Introduction

Organoids are complex three-dimensional (3D) cellular systems derived from pluripotent stem cells (PSCs) or adult stem cells [1, 2]. Concerning the nervous system’s organoids, when the originating cells are cultured in suspension under specific conditions that reproduce embryonic development, they form structures that mimic the organ architectures and functions, including the central nervous system (CNS) as a whole (whole-brain organoids) or specific CNS areas (regional organoids) [3]. As cells develop within brain organoids, they follow a developmental timeline that is similar to in vivo neurogenesis [3]. Brain organoids are able to generate spontaneous neural activity, form functional synapses, and support interneuron migration or axonal projection, as well as interact and fuse with adjacent organoids giving rise to assembloids, mimicking the architecture and complex interactions of CNS tissues [4, 5]. Additionally, transcriptomic and epigenetic studies confirmed that brain organoids recapitulate the key molecular features of the human embryonic/fetal brain [4].

The main source of cells used for brain organoid generation is induced pluripotent stem cells (iPSCs) [6] and embryonic stem cells (ESCs) [7], both widely employed. iPSCs are derived from somatic cells primarily of the skin or of the blood, which are reprogrammed into embryonic-like states by administration of specific pluripotency transcription factors. Unlike ESCs, iPSCs do not carry the ethical concerns associated with ESCs while fully reflecting the patient’s genetic background [6, 8, 9]. Notably, iPSCs are able to differentiate in several cell types, including neuronal cells, making them particularly suitable for studying neurodegenerative disorders.

To date, disease modeling has largely relied on two-dimensional (2D) cell cultures, human biopsy specimens, and animal models. Nevertheless, the majority of human tissues are difficult to biopsy, and their use is strictly regulated, while 2D cell cultures do not fully mimic the structural organization and functions of human tissues, poorly attempting to replicate the real in vivo conditions [8]. As regards in vivo animal models, despite their ability to replicate the complexity of living organisms better than in vitro ones, their utility is limited by interspecies differences.

Recently, iPSC-derived 3D models, grown in a way that cells interact with each other and their surroundings more naturally, mimicking the in vivo conditions to a greater extent, have become key tool for disease modeling and for the development of new therapeutic strategies, closely resembling the in vivo microenvironment. In this context, iPSC-derived organoids, free floating 3D models, offer the chance to deeply investigate physiological and pathological mechanisms in a specific genetic background. Particularly, brain organoids have attracted huge interest to study neural development and as innovative tools for drug discovery and regenerative medicine [10]. The exploitation of organoid potential can be further extended to the generation of multi-unit structure called assembloid, a more sophisticated in vitro model, which attempts to recapitulate intercellular interactions among different organ-like structures [11]. Assembloids are self-organizing cellular entities emerging from the integration of distinct organoids or derived from the combination with specialized cell populations [5, 12].

So far, iPSC-derived brain organoids have been generated to model a large range of both developmental and degenerative brain disorders [13,14,15,16,17]. Nonetheless, their major limitation, as a model, is the lack of a vascular system for transporting nutrients or drugs, which in vivo occurs through microvascular cells and structures. The absence of a neurovascular system limits organoid growth, neurogenesis, and functions, restricting their potential applications [18,19,20,21,22].

Because of that, vascularization of brain organoids is one of the main sought-after advancements in the field, allowing their use for etiopathological studies and drug screening tests based on blood–brain barrier (BBB) permeability, as well as a platform for studying neurological disorders and particularly cerebrovascular diseases [14, 15, 23,24,25]. Here, we aim to review both the current state and future perspectives of vascularized human brain organoids.

Neural Organoid Generation

One of the first attempts to generate brain organoids that resemble the human brain in 3D was performed in 2013 by Lancaster and colleagues [13]. The original protocol relied on embedding PSCs in a basement membrane-like matrix to facilitate neuroepithelial development. The generation of brain organoids in this way primarily relied on intrinsic signals, thus requiring only a minimal number of growth factors and other substances for patterning, with basic fibroblast growth factor (bFGF) being used as the sole growth factor in the first 6 days. These self-develo** organoids resulted in a stochastic mixture of different brain cellular components, spanning from the retina to the hindbrain, implying low reproducibility and high variability. To overcome this aspect, subsequent protocols were developed progressively introducing inductive cues such as morphogens and signaling molecules, capable of directing the neurodevelopmental specification in a timely manner [26, 27]. Indeed, by using these protocols, 3D structures with a more specific regional identity such as the hypothalamus, midbrain, brainstem, and choroid plexus could be created [28, 29]. Organoids’ growth and maturation can continue over several months, reaching a width of several millimeters [16]. Eventually, they contain a variety of different cell populations of ectodermal origin, including multiple neuronal subtypes, astrocytes, oligodendrocytes, and outer radial glia cells [27, 30,31,32,33,34,35]. Notably, it has been described that microglia can develop within the brain organoid, simultaneously with neuroectodermal cell types, if dual SMAD inhibition is removed. Indeed, innately developed mesodermal progenitors are capable of differentiating into mature microglia under the influence of the CNS microenvironment provided by neuroectodermal cells [36].

Human brain organoids have the advantage of being able to model unique human-specific characteristics, such as the development of outer radial glial cells which largely contribute to the development of the human cerebral cortex and whose alteration can be responsible for pathological conditions [15]. Different patterned CNS organoids’ derivation protocols have been implemented over the past few years. Telencephalic aggregates were first developed to segregate GABAergic and glutamatergic neurons, while cortical glutamatergic neurons and astrocytes have been derived through the generation of cortical spheroids [26, 37]. By modifying the neurocortical induction protocol, the choroid plexus and the medial pallium-like tissues, precursors of the hippocampal telencephalic area, were established [38]. Finally, midbrain organoids containing dopaminergic neurons of the nigro-striatal pathway were generated [104, 105]. Research on rodent models has demonstrated that ECs could attract recently differentiated neurons to migrate along vessels and reach areas affected by ischemia [106]. The influence of neurovascular interactions on developmental (angiogenesis, BBB formation, mural and vascular cell development, regulation of neuronal and glial development) and reparative events in humans remains unclear since there is no relevant human model or there are models with poor human translatability. Indeed, in vitro conventional cell culture models [107], microphysiological systems [108, 109], tissue-engineered 3D models, or bioprinting [110] do not properly fulfill conditions of the human NVU microenvironment. Recreating the intricate microenvironment and the cellular interactions within the neurovascular unit becomes pivotal in comprehending its functioning and, further, in devising CNS-targeted pharmaceuticals that can effectively penetrate the brain for experimental testing. The development of vascularized organoids could overcome this obstacle. Additionally, gene expression analysis, including single-cell profiles, may reveal new players in neurovascular mechanisms.

Further, organoids could also prove extremely useful for regenerative therapies. Indeed, a differentiated human brain organoid, containing appropriate cell populations, active neural circuits, and an adequate vasculature may facilitate their use as a cell source for transplantation strategy for CNS tissue regeneration [73, 111]. Finally, vascularized human brain organoids hold the potential for application in drug screening, discovery of new potential disease biomarkers, and advancement of cutting-edge diagnostics and therapies [63, 73]. As clinical avatar, patient-specific organoids can be used for diagnostic interventions, tested with therapeutic strategies, and personalized medicine could eventually be achieved in the next future [97].

Overall, it appears clear that brain organoids, especially vascularized organoids, could prove to be a fundamental resource for understanding and treating neurological diseases.

Conclusions

Vascularized human brain organoids are still imperfect tools, and they need to be further optimized before they are able to precisely recapitulate the development, function, and pathology of the neurovascular system. Several studies demonstrated that vascularizing human brain organoids allowed for a better maturation and survival of neural cells. More specifically, better functional circuit and firing rate [61] and increased number of mature neurons [73] have been observed. Furthermore, some key aspects of the interactions between vascular cells and neural cells in health and pathology have been clarified [53, 69], including the regulation of the BBB maturation by neural cues [72].

Current research suggests that vascularized brain organoids provide better pathophysiological models compared to non-vascularized ones [61, 68]. Indeed, compared to other in vitro models, neurovascular organoids can better reproduce the cytoarchitecture of the brain, providing functional and synaptic connectivity data. Nevertheless, the generation of vascularized brain organoids requires more resources with consequent lower accessibility than non-vascularized organoids, hindering their applicability. For an extensive use of brain organoids for disease modeling and drug discovery, substantial advancements in automation and scaling up are needed.

The pathophysiology of brain diseases is influenced by blood flow, cellular composition, vascular cell-derived factors, and microglia. Recent advances in cell culture technology allowed the growth of brain organoids for long periods, paving the way to model late-onset diseases such as neurodegenerative diseases [17]. Indeed, long-term cultures of vascularized brain organoids may be very useful for modeling aging-associated diseases such as stroke or AD, while young organoids used to model fetal brain development may not accurately reproduce stroke-relevant phenotypes.

Since vascularized organoids can more easily integrate into the host tissues and may promote healing better than non-vascularized organoids, they may be an excellent source for cell transplantation in regenerative medicine approaches. Nonetheless, more studies are required to evaluate the chance to perfuse vascularized brain organoids without transplanting them in vivo. Indeed, a controlled perfusion method could assess the vessel permeability through a direct injection of blood cells into the organoid as well as reduce the characteristic necrotic core of organoids [112, 113]. Additionally, biomechanical properties of the brain tissue and its vascular system such as stiffness, viscoelasticity, and spatial organization influence physiological processes such as proliferation, migration, differentiation, and cell functions [114].

Overall, all these studies have emphasized the significance of vascularized organoids in faithfully recapitulating different aspects of the neurovascular microenvironment, paving the way for a deeper understanding of the molecular mechanisms underlying neurodegeneration in a forward-looking perspective of identifying new therapeutic strategies.