Temporal morphogen-driven neural induction spurs growth of expanded brain organoids with enhanced neuroepithelium formation

By Tarun Sai LomteReviewed by Benedette Cuffari, M.Sc.Dec 4 2023

In a recent study published in Nature Communications, researchers examinea the effects of temporal morphogen gradient during neural induction (NI) on the formation of brain organoids.

Study: Temporal morphogen gradient-driven neural induction shapes single expanded neuroepithelium brain organoids with enhanced cortical identity. Image Credit: sdecoret / Shutterstock.com

How is brain development studied?

The development of the human brain is unique as compared to other mammals and difficult to study due to the lack of suitable model systems. Nevertheless, in vitro human brain models, including spheroids or organoids, have emerged as powerful tools for studying brain development.

Brain organoids have been critical for revealing essential aspects of brain development; however, some biological features can be limitedly recapitulated. A distinctive morphological feature within each brain organoid is the spontaneous and uncontrolled development of rosettes, which represent independent neuroepithelium units that lead to in between- and within-organoid heterogeneity.

Study findings

After the dissociation of human embryonic stem cells and re-aggregation into embryoid bodies in stem cell medium, dual SMAD inhibition was employed for cortical NI and switched to an NI medium suddenly or gradually.

In the gradual and stepwise protocol, cells were exposed to a protracted and decreasing gradient of the stem cell medium and, concomitantly, the NI medium was increased stepwise. Spherical-shaped cortical organoids (COs) formed with numerous rosettes under sudden NI. By contrast, a convoluted shape was evident under stepwise NI that became more pronounced over time.

A distinct lighter border with folds and ridges at the apex was visible on day 14, which prolonged and became more prominent by day 24, thus suggesting expanded neuroepithelium structures with stepwise NI.

Circularity was strikingly reduced under gradual NI and organoid areas significantly increased at days 20 and 25. Organoids generated using a commercial protocol also exhibited spherical morphology.

Cellular organization was analyzed by immunostaining with N-cadherin (NCAD). COs contained NCAD⁺ cells as a collection of neural rosettes of various shapes and sizes on days 16 and 24.

By contrast, organoids generated under gradual NI formed a continuous, elongated, radially organized, and folded NCAD⁺ neuroepithelium that resembled a ventricular zone (VZ)-like structure. Organoids formed with gradient NI were named expanded neuroepithelium organoids (ENOs).

Transforming growth factor (TGF)-β and fibroblast growth factor 2 (FGF2) were the main morphogens in the stem cell medium. Comparatively, the NI medium contained inhibitors of TGF-β (SB-431542) and bone morphogenetic protein (BMP) (dorsomorphin).

The researchers then evaluated whether the controlled TGF-β gradient of decreasing TGF-β levels and increasing levels of SB-431542 was responsible for the ENO phenotype. To this end, organoids were generated under different levels of TGF-β signaling modulation during NI.

Organoids generated by gradually reducing TGF-β levels without counteracting it with SB-431542 were phenotypically identical to ENOs. When TGF-β in the medium was suddenly removed and replaced by SB-431542, organoids were morphologically more similar to COs. Additional experiments suggested that ENOs experienced neuronal differentiation after a protracted stem cell expansion phase.

Although both COs and ENOs were positive for cortical progenitor markers, empty spiracles homeobox 2 (EMX2) and paired box 6 (PAX6), ENOs exhibited a higher proportion of PAX6⁺ cells in the VZ at different time points. Moreover, PAX2 expression was homogenous and present in germinal areas of the expanded neuroepithelium in ENOs. Comparatively, PAX2⁺ cells were scattered in rosette structures in COs.

These data suggested enhanced cortical specification of ENOs. The neuroepithelium was significantly thicker in ENOs than in rosettes of COs. Rosette structures’ apical perimeter decreased between days 16 and 24, whereas ENOs did not exhibit this decline.

Cells along the apical side of VZ-like structures in ENOs had a significantly larger apical surface area than cells in the rosette structures. Notably, an enlarged cellular apical surface area has been linked to the delayed transition to neurogenic radial glia, a characteristic of human brain development. Apical progenitor cells in ENOs exhibited longer apical-basal processes and densely packed nuclei with an elongated shape in the VZ than nuclei of COs.

Conclusions

The study findings highlight that a temporal morphogen gradient-driven NI could influence organoid development. Specifically, the temporal gradient during NI determines the formation of organoids composed of an expanded apical-out neuroepithelium, rather than the uncontrolled appearance of rosettes.

Increased VZ thickness, elongated nuclei and cell shape, and larger apical cell surface were observed in ENOs, all of which are features of a developing human brain. Overall, ENOs represent a platform to study early human cortical brain development and the complex relationship between cellular states and tissue architecture in a developing human brain.