Human cortical spheroids with a high diversity of innately developing brain cell types

Abstract

Background Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms. Methods Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety. Results We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19–26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids. Conclusions Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms.

Human cerebral organoids: cellular composition and subcellular morphological features

“…In the last decade, the number of publications on hCOs has increased remarkably. However, ultrastructural studies dealing with specific cellular features in these hCOs are still scarce (Chandrasekaran et al, 2017 ; De Kleijn et al, 2023 ; Pavlinov et al, 2023 ). In this study, we delve into the cellular structural findings that we have observed in hCOs after culturing for 45 days.…”

“…Oligodendrocyte precursor cells (OPCs) (Marton et al, 2019 ; Cristobal and Lee, 2022 ) express markers, such as PDGFRA, and they differentiate into pre-oligodendrocytes or pre-myelinating oligodendrocytes (pre-OLs), which can be labeled for CNPase (Fard et al, 2017 ). The OPCs in the hCOs exhibit an accumulation of heterochromatin attached to the nuclear membrane and in their cytoplasm, many small mitochondria, and dilated short endoplasmic reticulum (Nahirney and Tremblay, 2021 ; De Kleijn et al, 2023 ). In comparison, a study conducted by Ulloa-Navas et al ( 2021 ) showed that the ultrastructure of the cell found in the hCOs is similar to mature human oligodendrocytes; however, we cannot see myelin sheaths close, although there is an expression of the myelin protein PLP1.…”

Blood–Brain Barrier Breakdown in Neuroinflammation: Current In Vitro Models

“…This issue could be solved by vascularization within the organoid. However, this is still a major challenge for researchers in this field [ 164 ]. A study by Wörsdörfer et al [ 165 ] suggested the implementation of mesodermal progenitor cells into organoid models in order to tackle the lack of vessels within the 3D structure.…”

Moral considerability of brain organoids from the perspective of computational architecture

“…Brain organoids recapitulate many aspects of human neural development [ 1 ], and may provide insights into disease pathology [ 2 ]. Towards this goal, researchers are generating in vitro brain organoids with cell-type diversity [ 3 , 4 ], areal identity [ 5–7 ], vasculature [ 8 ], and cytoarchitecture [ 9–11 ] that more closely resemble their in vivo counterparts, living human brains. These technological advances raise ethical questions about consent of donors [ 12 ], biobanking [ 13 ], medical and non-medical (e.g.…”