Conventional 2D cell cultures fail to represent the complexity of the brain and novel 3D systems are emerging as more realistic and representative models. However, available 3D brain models have important limitations due to the ¿self-assembling¿ process commonly used for their production, which generates a high degree of variability and reproducibility issues. 3D Bioprinting is an additive manufacturing technique that uses, as ink, a combination of biocompatible non-living materials and cells (bioink). This technique provides the possibility to combine cells in a controlled way, to build structures that closely mimic natural tissues. Human induced Pluripotent Stem Cells (iPSCs) can be derived from any individual by reprogramming body cells (e.g. skin, blood), which hence acquire the potential to be converted into any cell type of interest. Neurodevelopmental disorders such as Fragile X Syndrome (FXS) are widely studied using human iPSCs as a starting point to obtain specific neural population on which performing molecular and functional analysis. However, conventional culture fails to recapitulate the complex neural environment revealing itself a not reliable model system to fully characterize the pathology.
This project aims at generating novel 3D models of the human nervous system focusing on the brain cortex development and organization by using iPSC-derived neurons as `building block¿ of the model. Patient-derived 3D models of Fragile X Syndrome will be produced exploiting innovative 3D bioprinting methods in order to reproduce in vitro a more physiological condition to study the disorder. We will challenge these disease models to assess whether they can be used for drug testing and for developing new diagnostic tools. This work will fill an important gap in the pre-clinical development of new therapies for diseases of the nervous system, i.e. the inappropriateness of current in vitro models in terms of cellular cytoarchitecture and functional properties.
Application of 3D bioprinting to iPSCs is a field at its infancy. Only recently, advancements in biofabrication techniques have opened the possibility to apply these methodologies to human iPSCs.
Despite the enormous potential of human iPSCs for disease modeling and regenerative medicine, and despite the boost that biofabrication methods might provide to the cell system, only a handful of recent reports describe bioprinted human 3D constructs based on iPSCs. This gap is mainly due to some peculiar characteristics of human iPSCs, which make bioprinting challenging for this kind of cells. First, human iPSCs cannot survive in culture as single cells, and single cell dissociation is a necessary step in most bioprinting procedures. Second, iPSCs are highly responsive to environmental cues, due to their intrinsic nature of embryonic-like cells able to respond to developmental signals. Third, human iPSCs tend to form clusters, or colonies, due to their epithelial character. This propensity must be taken into consideration when the bioprinter includes microfluidic devices.
When the goal is to produce a human construct structurally and functionally mimicking a tissue of interest, two strategies can be undertaken if the cellular building block is provided by iPSCs. The first approach, which we define post-printing differentiation, starts from undifferentiated iPSCs that are bioprinted and then induced to differentiate within the construct. In the second approach, hereafter pre-printing differentiation, iPSCs are first induced to differentiate by conventional protocols towards the cell type of interest. Once obtained, the specific iPSC-derived differentiated cells are printed. Pre-printing differentiation can be also combined with a purification strategy (e.g. cell sorting) that allows generating 3D constructs made of a specific individual cell type or containing a controllable proportion of each cellular component.
Some recent reports show post-printing differentiation of human iPSCs. These works are based on different
bioprinting methods, including extrusion (Faulkner-Jones et al., 2015; Reid et al., 2016; Gu et al., 2017;
Nguyen et al., 2017) and laser (Koch et al., 2018). After printing, undifferentiated iPSCs maintained their developmental potential, allowing subsequent post-printing differentiation along hepatic (Faulkner-Jones et al., 2015), cartilage (Nguyen et al., 2017), cardiac (Koch et al., 2018) and neural (Gu et al., 2017) lineages. Some examples of pre-printing differentiation also exist in recent literature. Specifically, hepatocytes (Ma et al., 2016; Yu et al., 2019), cardiomyocytes (Ong et al., 2017; Yu et al., 2019), endothelial and smooth muscle cells (Moldovan et al., 2017) and limbal epithelial stem cells (Sorkio et al., 2018), derived by differentiating human iPSCs or ESCs, have all been used to produce 3D constructs. At present, to the best of our knowledge, iPSC-derived neural cells have never been bioprinted to produce a human 3D model of the nervous system.
The choice between post- and pre-printing differentiation is crucial. We believe that only pre-printing
differentiation will represent a valid method for possible applications of this technology. It should be taken
into consideration that due to their intrinsic plurilineage differentiation capacity, iPSC differentiation mostly
produces mixed populations containing different lineages. In each experiment, the fraction of each cell type in the mixed population could be highly variable. For this reason, in conventional 2D cultures, strategies for the purification of desired cell types during iPSC differentiation have been set up. These include fluorescence sorting, magnetic separation and differential adhesion. The same considerations are valid for iPSC-derived bioprinted constructs. Notably, purification of cell types of interest is not possible in post-printing differentiation, leaving little control on the composition of the construct. Moreover, during post-printing differentiation, iPSCs proliferate and migrate, resulting in the loss of the original printed pattern (Koch et al., 2018). Conversely, pre-printing differentiation allows control on the cells that are incorporated in the final construct. Moreover, it provides the possibility to combine different cell types with precise stoichiometry. Finally, the relative position of each cell type within the construct can be determined.
Definition of the proper bioprinting conditions for iPSC-derived cells, to model brain tissue, could be particularly challenging since a number of different neuronal and glial cell types are present in the nervous system. Nevertheless, we believe that our pre-printing differentiation approach is a crucial point to generate 3D neural constructs suitable for applications which require rigorous control, such as disease modeling, drug screening and regenerative medicine.