Multi-omic analysis of guided and unguided forebrain organoids reveals differences in cellular composition and metabolic profiles

Marie Sejberg Øhlenschlæger; Pia Jensen; Jesper Foged Havelund; Sissel Ida Schmidt; Fadumo Abdullahi Mohamed; Magdalena Sutcliffe; Sofie Blomberg Elmkvist; Lucrezia Criscuolo; Steven W Wingett; Ilaria Chiaradia; Elif Bayram; Jeppe Allen Abildsten Nicolaisen; Lene Andrup Jakobsen; Jonathan Brewer; Michael Eriksen Benros; Kristine Freude; Nils Joakim Færgeman; Madeline A Lancaster; Martin Røssel Larsen; Helle Bogetofte

doi:10.1016/j.crmeth.2025.101295

Multi-omic analysis of guided and unguided forebrain organoids reveals differences in cellular composition and metabolic profiles

Marie Sejberg Øhlenschlæger, Pia Jensen, Jesper Foged Havelund, Sissel Ida Schmidt, Fadumo Abdullahi Mohamed, Magdalena Sutcliffe, Sofie Blomberg Elmkvist, Lucrezia Criscuolo, Steven W Wingett, Ilaria Chiaradia, Elif Bayram, Jeppe Allen Abildsten Nicolaisen, Lene Andrup Jakobsen, Jonathan Brewer, Michael Eriksen Benros, Kristine Freude, Nils Joakim Færgeman, Madeline A Lancaster, Martin Røssel Larsen, Helle Bogetofte^*

^*Corresponding author for this work

Abstract

Neural organoids are invaluable model systems for studying neurodevelopment, generated by either guided or unguided approaches. Despite the importance for the field, the resulting differences between these models are unclear. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated in parallel with two widely applied guided and unguided protocols. The guided forebrain organoids contained a larger proportion of neurons, including GABAergic interneurons, whereas the unguided organoids contained significantly more choroid plexus, radial glia, and astrocytes at later stages. Substantial differences in metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid β-oxidation in the unguided forebrain organoids and a higher reliance on glycolysis in the guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences between these guided and unguided forebrain organoids and provides an important resource for the neural organoid field studying neurodevelopment and disease.

Original language	English
Article number	101295
Journal	Cell reports methods
Volume	6
Issue number	2
ISSN	2667-2375
DOIs	https://doi.org/10.1016/j.crmeth.2025.101295
Publication status	Published - 23 Feb 2026

Keywords

Animals
Metabolome
Mice
Multiomics
Neurons/metabolism
Organoids/metabolism
Prosencephalon/metabolism
cerebral organoids
brain organoids
proteomics
schizophrenia
neural organoids
CP: neuroscience
single-cell transcriptomics
forebrain
post-translational modifications
CP: stem cell

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Øhlenschlæger, M. S., Jensen, P., Havelund, J. F., Schmidt, S. I., Mohamed, F. A., Sutcliffe, M., Elmkvist, S. B., Criscuolo, L., Wingett, S. W., Chiaradia, I., Bayram, E., Nicolaisen, J. A. A., Jakobsen, L. A., Brewer, J., Benros, M. E., Freude, K., Færgeman, N. J., Lancaster, M. A., Larsen, M. R., & Bogetofte, H. (2026). Multi-omic analysis of guided and unguided forebrain organoids reveals differences in cellular composition and metabolic profiles. Cell reports methods, 6(2), Article 101295. https://doi.org/10.1016/j.crmeth.2025.101295

Øhlenschlæger, MS, Jensen, P, Havelund, JF, Schmidt, SI, Mohamed, FA, Sutcliffe, M, Elmkvist, SB, Criscuolo, L, Wingett, SW, Chiaradia, I, Bayram, E, Nicolaisen, JAA, Jakobsen, LA, Brewer, J, Benros, ME, Freude, K, Færgeman, NJ, Lancaster, MA, Larsen, MR & Bogetofte, H 2026, 'Multi-omic analysis of guided and unguided forebrain organoids reveals differences in cellular composition and metabolic profiles', Cell reports methods, vol. 6, no. 2, 101295. https://doi.org/10.1016/j.crmeth.2025.101295

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title = "Multi-omic analysis of guided and unguided forebrain organoids reveals differences in cellular composition and metabolic profiles",

abstract = "Neural organoids are invaluable model systems for studying neurodevelopment, generated by either guided or unguided approaches. Despite the importance for the field, the resulting differences between these models are unclear. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated in parallel with two widely applied guided and unguided protocols. The guided forebrain organoids contained a larger proportion of neurons, including GABAergic interneurons, whereas the unguided organoids contained significantly more choroid plexus, radial glia, and astrocytes at later stages. Substantial differences in metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid β-oxidation in the unguided forebrain organoids and a higher reliance on glycolysis in the guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences between these guided and unguided forebrain organoids and provides an important resource for the neural organoid field studying neurodevelopment and disease.",

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author = "{\O}hlenschl{\ae}ger, \{Marie Sejberg\} and Pia Jensen and Havelund, \{Jesper Foged\} and Schmidt, \{Sissel Ida\} and Mohamed, \{Fadumo Abdullahi\} and Magdalena Sutcliffe and Elmkvist, \{Sofie Blomberg\} and Lucrezia Criscuolo and Wingett, \{Steven W\} and Ilaria Chiaradia and Elif Bayram and Nicolaisen, \{Jeppe Allen Abildsten\} and Jakobsen, \{Lene Andrup\} and Jonathan Brewer and Benros, \{Michael Eriksen\} and Kristine Freude and F{\ae}rgeman, \{Nils Joakim\} and Lancaster, \{Madeline A\} and Larsen, \{Martin R{\o}ssel\} and Helle Bogetofte",

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AU - Øhlenschlæger, Marie Sejberg

AU - Jensen, Pia

AU - Havelund, Jesper Foged

AU - Schmidt, Sissel Ida

AU - Mohamed, Fadumo Abdullahi

AU - Sutcliffe, Magdalena

AU - Elmkvist, Sofie Blomberg

AU - Criscuolo, Lucrezia

AU - Wingett, Steven W

AU - Chiaradia, Ilaria

AU - Bayram, Elif

AU - Nicolaisen, Jeppe Allen Abildsten

AU - Jakobsen, Lene Andrup

AU - Brewer, Jonathan

AU - Benros, Michael Eriksen

AU - Freude, Kristine

AU - Færgeman, Nils Joakim

AU - Lancaster, Madeline A

AU - Larsen, Martin Røssel

AU - Bogetofte, Helle

PY - 2026/2/23

Y1 - 2026/2/23

N2 - Neural organoids are invaluable model systems for studying neurodevelopment, generated by either guided or unguided approaches. Despite the importance for the field, the resulting differences between these models are unclear. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated in parallel with two widely applied guided and unguided protocols. The guided forebrain organoids contained a larger proportion of neurons, including GABAergic interneurons, whereas the unguided organoids contained significantly more choroid plexus, radial glia, and astrocytes at later stages. Substantial differences in metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid β-oxidation in the unguided forebrain organoids and a higher reliance on glycolysis in the guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences between these guided and unguided forebrain organoids and provides an important resource for the neural organoid field studying neurodevelopment and disease.

AB - Neural organoids are invaluable model systems for studying neurodevelopment, generated by either guided or unguided approaches. Despite the importance for the field, the resulting differences between these models are unclear. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated in parallel with two widely applied guided and unguided protocols. The guided forebrain organoids contained a larger proportion of neurons, including GABAergic interneurons, whereas the unguided organoids contained significantly more choroid plexus, radial glia, and astrocytes at later stages. Substantial differences in metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid β-oxidation in the unguided forebrain organoids and a higher reliance on glycolysis in the guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences between these guided and unguided forebrain organoids and provides an important resource for the neural organoid field studying neurodevelopment and disease.

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KW - Metabolome

KW - Mice

KW - Multiomics

KW - Neurons/metabolism

KW - Organoids/metabolism

KW - Prosencephalon/metabolism

KW - cerebral organoids

KW - brain organoids

KW - proteomics

KW - schizophrenia

KW - neural organoids

KW - CP: neuroscience

KW - single-cell transcriptomics

KW - forebrain

KW - post-translational modifications

KW - CP: stem cell

UR - https://www.scopus.com/pages/publications/105029403909

U2 - 10.1016/j.crmeth.2025.101295

DO - 10.1016/j.crmeth.2025.101295

M3 - Journal article

C2 - 41643681

SN - 2667-2375

VL - 6

JO - Cell reports methods

JF - Cell reports methods

IS - 2

M1 - 101295

ER -

Multi-omic analysis of guided and unguided forebrain organoids reveals differences in cellular composition and metabolic profiles

Abstract

Keywords

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