In vitro culturing conditions for human pluripotent stem cell –derived neural cells : Tissue engineering applications for spinal cord injury repair

Human pluripotent stem cells (hPSCs) are capable of self-renewal and differentiation into any cell type found in the human body. hPSCs include embryonic stem cells (ECSs), which are derived from the embryonic inner cell mass, as well as induced pluripotent stem cells (IPSCs), derived from somatic cells and reprogrammed using specific transcription factors to induce pluripotency. hPSCs can be directed to differentiate into the main cell types of the central nervous system (CNS), neurons, astrocytes, and oligodendrocytes in vitro. hPSC-derived neural cells provide an excellent approach to model CNS development and deficits in vitro and furthermore treat injuries and degenerative conditions by replacing damaged tissues. Spinal cord injury (SCI) describes damage to the spinal cord that leads to the loss of muscle function, sensation, or autonomic functions to different extents. CNS cells have limited capacity for regeneration, and cell replacement therapies have been extensively studied for SCI repair, as no efficient treatment for SCI currently exists. This thesis focused on studying various aspects, including genetic background and chemical and mechanical inductions, of hPSC-derived neural cell differentiation and behavior in vitro. The results were aimed to be applicable for developing tissue engineering products for SCI repair. First, the neural differentiation potentials of several hPSC lines were compared in vitro in order to discover the presence of potential systematic differences between hESCs and hIPSCs. Previous studies have demonstrated that despite their considerable resemblance, the different genetic backgrounds of hESCs and hIPSCs may affect to their differentiation potential. Our results revealed cell line-specific variations in neural differentiation efficiency. However, the variation between individual hESC and hIPSC lines was higher than the variation between cell lines of different origins, indicating the lack of systematic differences between the two hPSC types. Next, chemical induction was studied for the differentiation of hPSC-derived oligodendrocytes. Differentiation was induced by a variety of growth factors and morphogens in a temporally defined manner to direct the differentiation through the neural precursor cell stage into oligodendrocyte precursor cells (OPCs), and finally to maturing oligodendrocytes. Although efficient differentiation of OPCs was achieved, the maturation of the cells into oligodendrocytes should be further stabilized and enhanced. The differentiation process was initially performed under xeno-free and defined culture conditions, enabling clinical applications of the produced cells. The protocol was also adapted into a more cost-effective researchgrade method. Cellular responses to different extracellular matrix (ECM) molecules in vitro was studied using hPSC-derived neurons and astrocytes. ECM is found at the intercellular space in vivo and it provides both structural support and chemical cues for cell survival, differentiation, migration, and growth. Neurons and astrocytes were cultured on defined substrates using different isoforms or fragments of ECM component laminin. hPSC-derived neurons and astrocytes were shown to attach and grow most efficiently on substrates containing laminin α5-chain. These substrates also enhanced the functional development of neuronal networks. The studied substrates were both defined and xeno-free and thus can be used as platforms for the efficient production of clinical-grade neural cells from hPSCs. Finally, the effects of mechanical alterations in the culture conditions were studied using hPSC-derived neurons, astrocytes, and OPCs. The cells were cultured on aligned nanofiber platforms mimicking the aligned cellular orientation in the spinal cord. All cell types were observed to orient according to the underlying fiber alignment. However, ECM protein-coating for the nanofibers was required for efficient growth of the neurons and astrocytes, and previously identified laminin α5chain substrates were successfully applied here. As the 3D cultures resemble in vivo conditions more accurately than traditional 2D culturing surfaces, orientation guidance of hPSC-derived neurons was also demonstrated in 3D culture conditions consisting of nanofiber platform and hydrogel component. In conclusion, the results of this thesis provide new insights for the culturing of hPSC-derived neural cells in vitro. Obtained knowledge can be utilized for tissue engineering applications, as the novel combination of hPSC-derived CNS cells, clinically relevant ECM substrates, 3D environment of the hydrogel and guiding features of the nanofibers would be beneficial for the development of functional cell grafts for SCI repair.