Human Neural Development group
The Kirkeby group studies the factors involved in human neural subtype specification in order to enable production of specific neurons for understanding and treating neurological diseases.
The human brain is a highly complex structure, consisting of hundreds of different subtypes of neural cells; each of which fulfil a specific function in the brain network. However, experimental evidence regarding the development of the human brain is highly restricted due to the limited availability of fetal brain tissue – instead, smaller model organisms have classically been applied for neurodevelopmental studies. In the Kirkeby lab, we apply advanced human stem cells models to understand how hundreds of human neuronal subtypes are formed during embryo development. This knowledge enables us with new tools to produce and study human neurons in the lab, for disease modelling, drug screening and transplantation therapies towards brain diseases.
For more information, see below for publications and ongoing projects in the lab. Further information can also be found here:
Creating a human fetal brain in the dish with microfluidics (the MiSTR model)
This project is an interdisciplinary collaboration joining forces from the fields of bioengineering and stem cell biology with the aim of producing a novel model of early human neural tube patterning. 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 technique, we produce an anatomically relevant 3D in vitro model of the developing human neural tube corresponding to around weeks 2-10 of fetal development – read more here.
Mapping human neural subtype identities through single cell RNAseq
Using the MiSTR model described above, we apply single cell RNA sequencing to map the complexity of human neural subtypes present during early and late neural specification. By mapping cells at a single cell level at several different time points, we identify the unique molecular signatures of different human neural subtypes and aim to dissect the trajectories underlying the regionalisation and subregionalisation of human neural progenitors and neurons during embryo development.
Identifying secreted factors from different human brain regions through proteomics
For this project, we use our MISTR model to search for human-specific patterns of developmental growth factors through unbiased mass-spec (MS) proteomic analysis of the secreted proteins produced from different neural regions during development. Novel candidates identified from this approach are explored for their biological function in directing neural cells towards specific neuronal subtypes and for inducing neuronal subtype maturation.
Studying the functions of long non-coding RNAs in human neural cells
Long non-coding RNAs (lncRNAs) are highly abundant in the human genome, and for the vast majority of these their functions are unknown. Here, we apply CRISPR gene editing techniques in human pluripotent cells (CRISPR knockout, CRISPR activation and CRISPR inhibition) to investigate the functions of novel lncRNAs during human neural specification and differentiation. We aim to uncover both general functions in neural differentiation as well as more specific functions in regional subtype specification.
Generating human hypothalamic neurons for studying neuronal control of appetite and metabolism
The hypothalamus is important for regulating appetite, metabolism and sleep, and hypothalamic neuronal dysfunction is coupled to obesity, type 2 diabetes and narcolepsy. As such, 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, partly due to a lack of knowledge on the developmental trajectories giving rise to each specific neuronal subtype of the hypothalamus. In this project, we generate protocols for subtype-specific differentiation of hESCs into different subregions of the hypothalamus, and we investigate the neuronal lineages through single cell RNA sequencing. We focus in particular on hypothalamic neuronal subtypes which are important for the regulation of appetite, metabolism and sleep
Generating human hypocretin (HCRT) neurons for treatment of narcolepsy
Narcolepsy is a chronic, neurological disease affecting approximately 20-50 out of 100,000 individuals. The disease onset is typically during adolescence or early adulthood, and symptoms include excessive daytime sleepiness, cataplexy, hypnagogic hallucinations and sleep paralysis. The most common form of the disease, Type 1 narcolepsy, is caused by the selective loss of HCRT neurons in the hypothalamus of the patients. In this project, we generate protocols for production of HCRT neurons from hESCs, and we investigate in animal models the potential of these neurons to be used as a transplantation therapy for narcolepsy, by regenerating the HCRT sleep circuit
Generating human basal forebrain cholinergic neurons for treatment of dementia
Neurodegenerative disorders such as Alzheimer’s Disease, 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 and 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 part of the EU H2020 consortium NSC-Reconstruct.
Studying neuroinflammation in in vitro stem cell models of Parkinson’s Disease
Recent studies in the field of Parkinson’s Disease has put increased focus on the role of neuroinflammation in the pathogenesis of the disease. In this project, we study the factors involved in neuroinflammation in Parkinson’s Disease through the use of complex in vitro models containing human neurons, astrocytes and microglia. This work is funded as a Michael J Fox Foundation/ASAP Research consortium
STEM-PD: developing a stem cell therapy for treatment of Parkinson’s Disease
In our lab at Lund University, together with the lab of Malin Parmar, we work in a long-standing collaboration to develop a cell therapy for treatment of Parkinson’s Disease. In this project, we produce human dopaminergic progenitor cells from human embryonic stem cells for transplantation therapy to replace the dopamine neurons which are lost in the brains of Parkinson’s Disease patients. This work has resulted in the development of a clinical stem cell product (STEM-PD), which is anticipated to go into clinical trial in Parkinson’s Disease patients in late 2021/early 2022.
The projects above have received funding from the following sources:
- 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
- Knut and Alice Wallenberg Foundation, WCMM Fellowship
- 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). . 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). . Cell Stem Cell 20, 135-148, doi:10.1016/j.stem.2016.09.004.
- Kee, N., Volakakis, N., Kirkeby, A., Dahl, L., Storvall, H., Nolbrant, S., Lahti, L., Björklund, Å.K., Gillberg, L., Joodmardi, E., Sandberg, R., Parmar, M., and Perlmann, T. (2017). . Cell Stem Cell 20, 29-40, doi:10.1016/j.stem.2016.10.003.
- 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). , doi: 10.1016/bs.pbr.2016.11.011.
- Grealish, S., Diguet, E., Kirkeby, A., Mattsson, B., Heuer, A., Bramoulle, Y., Van Camp, N., Perrier, A.L., Hantraye, P., Björklund, A., and Parmar, M. (2014). . Cell Stem Cell 15, 653-665, doi:10.1016/j.stem.2014.09.017.
- Kirkeby, A., Grealish, S., Wolf, D.A., Nelander, J., Wood, J., Lundblad, M., Lindvall, O., and Parmar, M. (2012). . Cell Reports 1, 703-714, doi:10.1016/j.celrep.2012.04.009
- . Proceedings of the National Academy of Sciences 108, 10343-10348, doi:10.1073/pnas.1105135108. *contributed equally
Google Scholar https://scholar.google.com/citations?hl=en&user=XTPTxw4AAAAJ
Agnete Kirkeby has founded her research on applying human pluripotent stem cells to generate subtype-specific neural cells for developmental studies and regenerative therapy. During her time at Lund University, Agnete has been heavily involved in developing protocols for producing dopaminergic progenitor cells as a cell therapy for Parkinson’s Disease patients. This work has led to the development of a stem cell product which is currently in preparation for clinical trial. Moreover, Agnete has focused on producing novel tools for studying human neural development through modelling of neural tube patterning with microfluidic morphogenic gradients. The main focus of Agnete Kirkeby’s group is to use these 3D in vitro models of human brain development to map and understand human neural subtype specification and maturation.
2019 - : Group leader, Associate Prof., Department of Neuroscience, University of Copenhagen
2019 - : Group leader, WCMM fellow, Wallenberg Center for Regenerative Medicine, Lund University, Sweden
2017-19: Group leader, Associate Prof., Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, University of Copenhagen
2015-2019 : Group leader, Research Scientist, Medical Faculty, Lund University, Sweden
2009-2015: Postdoc/Research scientist, Lund University, Dept. Developmental and Regenerative Neurobiology. (Malin Parmar group), Lund University, Sweden
2006-2009: Ph.D in Neurobiology, Performed at Sloan Kettering Institute, New York and H. Lundbeck A/S. Title: Studying the effects of low oxygen on stem cells. PhD Institution: Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Supervisors: Prof. Jan Sap and Dr. Lorenz Studer. Doctoral degree obtained Sept. 2009.
2003-2006: MSc in Human Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Masters project performed at H. Lundbeck A/S.
- Human pluripotent stem cells: Culturing and neural differentiation of human pluripotent stem cells to subtype-specific neural cells.
- 3D stem cell models: Applying 3D models for neural differentiation of human stem cells into regionalised neural subtypes.
- Microfluidic cell culturing: Development and use of microfluidic culturing devices to control regionalisation of human stem cells during differentiation
- Proteomics: Shotgun proteomics (LC-MS/MS) for identifying proteins in cells and cell culture medium from human stem cells.
- Molecular biology: Generation of CRISPR/Cas9 reporter cell lines, generation of lentiviral constructs, high-throughput qRT-PCR analysis
- Bioinformatics: Single cell RNAsequencing using the 10X Chromium platform and lineage trajectory analysis by bioinformatics
- In vivo transplantation: Rat models for stereotaxic xeno-transplantation of human neural cells
- Imaging: Confocal fluorescence microscopy and immunohistochemistry
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
Tiklova K, Nolbrant S, Björklund Å, Fiorenzano A, Heuer A, Gillberg L, Hoban D, Cardoso T, Adler A, Lundén-Miguel H, Volakakis N, Kirkeby A, Perlmann T, Sharma Y, Birtele M and Parmar M. Single Cell Transcriptomics Identifies Stem Cell-Derived Graft Composition in a Model of Parkinson’s Disease. Nature Communications, May 2020, https://doi.org/10.1038/s41467-020-16225-5
Korshunova I, Rhein S, García-González D, Stölting I, Pfisterer U, Barta A, Dmytriyeva O, Kirkeby A, Schwaninger M and Khodosevich K. Genetic modification increases the survival and the neuroregenerative properties of transplanted neural stem cells. JCI Insight, Jan 2020, doi: 10.1172/jci.insight.126268,
Kirkeby, A., Barker, R.A. (2019). Parkinson disease and growth factors - is GDNF good enough?. Nature Reviews Neurology, doi: 10.1038/s41582-019-0180-6.
Cardoso, T., Adler, A.F., Mattsson, B., Hoban, D.B., Nolbrant, S., Wahlestedt, J.N., Kirkeby, A., Grealish, S., Bjorklund, A., and Parmar, M. (2018).. Journal of Comparative Neurology 526, 2133-2146,doi: 10.1002/cne.24500
Porrello, E.R. and Kirkeby, A. (2018).. Development 145, dev163501,doi: 10.1242/dev.163501
Abbot, S., Agbanyo, F., Ahlfors, J.E., Baghbaderani, B.A., Bartido, S., Bharti, K., Burke, C., Carlsson, B., Cavagnaro, J., Creasey, A., DiGiusto, D., Francissen, K., Gaffney, A., Goldring, C., Gorba, T., Griffiths, E., Hanatani, T., Hayakawa, T., Heki, T., Hoogendoorn, K., Kawamata, S., Kimura, H., Kirkeby, A., Knezevic, I., Lebkowski, J., Lin, S., Lin-Gibson, S., Lubiniecki, A., O'Shea, O., Pera, M., Petricciani, J., Pigeau, G., Ratcliffe, A., Sato, Y., Schumann, G.G., Shingleton, W., Stacey Chair, G., Sullivan, S., Svendsen, C.N., Trouvin, J.H., Vandeputte, J., Yuan, B.Z., and Zoon, K. (2018). Report of the international conference on manufacturing and testing of pluripotent stem cells. Biologicals, doi:10.1016/j.biologicals.2018.08.004.
Mondal, T., Juvvuna, P.K., Kirkeby, A., Mitra, S., Kosalai, S.T., Traxler, L., Hertwig, F., Wernig-Zorc, S., Miranda, C., Deland, L., Volland, R., Bartenhagen, C., Bartsch, D., Bandaru, S., Engesser, A., Subhash, S., Martinsson, T., Caren, H., Akyurek, L.M., Kurian, L., Kanduri, M., Huarte, M., Kogner, P., Fischer, M., and Kanduri, C. (2018).Cancer Cell 33, 417-434.e417, doi:10.1016/j.ccell.2018.01.020.
Lehnen D, Barral S, Cardoso T, Grealish S, Heuer A, Smiyakin A, Kirkeby A, Kollet J, Cremer H, Parmar M, Bosio A and Knöbel S. (2017)., Stem Cell Reports 9, 1207-1220 doi:10.1016/j.stemcr.2017.08.016.
Nolbrant, S., Heuer, A., Parmar, M., and Kirkeby, A. (2017).Nature Protocols 12, 1962-1979, doi:10.1038/nprot.2017.078.
Moraghebi, R., Kirkeby, A., Chaves, P., Ronn, R.E., Sitnicka, E., Parmar, M., Larsson, M., Herbst, A., and Woods, N.B. (2017).Stem Cell Research and Therapy 8, 190, doi:10.1186/s13287-017-0582-6.
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).Cell Stem Cell 20, 135-148, doi:10.1016/j.stem.2016.09.004.
Kee, N., Volakakis, N., Kirkeby, A., Dahl, L., Storvall, H., Nolbrant, S., Lahti, L., Bjorklund, A.K., Gillberg, L., Joodmardi, E., Sandberg, R., Parmar, M., and Perlmann, T. (2017).Cell Stem Cell 20, 29-40, doi:10.1016/j.stem.2016.10.003.
Kirkeby, A., Parmar, M., and Barker, R.A. (2017).Progress in Brain Research, book series: Functional Neural Transplantation – IV, Elsevier, doi: 10.1016/bs.pbr.2016.11.011.
Barker, R.A., Parmar, M., Kirkeby, A., Bjorklund, A., Thompson, L., and Brundin, P. (2016).Journal of Parkinson’s Disease 6, 57-63, doi:10.3233/jpd-160798.
Heuer, A., Kirkeby, A., Pfisterer, U., Jonsson, M.E., and Parmar, M. (2016).Experimental Neurology 282, 78-85, doi:10.1016/j.expneurol.2016.05.027.
Kirkeby, A., Perlmann, T., and Pereira, C.F. (2016).Development 143, 2877-2881, doi:10.1242/dev.140095.
Grealish, S., Heuer, A., Cardoso, T., Kirkeby, A., Jonsson, M., Johansson, J., Bjorklund, A., Jakobsson, J., and Parmar, M. (2015).Stem Cell Reports 4, 975-983, doi:10.1016/j.stemcr.2015.04.011.
Jönsson, M.E., Nelander Wahlestedt, J., Åkerblom, M., Kirkeby, A., Malmevik, J., Brattaas, P.L., Jakobsson, J., and Parmar, M. (2015).Development 142, 3166-3177, doi:10.1242/dev.122747.
Grealish, S., Diguet, E., Kirkeby, A., Mattsson, B., Heuer, A., Bramoulle, Y., Van Camp, N., Perrier, A.L., Hantraye, P., Bjorklund, A., and Parmar, M. (2014).Cell Stem Cell 15, 653-665, doi:10.1016/j.stem.2014.09.017.
Kirkeby, A., Nelander, J., and Parmar, M. (2013).. Frontiers in Cellular Neuroscience 6, 64, doi:10.3389/fncel.2012.00064
Kirkeby, A., Grealish, S., Wolf, D.A., Nelander, J., Wood, J., Lundblad, M., Lindvall, O., and Parmar, M. (2012).Cell Reports 1, 703-714, doi:10.1016/j.celrep.2012.04.009.
Kirkeby, Agnete & Malin Parmar (2012)(Review). Translational Neuroscience, 3(4), 314-319, doi: 10.2478/s13380-012-0041-x.
Pfisterer, U.*, Kirkeby, A.*, Torper, O.*, Wood, J., Nelander, J., Dufour, A., Bjorklund, A., Lindvall, O., Jakobsson, J., and Parmar, M. (2011).Proceedings of the National Academy of Sciences 108, 10343-10348, doi:10.1073/pnas.1105135108. *contributed equally
Kirkeby, A., Parmar, M., Jakobsson, J. (2011).Human embryonic and induced pluripotent stem cells – lineage-specific differentiation protocols, Springer Protocols, doi:1007/978-1-61779-267-0_34.
Sachdeva, R., Jonsson, M.E., Nelander, J., Kirkeby, A., Guibentif, C., Gentner, B., Naldini, L., Bjorklund, A., Parmar, M., and Jakobsson, J. (2010).Proceedings of the National Academy of Sciences 107, 11602-11607, doi:10.1073/pnas.1006568107.
Lapchak, P.A., Kirkeby, A., Zivin, J.A., and Sager, T.N. (2008).Brain Research 1238, 208-14, doi:10.1016/j.brainres.2008.08.017.
Kirkeby, A., Torup, L., Bochsen, L., Kjalke, M., Abel, K., Theilgaard-Monch, K., Johansson, P.I., Bjorn, S.E., Gerwien, J., and Leist, M. (2008).Thrombosis and Haemostasis 99, 720-8, doi:10.1160/TH07-03-0208.
Leist, M., Bremer, S., Brundin, P., Hescheler, J., Kirkeby, A., Krause, K.H., Poerzgen, P., Puceat, M., Schmidt, M., Schrattenholz, A., Zak, N.B., and Hentze, H. (2008).Altex 25, 163-190.
Montero, M., Poulsen, F.R., Noraberg, J., Kirkeby, A., van Beek, J., Leist, M., and Zimmer, J. (2007).Experimental Neurology 204, 106-117, doi:10.1016/j.expneurol.2006.09.026.
Kirkeby, A., van Beek, J., Nielsen, J., Leist, M., and Helboe, L. (2007).Journal of Neuroscience Methods 164, 50-58, doi:10.1016/j.jneumeth.2007.03.026.
|Agnete Kirkeby||Associate Professor, Group Leader||Kirkeby Lab|
|Alison Salvador||Master-Student||Kirkeby Lab|
|Amalie Holm||Master-Student||Kirkeby Lab|
|Arun Thiruvalluvan||Postdoc||Kirkeby Lab|
|Gaurav Singh Rathore||PhD Fellow||Kirkeby Lab|
|Jens Bager Christensen||Master-Student||Kirkeby Lab|
|Martina Cardinali||Master-Student||Kirkeby Lab|
|Matias Heide Ankjær||Master-Student||Kirkeby Lab|
|Patrick Anders Aldrin Kirk||Postdoc||Kirkeby Lab|
|Pedro Goncalo Reis Rifes||Assistant Professor||Kirkeby Lab|
|Sarah Kieler Saietz||Master-Student||Kirkeby Lab|
|Tobias Overlund Stannius||Master-Student||Kirkeby Lab|
|Ugne Dubonyte||Research Assistant||Kirkeby Lab|
|Yuan Li||Guest Researcher||Kirkeby Lab|
|Zehra Caldwell Abay-Nørgaard||PhD Student||Kirkeby Lab|