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Microglia integration into human midbrain organoids leads to increased neuronal maturation and functionality

Sonia Sabate-Soler, Sarah Louise Nickels, Cláudia Saraiva, Emanuel Berger, Ugne Dubonyte, Kyriaki Barmpa, Yan Jun Lan, Tsukasa Kouno, Javier Jarazo, Graham Robertson, Jafar Sharif, Haruhiko Koseki, Christian Thome, Jay W. Shin, Sally A. Cowley, Jens C. Schwamborn

Preprint posted on January 22, 2022 https://www.biorxiv.org/content/10.1101/2022.01.21.477192v1

Article now published in Glia at http://dx.doi.org/10.1002/glia.24167

Ever wondered how we can model the human brain in a dish? Organoids and assembloids mimic the structure and complexity of the human brain!

Selected by Emma Wilson

Categories: cell biology, neuroscience

Introduction 

The human brain is a highly complex organ in terms of structure molecular and cellular composition, making neurological disorders difficult to model. The recent emergence of 3D cellular cultures called organoids has been revolutionary in mimicking the spatial and functional complexity of the human brain, and a variety of organoid differentiation protocols have been developed to try and model the diverse range of neurological disorders1,2. Interestingly, many labs have been focusing on generating midbrain organoids. Currently, midbrain organoids have been generated in both patient and non-patient cells and have been able to recapitulate cardinal features of Parkinson’s disease (PD)3,4. These midbrain organoids have key hallmarks of PD including loss of dopaminergic neurons and protein aggregation, highlighting these as a potential model.

One of the major disadvantages of the current midbrain organoids differentiation protocols is the lack microglia. Microglia are the largest population of immune cells in the brain. They are tissue-resident macrophages and represent 5-15% of all adult brain cells. Microglia function to establish contacts with neural progenitors to support neurogenesis during development and interact with neurons, astrocytes, and oligodendrocytes to help brain homeostasis and immune defence5. Organoids lacking microglia are not able to model neurological disorders appropriately as neuroinflammation is a major feature of many disorders including Parkinson’s Disease (PD)6.

In this preprint, the authors describe a protocol to generate assembloids; organoids which have incorporated microglia to allow a truer model of PD and other neurological disorders.

Generating Assembloids

The authors of this preprint combined protocols for microglia differentiation and midbrain organoid differentiation. First, they tested midbrain organoid differentiation media, microglia media and a combination of microglia media with essential midbrain organoid differentiation factors (co-culture medium)(Figure 1a). They found that the co-culture medium showed no differences in the viability of the microglia compared to the microglia media alone, therefore suggesting that they can be co-cultured with midbrain organoids without loss of microglia viability or changes in midbrain maturation.

During the co-culture protocol the macroglia precursors first aggregate to form smaller, round colonies that attached to the surface of the organoid. The microglia are then incorporated within the structure.  After 20 days of co-culture, the incorporation of microglia into the midbrain organoid was assessed via immunofluorescence of IBA1, a reliable microglia marker. The authors found 6.4% of cells were IBA1 positives, which is within the normal physiological range for this cell type. Neuronal populations within the assembloid were confirmed by pan-neuronal markers MAP2 and TUJ1 and midbrain identity was confirmed via FOX2A, a dopaminergic precursor marker and TH, a dopaminergic neurons marker (Figure 1b). Interestingly, after 70 days of culture the presence of GFAP, an astrocytic marker, was found in the population. This suggests that microglia have integrated into the midbrain organoid and all expected cell types are present. This was further confirmed using single cell RNA (scRNA) sequencing. scRNA sequencing showed a range of cell populations (8 in total) in the assembloid including mature dopaminergic, GABAergic neurones as well as microglia (Figure 1c). However, the authors found only 1% of cells were microglia, which was lower than the expected 6% as shown previously via immunofluorescence. They authors reasoned this was due to the loss of cells in the extraction of RNA for RNA sequencing. The 1% of microglia that were present did have a distinct RNA expression pattern, different from the neuronal or precursor populations and the top 100 microglia markers were present, giving confidence that these were true microglia.

Next, they examined the function of the microglia. Activated microglia at the site of inflammation change their morphology and become phagocytic. They release inflammatory cytokines that amplify the inflammatory response by activating and recruiting other cells to the brain lesion7. The media in which the assembloids were growing contained excreted cytokines and chemokines, suggesting that the microglia were functional. This data suggests that midbrain organoids can reliability incorporate microglia and are functional in assembloids.

Figure 1: a) Schematic diagram showing the process of generating assembloids and testing the various medias. B) immunofluorescent images of assembloids showing nucleoid (blue) microglia marker IBA1 (green) and FOXA2 or TH (red) OR MAP2 and TUJ1 (white). C) single cell RNA sequencing data showing the different cell types present in midbrain organoids and assembloids as shows as a percentage.

What is the effect of microglia on midbrain organoids?

After generating a reliable model for assembloids the authors then assessed what the impact of the microglia were on the neural cells within the assembloids. Firstly, the assembloids were shown to express phagocytic genes, indicating that they are functional. The assembloids also appeared smaller with a significantly reduced amount of dead cells compared to organoids, implying that the microglia are removing dead cells. Next, the authors examined differentially expressed genes in assembloids compared to midbrain organoids. They found 12 differentially expressed pathways that included changes to oxidative stress genes, immunoresponse genes, and neurogenesis and axonal guidance genes. Of particular interest were differentially expressed genes involved with synaptic vesicle exocytosis, synaptic contact or synaptogenesis. General synaptic markers such as synaptotagmin and synaptophysin were down regulated across all the cell types in the assembloids, as were dopaminergic neuronal circuit formation genes ROBO1 and DCC. This suggests that axonal remodelling is influenced by the presence of microglia. To examine this further the authors used patch clamping to assess the functional impact of these alterations in gene and protein expression. They found that neurons in midbrain organoids and assembloids exhibit similar

  • Resting membrane potentials and input resistance
  • Reliable fired repetitive action potentials
  • Amplitude of these currents were not different between both groups.

The voltage threshold for action potential generation, however, was more negative in the group of assembloids neurons, which is common and a strong indicator of increased neuronal excitability as seen in mature neurons. They followed this up using a multi electrode array platform (MEA) which can recode action potential across a population of cells. The MEA data showed a lower inter spike interval, meaning the distance between action potentials is smaller and supporting what was observed in the patch clamp experiments.

In summary, neurons in assembloids develop fully mature electrophysiological properties with a lower threshold for action potential generation than in midbrain organoids.

Why I chose this preprint

As a researcher into neurodegenerative diseases, I found it highly intriguing that we may be able to model Parkinson’s disease and other neurodegenerative disease in a 3D cell culture model. This will allow us researchers to test new drugs and treatments in vitro in a human model and only send those which are most promising to human trial. This would hopefully reduce the number of mice used in research as well increase the amount of drug targets which transfer to patients. This is another great tool in our scientific tool kit. The incorporation of other cell types also opens interesting fields or research about combining different organoids.

Questions

  1. Why did you use nuclei rather than whole cells for your single cell RNAseq? Was there an experimental issue with making them single cells? scRNA also showed less microglia than expected? Why do you think this was?
  2. After 70 days in culture you got astrocytes, was this expected and what does that mean?
  3. Why do you think having microglia reduces the formation of synapses or alters synapse formation?
  4. You showed that six of the strongly downregulated genes were involved with oxidative stress? Do you have a theory as to why these were downregulated?

References

  1. Choi, S. H. et al. A three-dimensional human neural cell culture model of Alzheimer’s disease. Nature 515, (2014).
  2. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, (2013).
  3. Smits, L. M. et al. Modeling Parkinson’s disease in midbrain-like organoids. npj Park. Dis. 5, (2019).
  4. Kim, H. et al. Modeling G2019S-LRRK2 Sporadic Parkinson’s Disease in 3D Midbrain Organoids. Stem Cell Reports 12, (2019).
  5. Thion, M. S., Ginhoux, F. & Garel, S. Microglia and early brain development: An intimate journey. Science 362, (2018).
  6. Kouli, A., Camacho, M., Allinson, K. & Williams-Gray, C. H. Neuroinflammation and protein pathology in Parkinson’s disease dementia. Acta Neuropathol. Commun. 8, (2020).
  7. Kim, Y. S. & Joh, T. H. Microglia, major player in the brain inflammation: Their roles in the pathogenesis of Parkinson’s disease. Experimental and Molecular Medicine 38, (2006).

Tags: cellular models, ipscs, microglia, organoids, parkinson's disease

Posted on: 8th March 2022

doi: https://doi.org/10.1242/prelights.31489

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Author's response

Sonia Sabate-Soler shared

Why did you use nuclei rather than whole cells for your single cell RNAseq? Was there an experimental issue with making them single cells? scRNA also showed less microglia than expected? Why do you think this was?

We used single nuclei instead of single cell RNAseq for different reasons. In past analysis, we experienced background noise that made the clustering and pathway identification difficult. In addition, past experiences with single cell sequencing led to a substantial loss of neurons, maybe because single cells tend to be more sensitive to technical manipulation than nuclei. Furthermore, single cell RNAseq requires fresh material, which represents a technical limitation, since obtaining a fresh single cell suspension could lead to sample loss during preparation and transport. Single nuclei extraction can be done from frozen material, and single nuclei can be frozen for sequencing. Overall, we believed that snRNA-Seq would lead to less technical risks and a reliable data outcome.

Concerning the proportion of microglia in the system, we believe there was a sample loss during the nuclei purification that altered the microglia proportion. We demonstrated with immunofluorescence analysis (done in multiple batches and with three cell lines) that the percentage of microglia in the system is around 6.4%. In order to proceed to sectioning and immunofluorescence staining, we fix the organoids quickly after culture, which avoids sample degradation and loss. However, the nuclei extraction was performed from frozen material that had to be thawed and processed, which we assume that implies a higher risk of sample loss.

After 70 days in culture you got astrocytes, was this expected and what does that mean?

The presence of astrocytes in later time points is expected in midbrain organoids (Monzel et al., 2017). Since during development, astrocytes differentiate after neurogenesis, it makes sense that in our midbrain organoid model we see the same pattern. We speculate that the small molecules in the medium, together with signalling from neurons, could promote astrocyte differentiation in the system.

Why do you think having microglia reduces the formation of synapses or alters synapse formation?

Microglia participate in a process called synaptic pruning in the brain. They phagocytose excessive synapse during human childhood and early adulthood, which leads to a strong synapse network with highly functional synapses. We believe the lower gene expression of synaptic genes and protein levels of VAMP2, together with higher expression of action-potential-related genes and enhanced electrophysiological properties indicate that microglia could be performing synapse pruning in organoids. This happens through recognition of molecules – such as CX3CL1 – by receptors in microglia in astrocytes – like CX3CR1. This could be the ongoing mechanism in assembloids, since the ligand was detected in assembloid supernatants and in the sequencing data.

You showed that six of the strongly downregulated genes were involved with oxidative stress? Do you have a theory as to why these were downregulated?

Microglia in the brain phagocytose cell debris and metabolic waste products in order to maintain homeostasis in the brain. We believe apoptotic and necrotic cells in the system may be producing factors that increase oxidative stress in organoids. Metabolic pathways can also produce ROS and factors that increase the overall oxidative stress levels in the system. We speculate that functional microglia in assembloids could phagocytose cell debris and metabolic waste products, reducing oxidative stress in the cells and therefore the expression of stress-related genes.

 

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