Abstract
The dopaminergic system has key roles in human physiology and is implicated in a broad range of neurological and neuropsychiatric conditions that are increasingly investigated using induced pluripotent stem cell-derived midbrain models. To determine similarities of such models to human systems, here we undertake single-cell and spatial profiling of first and second trimester fetal midbrain and compare it to in vitro midbrain models. Histological examination reveals that, by the second trimester, fetal midbrain tissue exhibits structural complexity comparable to that of adults. At the molecular level, single-cell profiling uncovers differences in cellular composition across models, with brain organoids most closely resembling late first trimester tissue — an observation supported by meta-integration of existing midbrain datasets. By reconstructing developmental trajectories of neuronal and astrocytic lineages, we map gene expression dynamics associated with maturation. Importantly, integration of spatial transcriptomics provides critical context for aligning organoid models, revealing that their spatial organization and intercellular signaling resemble the architecture and microenvironment of the second trimester midbrain. Ultimately, we leverage our findings to study Dopamine Transporter Deficiency Syndrome progression in patient-derived midbrain organoids, validating their relevance. Understanding the extent of human tissue recapitulation in midbrain laboratory models is essential to justify their use as biological proxies.
Data availability
Raw FASTQ files for both single-cell and spatial transcriptomics generated in this study have been deposited in Gene Expression Omnibus (GEO) under accession code GSE277032, accessible at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE277032. The four supplemental data files generated in this study are available in a Zenodo repository (https://zenodo.org/records/13765496), detailed as follows: Files S1, S2: VCF files with donor genotypes to deconvolute pooled scRNA-seq data. File S3: Single-cell data generated in our study (RDS object: Seurat object with counts and metadata). File S4: Integrated datasets (RDS object: Seurat object with counts and metadata). Additionally, the images from the immunohistochemistry analysis on midbrain tissues are available in the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) Atlas (https://hdbratlas.org/gene-expression/midbrain.html). Source data for graphs and tables are included with the manuscript. Source data are provided with this paper.
Code availability
Two GitHub repositories are available for reproducing the analysis and figures of the manuscript: one for single-cell transcriptomics (https://github.com/paupuigdevall/MLOscRNAseq, archived at https://doi.org/10.5281/zenodo.17541380) and another for spatial transcriptomics (https://github.com/george-hall-ucl/mlo_spatial_transcriptomics, archived at https://doi.org/10.5281/zenodo.17550243). These repositories include scripts for processing steps, data analysis and figure generation. Additionally, the Docker images used for spatial transcriptomics are available on Dockerhub (main pipeline: georgehallucl/midbrain_tissues_organoids_docker; commot: georgehallucl/commot_docker; cell2location: georgehallucl/cell2location_docker). An archive to reproduce the spatial transcriptomics analysis is available at https://doi.org/10.5522/04/27044423.
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Acknowledgements
We sincerely thank our patients and their families for participating in this study. D.B. was supported by the Sir Jules Thorn Trust. P.P.C. and H.K. are currently funded by the Sigrid Jusélius Foundation and previously by the NIHR Great Ormond Street Hospital Biomedical Research Centre. G.T.H. is funded by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.J. was supported by a postdoctoral fellowship from OpenTargets. F.P. is supported by the UK Medical Research Council (MRC) [UKRI222 – PI S.B.]. M.A.K. is supported by the NIHR Professorship, the Sir Jules Thorn Award for Biomedical Research and the Rosetrees Trust. S.B. was supported by the Great Ormond Street Hospital Children’s Charity. We thank UCL Genomics (UCL GOS Institute of Child Health) and the Wellcome Sanger Institute for undertaking scRNA and spatial sequencing. We thank the Human Developmental Biology Resource (HDBR) for providing human midbrain fetal tissue. We thank the UCL Imaging Facility and the facility manager, Dr Dale Moulding, for the support with immunofluorescence analysis. This research was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Graphical schemes have been created in BioRender. https://BioRender.com/.
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D.B., S.B., and M.A.K. conceived and designed the study. S.B. designed and performed experiments and data analysis for the in vitro and in vivo study. D.B. designed and performed experiments and data analysis for the in vitro and in vivo study. P.P.C. designed and performed data analysis for the scRNA-seq study. G.T.H. designed and performed data analysis for the spatial transcriptomic study. C.R. performed experiments for the in vitro study. T.X. designed and performed experiments for the scRNA-seq and spatial transcriptomic studies. E.M. performed experiments for the spatial transcriptomics study. J.J. performed experiments for the scRNA-seq study. A.D.D. performed experiments for the in vitro study. F.P. performed experiments for the in vivo study. H.K. supervised the scRNA-seq study. S.C. supervised scRNA-seq and spatial transcriptomic studies. D.B., P.P.C., G.T.H., S.C., S.B., and M.A.K. drafted the manuscript. C.R. and T.X. contributed to the written sections of the manuscript. All authors reviewed the manuscript prior to submission.
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M.A.K. is a founder of and consultant to Bloomsbury Genetic Therapies. She has received honoraria from PTC for sponsored symposia and provided consultancy. The remaining authors declare no competing interests.
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Budinger, D., Puigdevall, P., Hall, G.T. et al. An in vivo and in vitro spatiotemporal profile of human midbrain development. Nat Commun (2026). https://doi.org/10.1038/s41467-025-67779-1
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DOI: https://doi.org/10.1038/s41467-025-67779-1
