Brain organoids, also known as brain spheroids, are three-dimensional cultures of neural cells derived from pluripotent stem cells (PSCs) that mimic the brain’s organization, development, and activity in a dish (Lancaster and Knoblich, 2014). The technology available to develop brain organoids has advanced considerably in recent years, providing an unprecedented opportunity to examine brain development and disease (Pasca, 2018). However, existing brain organoids still have limitations: most notably, they do not contain vascular cells, immune cells and other non-neural cells.
The neurovascular system is responsible for the delivery of oxygen and nutrients to the brain, the growth and development of neural tissue, and allowing the brain to perform its roles normally (Jin et al., 2002). Thus, a functional vasculature is critical to obtaining brain organoids with an architecture similar to the mature brain, and cells that have differentiated appropriately.
Several strategies have been devised to generate vascularized brain organoids (Figure 1). The simplest and most straightforward method is to co-culture brain organoids with endothelial cells which line blood vessels (Pham et al., 2018). Another approach is to genetically induce blood vessels by expressing a transcription factor that converts PSCs into endothelial cells as the brain organoids develop (Cakir et al., 2019). Yet another way to obtain vascularized brain organoids is to fuse PSC-derived brain organoids with endothelial cell organoids (Song et al., 2019).
Similar strategies have been applied to add microglia, another non-neural cell type, to brain organoids (Abud et al., 2017; Xu et al., 2021; Cakir et al., 2022). Although each method has its limitations, all aim to incorporate a single type of non-neural cells into brain organoids. Now, in eLife, Zhen-Ge Luo from ShanghaiTech University, Xiang-Chun Ju from the Chinese Academy of Sciences, and co-workers – including Xin-Yao Sun as first author – report on the generation of fusion vessel brain organoids (fVBOrs) with both vascular- and microglia-like cells obtained by fusing vessel organoids with brain organoids (Sun et al., 2022).
Sun et al. first focused on generating vessel organoids. To do this, they activated Wnt signaling in embryonic bodies made of human embryonic stem cells (hESCs). The Wnt signaling pathway triggers hESCs to differentiate into mesodermal cells, which give rise to muscles, blood vessels, and connective tissue during development. Once the cells in the embryoid bodies had differentiated into the mesoderm, Sun et al. added vascular endothelial growth factor (VEGF) to further differentiate them into endothelial cells.
At this point, the embryoid bodies were embedded in Matrigel, a culture substrate containing many extracellular matrix components that cells encounter in vivo. Next, Sun et al. added neurotrophic factors – molecules that promote the growth and maturation of neurons – to the embryoid bodies to reproduce the brain trophic environment and facilitate the differentiation of the vessel organoids into brain vessels. As a result, the vessel organoids acquired complex vascular structures.
The next step was to characterize these vessel organoids by performing single-cell RNA sequencing (scRNA-seq). The results showed that the vessel organoids have characteristics unique to the vascular system, including the expression of groups of genes that are match the ones active in endothelial cells, vascular progenitors, fibroblasts, pericytes, and smooth muscle cells. Interestingly, a type of immune cells in the brain called microglia was also detected in these vessel organoids, likely due to the neurotrophic factors in the maturation media.
Once the vessel organoids had been developed and characterized, Sun et al. applied a co-culture approach to develop brain organoids with vascular systems (Figure 1). First, they made brain organoids using unguided protocols of intrinsic neuroectoderm differentiation, where hESCs are cultured together in signal-free media and spontaneously differentiate into neural cells (Lancaster and Knoblich, 2014). In previous studies, a single brain organoid had been fused with a single vessel organoid to achieve vascularization (Song et al., 2019). In the current study, however, each brain organoid was fused with two vessel organoids within Matrigel to generate ffVBOrs by entirely surrounding the neural tissue with the invading vascular structures.
Notably, neural tissue from these fVBOrs also contained functional microglia-like cells, which take part in the maturation of neural networks by engulfing synapses and regulating neuronal activity. Therefore, incorporating vascular and immune cells into the brain organoids might further promote the survival of neuronal progenitors by providing them with the growth factors they need. However, the spontaneous and stochastic differentiation of mesodermal progenitors into different cell types in vessel organoids can lead to high variability in the generation of microglia in fVBOrs, which could become an issue.
Despite the potential limitations, the fVBOrs reported by Sun et al. offer an excellent opportunity to examine the interaction between neural tissue and vascular structures during early brain development. Notably, this model differs from previously published approaches by acquiring endothelial and immune cells, the major non-neural cells missing in brain organoids. In the future, this study could be extended into other organoid models that mimic specific regions of the human brain and require a greater cellular diversity.
Throughout development, the brain transits from early highly synchronous activity patterns to a mature state with sparse and decorrelated neural activity, yet the mechanisms underlying this process are poorly understood. The developmental transition has important functional consequences, as the latter state is thought to allow for more efficient storage, retrieval and processing of information. Here, we show that, in the mouse medial prefrontal cortex (mPFC), neural activity during the first two postnatal weeks decorrelates following specific spatial patterns. This process is accompanied by a concomitant tilting of excitation-inhibition (E-I) ratio towards inhibition. Using optogenetic manipulations and neural network modeling, we show that the two phenomena are mechanistically linked, and that a relative increase of inhibition drives the decorrelation of neural activity. Accordingly, in mice mimicking the etiology of neurodevelopmental disorders, subtle alterations in E-I ratio are associated with specific impairments in the correlational structure of spike trains. Finally, capitalizing on EEG data from newborn babies, we show that an analogous developmental transition takes place also in the human brain. Thus, changes in E-I ratio control the (de)correlation of neural activity and, by these means, its developmental imbalance might contribute to the pathogenesis of neurodevelopmental disorders.
Humans and animals make predictions about the rewards they expect to receive in different situations. In formal models of behavior, these predictions are known as value representations, and they play two very different roles. Firstly, they drive choice: the expected values of available options are compared to one another, and the best option is selected. Secondly, they support learning: expected values are compared to rewards actually received, and future expectations are updated accordingly. Whether these different functions are mediated by different neural representations remains an open question. Here we employ a recently-developed multi-step task for rats that computationally separates learning from choosing. We investigate the role of value representations in the rodent orbitofrontal cortex, a key structure for value-based cognition. Electrophysiological recordings and optogenetic perturbations indicate that these representations do not directly drive choice. Instead, they signal expected reward information to a learning process elsewhere in the brain that updates choice mechanisms.