The process of engineering intricate layered neural tissue with a myriad of diverse cell types has been a formidable challenge. Professor Zoltán Molnár said:
“Over millions of years of brain evolution, nature selected the best developmental mechanisms to generate over 200 different types of cortical neurones from various progenitors and organised them into complex circuits that can help us to detect and respond to external signals, to communicate with each other and to predict and plan for the future. Our brain consists of billions of cells that are interconnected with trillions of connections. Human brain development is highly delicate and elaborate process that we do not fully understand. There is a lengthy and complex choreography to generate different subtypes of neurones, deliver them to the cortical plate through tangential and radial migration, and assemble them into transient and then more permanent circuits, and finally to link them up with other elements of brain tissue such as astrocytes, vasculature, microglia and meninges."
Despite the devastating impact of brain damage through injury or disease, existing treatments have proved insufficient with few potential therapies making it past clinical trials. In a ground-breaking project, a team of Oxford University scientists, comprising DPAG’s Zoltán Molnár and Francis Szele, the Department of Chemistry’s Hagan Bayley and Oxford Martin Fellow Dr Linna Zhou are pioneering a radical new therapeutic approach to brain repair: 3D printing cerebral cortical tissue from human stem cells.
Francis states: “We started the collaboration with Hagan and Linna with a John Fell Fund grant” and published a paper in 2019 in Advanced Materials (IF-29, PMID: 32537827) showing the power and flexibility of 3D printing cortical cells generated from human induced pluripotential stem cells (hiPSCs). We then asked Zoltán Molnár to join the team and soon acquired a four-year grant from the OMS.” Over the past three years, the collaborative team have been using droplet printing to fabricate simple neural tissues that mimic the basic structure of cerebral cortex columns. However, instead of allowing the cells to self-organise, they are 3D printed in vitro. With these manufactured basic cortical units, they intend to replace damaged cells in brain injury. They are presently investigating how to transplant the printed tissue into mouse models of traumatic brain injury, a condition affecting more than 5 million people globally. Their latest results show efficient integration of printed cortical tissue into living slices and are now published in Nature Communications (IF-17, PMID: 37794031.
The human cerebral cortex has a uniform structure of six distinct cortical layers, each with a different cell composition, different circuits and physiological properties, but with the same basic modules and cell types. Using hiPSCs generated from skin cells, the team have successfully collectively reproduced the top four layers “upper layers” and the bottom two layers “deep layers”. While they can print and differentiate all six layers together in principle (Fig. 1), they first need to understand why different cortices have different properties. Once the team understand one cortex, they can apply this knowledge to the others.
Zoltán further explained “It would be naïve to think that we can perfectly recreate cortical structure in living tissue. However, our 3D printing project represents huge progress to start to control the fate and arrangements of human induced pluripotent stem cells to form the basic functional units of the cerebral cortex. We consider the generation of these two layered printed neuronal assemblies as the first step. In comparison to the cerebral cortical columns in our brains, our 3D printed cortical constructs that contain neurones with characteristics of upper and lower cortical layers is much more simple and much less sophisticated. However, our study demonstrated that even these assemblies develop activity patterns and can interact with host tissue in vitro.”
Following their breakthrough, the team are currently exploring whether these printed constructs are more efficient in cell replacement therapies in vivo mouse models than conventional stem cell suspensions or organoids. To do so, Dr Luana Campos Soares is implanting the printed human cells into immunosuppressed mice to observe the impact on their behaviour. Concurrently, DPhil student Mona Barkat is recording the activity of these neural assemblies to see how the cells communicate along with the kind guidance of Prof Pawel Swietach. For the past year, a visiting PhD student, Elisa Marozzi, is testing how astrocytes can augment connectivity and function in the 3D printed constructs.