorders in vitro. A prerequisite for such studies are robust protocols that efficiently yield standardized populations of neural cell types.

Recently we have established protocols for the derivation of long-term self-renewing neuroepithelial stem (lt-NES) cells from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). Lt-NES represent a stable, rosette-forming population, which can still be patterned to generate distinct neural subpopulations enabling direct access to metabolic pathways and signalling cascades relevant for the pathogenesis of neurodegenerative disorders. Derived from disease-specific iPSC, lt-NES cells can be exploited for studying key pathogenic mechanisms such as protein aggregation in polyglutamine disorders directly in human neurons and without resorting to overexpression paradigms.

We applied this strategy to Machado-Joseph Disease (MJD; syn. spinocerebellar ataxia type 3; SCA3), a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene. Proteolytic cleavage of the MJD1 gene product, ataxin-3 (ATXN3), is believed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD.

Our data show that aggregate formation can be recapitulated in MJD iPS cell-derived neurons, and that this phenomenon can be used to dissect the molecular pathogenesis of pathological protein aggregation. These data indicate that developmentally early hESC and iPSC-derived neurons provide a unique window of opportunity to study pathogenic driver mechanisms underlying age-related neurodegenerative diseases in a pre-symptomatic phase preceding neuronal degeneration.

Amyloid generation and axonal tau pathology in human pluripotent stem cell-derived neurons expressing Alzheimer’s disease-associated mutant proteins

Jerome Mertens¹, Philipp Koch¹, Irfan Tamboli², Kathrin Stüber¹, Julia Ladewig¹, Daniel Poppe¹, Jonas Doerr¹, Jochen Walter², Oliver Brüstle¹

¹Institute of Reconstructive Neurobiology, Life&Brain Center, University of Bonn and Hertie Foundation, Germany; ²Institute for Neurology, Sigmund-Freud-Straße 25, D-53127 Bonn, Germany

Alzheimer’s disease (AD) is the most frequent cause of dementia, and there is strong evidence that altered proteolytic generation of amyloid-beta peptides (Aβ) as well as aberrant behavior of the microtubule-associated protein tau play critical roles in AD pathogenesis. However, the effective mechanisms that conspire to trigger the degeneration of neurons remain obscure. Although amyloidogenesis and tau phosphorylation are known to be highly cell type and species-specific, most studies conducted so far have, due to the limited access to vital human neuronal tissue, been conducted using heterologous overexpression systems in a non-human background. We set out to explore whether mature human neurons generated from human pluripotent stem cell-derived neural stem cells (hNSC) could serve as in vitro model for AD. We found that neurons generated from differentiating hNSC express the neuron-specific APP695 isoform, β- and γ-secretases as well as the six tau isoforms typically found in the adult human brain. The neurons recapitulate key steps in proteolytic APP processing, exhibit a differentiation-dependent increase in Aβ secretion and BACE1 maturation and can be directly used to read out pharmacotherapeutic effects of compunds targeting γ-secretase including non-steroidal anti-inflammatory drugs (NSAIDs). Expression of mutant presenilin-1 (L166P) in these cells results in a partial loss of γ-secretase function and an increased Aβ42/40 ratio, which is resistant to γ-secretase modulation. Given the involvement of tau in cytoskeletal stabilization and axonal transport, we investigated whether an overload of normal tau or aberrantly pseudo-hyperphosphorylated tau influence axonal trafficking and resistance of neurons to oxidative stress. Comparative analyses of neurons expressing normal or pseudo-hyperphosphorylated tau revealed that only excess of aberrant tau but not normal tau leads to the pathological MC-1 conformation and suffices to impair axonal transport of mitochondria without inducing neuronal death. As a consequence, in non-redox-protected conditions, tau-induced malfunctions culminated in the development of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Human pluripotent stem cell-derived neurons thus represent a unique cell-based tool for assessing cytopathological processes relevant for AD pathogenesis as well as for the evaluation of potential drugs directly in a human neuronal system.

TIMP-1 binds to CD63 and enhances murine hematopoietic stem and progenitor cell homing in vivo

C. Matthias Wilk¹, Frank A. Schildberg², Marcel A. Lauterbach³, Ron-Patrick Cadeddu¹, Julia Fröbel¹, Sebastian Buest¹, Volker Westphal³, René H. Tolba⁴, Stefan W. Hell³, Akos Czibere⁵, Ingmar Bruns¹, Rainer Haas¹

¹Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University, Düsseldorf, Germany; ²Institute for Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-University, Bonn, Germany; ³Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Germany; ⁴Institute for Laboratory Animal Science and Experimental Surgery, RWTH-Aachen University, Aachen, Germany; ⁵Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Tissue Inhibitors of Metalloproteinases (TIMPs) like TIMP-1 are soluble serum proteins that are known for their dual functions. While TIMPs inhibit matrix metalloproteinases through their n-terminus, they also exert direct effects on different cell types such as on bone marrow derived cells. These cytotropic functions are mediated by a variety of cell surface proteins binding to the n-terminal region of TIMPs. The TIMP-1 protein is a recently identified tetraspanin interacting cell surface protein in the immortalized human breast epithelial cell line MCF10A. Tetraspanins like CD63 are proteins that consist of four transmembrane domains and are known to interact with integrins. Integrins play a crucial role in hematopoietic stem and progenitor cell (HSPC) homing and mobilization.

In this study we show that TIMP-1 co-localizes with the tetraspanin CD63 and beta-integrin on human CD34+ HSPCs. We found a functional interaction of TIMP-1 with this receptor complex resulting in a higher amount of activated beta-1 integrin on the cell surface. This leads to strengthened migratory capacities towards a SDF-1a gradient and to enhanced adhesion to fibronectin. As also described for other cell types, TIMP-1 prevents CD34+ HSPCs from apoptosis. This antiapoptotic effect stands in close relation to the finding that TIMP-1 stimulation of CD34+ HSPCs leads to increased BFU-E counts in clonogenic assays. In mice treated with TIMP-1 we found altered erythropoiesis parameters such as increased red blood cell counts and hemoglobin and higher numbers of reticulocytes. In a transplantation model based on congenic mice strains, we found strengthened homing parameters in host mice that received bone marrow transplants from previously TIMP-1 stimulated donor mice.

Taken together, these results reveal that TIMP-1 exerts direct effects on human and murine hematopoetic stem- and progenitor cells.

Mesenchymal Stem Cells from Osteoporotic Patients: A Novel Niche for In-vitro Disease Modelling

Thomas Martin Randau¹, Frank Alexander Schildberg², Margit Zweyer³, Sascha Gravius¹, Robert Pflugmacher¹, Dieter Christian Wirtz¹, Dieter Swandulla³, El-Mustapha Haddouti¹

¹Department of Orthopedics and Trauma Surgery, University Clinic of Bonn, Germany; ²Institute of Molecular Medicine, University Clinic of Bonn, Germany; ³Institute of Physiology II, University Clinic of Bonn, Germany

Osteoporosis is among the 10 most crucial global diseases. It is defined as a systemic deterioration of bone mass and micro-architecture, disturbing the delicate coupling of bone-forming (osteoblasts) and bone-resorbing cells (osteoclasts). Bone marrow harbours mesenchymal stem cells (MSCs), the progenitors of osteoblasts, but the vertebral body as a stem cell niche is quite poorly described. Whether dysfunction or deficiency of MSCs contributes to the pathogenesis of osteoporosis and the associated delayed bone healing is unclear. The aim of this project was to establish the first human cell culture model originating from the vertebral compression fracture. As the spine is the most commonly affected osteoporotic fracture, we wanted to prove that fractured vertebral bodies harbour a population of MSCs, and show how these cells might differ from those obtained from healthy donors.

Bone marrow aspirate was taken from the vertebral bodies of osteoporotic and healthy donors undergoing spinal surgery. MSCs were isolated and expanded. All isolated cells were plastic adherent, proliferating, and showed a typical phenotype of MSCs. FACS analysis showed typical stem cell-like expression patterns of CD11b (neg), CD45 (neg), CD73 (pos) and CD106 (pos). MSCs were able to effectively suppress the proliferation of activated lymphocytes, and no differences were found between the groups. MSCs were successfully differentiated toward osteogenic, adipogenic and chondrogenic lineages as assessed by corresponding stainings and RT-PCR. Healthy and osteoporotic donors showed differences on the mRNA level for osteogenic differentiation markers. Interestingly, differential expression of regulatory receptors such as the endocannabinoid receptor 2 (CB2R) could be detected.

In summary, we were able to isolate, expand and characterize MSCs from vertebral bodies with osteoporotic compression fractures. These MSCs possess a similar phenotype and have similar characteristics, surface markers and immunomodulatory capacity compared to MSCs of healthy donors. Previous studies showed that the CB2R is a key player in the regulation of skeletal remodelling. As our results demonstrated clear differences in the expression of CB2R, this could explain the divergent differentiation potential of MSCs isolated from healthy and diseased donors.