Single cell transcriptomics identifies stem cell-derived graft composition in a model of Parkinson's disease
Katarína Tiklová, Sara Nolbrant, Alessandro Fiorenzano, Åsa K Björklund, Yogita Sharma, Andreas Heuer, Linda Gillberg, Deirdre B Hoban, Tiago Cardoso, Andrew F Adler, Marcella Birtele, Hilda Lundén-Miguel, Nikolaos Volakakis, Agnete Kirkeby, Thomas Perlmann, Malin Parmar, Katarína Tiklová, Sara Nolbrant, Alessandro Fiorenzano, Åsa K Björklund, Yogita Sharma, Andreas Heuer, Linda Gillberg, Deirdre B Hoban, Tiago Cardoso, Andrew F Adler, Marcella Birtele, Hilda Lundén-Miguel, Nikolaos Volakakis, Agnete Kirkeby, Thomas Perlmann, Malin Parmar
Abstract
Cell replacement is a long-standing and realistic goal for the treatment of Parkinson's disease (PD). Cells for transplantation can be obtained from fetal brain tissue or from stem cells. However, after transplantation, dopamine (DA) neurons are seen to be a minor component of grafts, and it has remained difficult to determine the identity of other cell types. Here, we report analysis by single-cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from human embryonic stem cells (hESCs) and fetal tissue after functional maturation in a pre-clinical rat PD model. We show that neurons and astrocytes are major components in both fetal and stem cell-derived grafts. Additionally, we identify a cell type closely resembling a class of recently identified perivascular-like cells in stem cell-derived grafts. Thus, this study uncovers previously unknown cellular diversity in a clinically relevant cell replacement PD model.
Conflict of interest statement
M.P. is the owner of Parmar Cells AB and co-inventor of the U.S. patent application 15/093,927 owned by Biolamina AB and EP17181588 owned by Miltenyi Biotec. All other authors declare no competing interests.
Figures
References
- Cooper O, et al. Differentiation of human ES and Parkinson’s disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid. Mol. Cell Neurosci. 2010;45:258–266. doi: 10.1016/j.mcn.2010.06.017.
- Kriks S, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature. 2011;480:547–551. doi: 10.1038/nature10648.
- Barker RA, Parmar M, Studer L, Takahashi J. Human trials of stem cell-derived dopamine neurons for Parkinson’s disease: dawn of a new era. Stem Cell. 2017;21:569–573.
- Nolbrant S, Heuer A, Parmar M, Kirkeby A. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat. Protoc. 2017;12:1962–1979. doi: 10.1038/nprot.2017.078.
- Barker, R. A. TRANSEURO consortium. Designing stem-cell-based dopamine cell replacement trials for Parkinson’s disease. Nat. Med.25, 1045–1053 (2019).
- Thompson L, Barraud P, Andersson E, Kirik D, Björklund A. Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections. J. Neurosci. 2005;25:6467–6477. doi: 10.1523/JNEUROSCI.1676-05.2005.
- Grealish S, et al. Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson’s disease. Cell Stem Cell. 2014;15:653–665. doi: 10.1016/j.stem.2014.09.017.
- Kirkeby A, et al. Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep. 2012;1:703–714. doi: 10.1016/j.celrep.2012.04.009.
- Kee N, et al. Single-cell analysis reveals a close relationship between differentiating dopamine and subthalamic nucleus neuronal lineages. Cell Stem Cell. 2017;20:29–40. doi: 10.1016/j.stem.2016.10.003.
- Nelander J, Hebsgaard JB, Parmar M. Organization of the human embryonic ventral mesencephalon. Gene Expr. Patterns. 2009;9:555–561. doi: 10.1016/j.gep.2009.10.002.
- Kirkeby A, et al. Predictive markers guide differentiation to improve graft outcome in clinical translation of hESC-based therapy for Parkinson’s disease. Cell Stem Cell. 2017;20:135–148. doi: 10.1016/j.stem.2016.09.004.
- Kikuchi T, et al. Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model. Nature. 2017;548:592–596. doi: 10.1038/nature23664.
- Li J, Tibshirani R. Finding consistent patterns: a nonparametric approach for identifying differential expression in RNA-Seq data. Stat. Methods Med. Res. 2013;22:519–536. doi: 10.1177/0962280211428386.
- Zhang Y, et al. Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse. Neuron. 2016;89:37–53. doi: 10.1016/j.neuron.2015.11.013.
- Zeisel A, et al. Molecular architecture of the mouse nervous system. Cell. 2018;174:999–1014.e22. doi: 10.1016/j.cell.2018.06.021.
- Marques S, et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science. 2016;352:1326–1329. doi: 10.1126/science.aaf6463.
- La Manno G, et al. Molecular diversity of midbrain development in mouse, human, and stem cells. Cell. 2016;167:566–580.e19. doi: 10.1016/j.cell.2016.09.027.
- Doi D, et al. Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation. Stem Cell Rep. 2014;2:337–350. doi: 10.1016/j.stemcr.2014.01.013.
- Kirkeby, A. Generating regionalized neuronal cells from pluripotency, a step-by-step protocol. Front Cell Neurosci. 1–4, 10.3389/fncel.2012.00064/abstract (2012).
- Chen, Y. et al. Chemical control of grafted human PSC-derived neurons in a mouse model of Parkinson’s disease. Stem Cell 1–11, 10.1016/j.stem.2016.03.014 (2016).
- Nguyen QH, Pervolarakis N, Nee K, Kessenbrock K. Experimental considerations for single-cell RNA sequencing approaches. Front Cell Dev. Biol. 2018;6:108. doi: 10.3389/fcell.2018.00108.
- Vanlandewijck M, et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature. 2018;554:475–480. doi: 10.1038/nature25739.
- Broadwell RD, et al. Angiogenesis and the blood-brain barrier in solid and dissociated cell grafts within the CNS. Prog. Brain Res. 1990;82:95–101. doi: 10.1016/S0079-6123(08)62595-9.
- Baker-Cairns BJ, Sloan DJ, Broadwell RD, Puklavec M, Charlton HM. Contributions of donor and host blood vessels in CNS allografts. Exp. Neurol. 1996;142:36–46. doi: 10.1006/exnr.1996.0177.
- Lehnen D, et al. IAP-based cell sorting results in homogeneous transplantable dopaminergic precursor cells derived from human pluripotent stem cells. Stem Cell Rep. 2017;9:1207–1220. doi: 10.1016/j.stemcr.2017.08.016.
- Doi D, et al. Stem cell reports. Stem Cell Rep. 2014;2:337–350. doi: 10.1016/j.stemcr.2014.01.013.
- Stuart T, et al. Comprehensive integration of single-cell data. Cell. 2019;177:1888–1902.e21. doi: 10.1016/j.cell.2019.05.031.
- Thompson L, Björklund A. Survival, differentiation, and connectivity of ventral mesencephalic dopamine neurons following transplantation. Prog. Brain Res. 2012;200:61–95. doi: 10.1016/B978-0-444-59575-1.00004-1.
- Nowakowski TJ, et al. Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex. Science. 2017;358:1318–1323. doi: 10.1126/science.aap8809.
- Freed CR, et al. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N. Engl. J. Med. 2001;344:710–719. doi: 10.1056/NEJM200103083441002.
- Olanow CW, et al. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann. Neurol. 2003;54:403–414. doi: 10.1002/ana.10720.
- Heuer A, Lelos MJ, Kelly CM, Torres EM, Dunnett SB. Dopamine-rich grafts alleviate deficits in contralateral response space induced by extensive dopamine depletion in rats. Exp. Neurol. 2013;247:485–495. doi: 10.1016/j.expneurol.2013.01.020.
- Lancaster MA, et al. Cerebral organoids model human brain development and microcephaly. Nature. 2013;501:373–379. doi: 10.1038/nature12517.
- Zeisel A, et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science. 2015;347:1138–1142. doi: 10.1126/science.aaa1934.
- Picelli, S. et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods10.1038/nmeth.2639 (2013).
- Picelli S, et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 2014;9:171–181. doi: 10.1038/nprot.2014.006.
- Dobin A, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635.
- Ramsköld D, Wang ET, Burge CB, Sandberg R. An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data. PLoS Comput. Biol. 2009;5:e1000598. doi: 10.1371/journal.pcbi.1000598.
- Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 2018;36:411–420. doi: 10.1038/nbt.4096.
- McInnes, L. & Healy, J. UMAP: Uniform manifold approximation and projection for dimension reduction. Preprint at (2018).
- Tirosh I, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016;352:189–196. doi: 10.1126/science.aad0501.
Source: PubMed