Human neural development - Kirkeby Group

Mapping human neural subtype identities in a microfluidic model of the human neural tube (the MiSTR model)

Through microfluidic engineering techniques, we expose hESCs to morphogenic gradients in vitro, thereby building a microenvironment in which the stem cells are patterned into structures resembling the early stages of the rostral-to-caudal regionalised human neural tube. With this we apply single cell RNA sequencing to identify the unique molecular signatures of different regional human neural subtypes and dissect the trajectories underlying the regionalisation and subregionalisation of the brain during embryo development.

Generating human hypothalamic neurons for studying neuronal control of appetite, metabolism and sleep

Access to functional human hypothalamic neurons is of great interest to pharma, biotech and academia for use in disease modelling, drug screening and cell replacement therapies. However, protocols for generation of subtype-specific hypothalamic neurons are still suboptimal. In this project, we generate protocols for subtype-specific differentiation of hESCs into different subregions of the hypothalamus for molecular studies. We focus in particular on neuronal subtypes which are important for the regulation of appetite, metabolism and sleep, and we investigate the use of HCRT neurons as a possible transplantation therapy for narcolepsy.

Generating human basal forebrain cholinergic neurons for treatment of dementia

Neurodegenerative disorders such as Parkinson’s Disease and Lewy body disease are associated with the loss of basal forebrain cholinergic neurons, which can lead to memory loss and dementia. In this project, we develop protocols for generating cultures of human basal forebrain cholinergic neurons from stem cells. Through animal models, we assess the potential of these cells to be used as a transplantation cell therapy for treatment of cognitive deficits. This project is partly funded by the EU H2020 consortium NSC-Reconstruct. 

Developing in vitro stem cell models of Parkinson’s Disease and Alzheimer’s disease

Recent studies in the field of neurodegenerative diseases has put increased focus on the role of neuroinflammation and glial in the pathogenesis of disease progression. Through several projects, we develop models of neuropathology including neuroinflammation in PD and AD through the use of complex in vitro models containing human neurons, astrocytes and microglia. The aim of these models is to perform high-throughput drug screening to identify novel diease-modifying targets. Part of this work is funded as a Michael J Fox Foundation/ASAP Research consortium

Studying schizophrenia pathology in the dish

Through a collaborative research project with Prof. Kasper Lage and Prof. Thomas Werge, we use stem cell models of human inhibitory interneurons and cortical excitatory neurons to study the neuronal pathology underlying schizophrenia in monogenic and polygenic forms of the disease. This project is funded by the Lundbeck Foundation.

STEM-PD: A clinical trial of developing a stem cell therapy for treatment of Parkinson’s Disease

Through our work at Lund University, we have in a longstanding collaboration together with the lab of Malin Parmar developed a cell transplantation therapy to replace the dopamine neurons which are lost in the brains of Parkinson’s Disease patients. This work has resulted in a clinical stem cell product (STEM-PD), which has been approved by the regulatory authorities, and which was the first pluripotent stem cell product to enter clinical trial for brain transplantation in the EU. Read more here: https://stem-pd.org/. 

Full list of publications by Agnete Kirkeby

 
Selected publications

• Dubonyte U, Asenjo-Martinez A, Werge T, Lage K, Kirkeby A. Current advancements of modelling schizophrenia using patient‑derived induced pluripotent stem cells. Acta Neuropathologica Communications, Dec 2022. doi: 10.1186/s40478-022-01460-2  

• Rifes P, Isaksson M, Rathore GS, Aldrin—Kirk P, Møller OM, Barzaghi G, Lee J, Egerod KL, Rausch DM, Parmar M, Pers TH, Laurell T and Kirkeby A. Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient. Nature Biotechnology, May 2020, DOI: 10.1038/s41587-020-0525-0
• Nolbrant, S., Heuer, A., Parmar, M., and Kirkeby, A. (2017). Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nature Protocols 12, 1962-1979, doi:10.1038/nprot.2017.078.
• Kirkeby, A., Nolbrant, S., Tiklova, K., Heuer, A., Kee, N., Cardoso, T., Ottosson, D.R., Lelos, M.J., Rifes, P., Dunnett, S.B., Grealish, S., Perlmann, T., and Parmar, M. (2017). Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease. Cell Stem Cell 20, 135-148, doi:10.1016/j.stem.2016.09.004.
• Kirkeby, A., Parmar, M., and Barker, R.A. (2017). Strategies for bringing stem cell-derived dopamine neurons to the clinic: A European approach (STEM-PD). Progress in Brain Research, book series: Functional Neural Transplantation – IV, Elsevier, doi: 10.1016/bs.pbr.2016.11.011.

• STEM-PD consortium and Novo Nordisk A/S: Conducting a clinical trial with a pluripotent stem cell product for treatment of Parkinson’s Disease (stem-pd.org)
• Malin Parmar (Lund University, SE): Improving dopamine cell products for treatment of Parkinson’s Disease
• Kasper Lage (Broad Institute, USA) and Thomas Werge (Copenhagen, DK): Investigating key protein networks in schizophrenia
• Barbara Treutlein (ETH Zurich, Basel, CH): Single cell mapping of a microfluidic morphogenic model of the human neural tube.
• Johan Jakobsson (Lund University, SE) and Roger Barker (Cambridge University, UK): Studying the role of transposable elements in Parkinson’s Disease (ASAP consortium)
• Tune Pers (University of Copenhagen, DK): Studying human hypothalamic neurons from stem cells through single cell bioinformatics
• Bioneer (Copenhagen): Improving neuron-glial co-culture systems for modelling neurodegenerative Disease
• NSCReconstruct EU consortium: Developing second-generation cell products for neurodegenerative diseases

• Novo Nordisk Foundation, Hallas Møller Emerging Investigator fellowship
• Independent Research Fund Denmark, Sapere Aude Research Leader fellowship
• EU Horizon 2020 Research Consortium NSC-Reconstruct
• Michael J Fox Foundation/ASAP, Research consortium
• Lundbeck Foundation, Collaborative project & Postdoc Fellowship
• Bioneer, DigitSTEM project
• Knut and Alice Wallenberg Foundation, WCMM Fellowship

• Human pluripotent stem cells: Culturing and neural differentiation of human pluripotent stem cells to subtype-specific regionalised neural cells.
• 2D and 3D stem cell models: Generating mixed neuronal and glial 2D and 3D models for studying pathology of neurodegenerative diseases
• Microfluidic cell culturing: Development and use of microfluidic culturing devices to control regionalisation of human stem cells during differentiation
• Gene editing: Generation of CRISPR/Cas9 reporter cell lines, knockout lines and lentiviral constructs for transgene exression
• Bioinformatics: High-throughput qRT-PCR analysis and Single cell RNAsequencing using the 10X Chromium platform
• In vivo xenotransplantation: Rat models for stereotactic xeno-transplantation of human neural cells
• Imaging: Confocal fluorescence microscopy, live imaging and immunohistochemistry