Differentiation & Purification of Human Pluripotent
Stem Cell-Derived Neuronal & Glial Cells
Graft Designing for Spinal Cord Injury Repair
Acta Universitatis Tamperensis, No. 1593
By Maria Sundberg
Tampere University Press
Distributed by Coronet Books
$87.50 Paper Original
Human embryonic stem cells (hESCs) are pluripotent cells that can be differentiated into all three germ layer cell types of the human body. The potential of these cells to efficiently proliferate and differentiate into neuronal and glial cells makes them a desirable cell source to be used for neural graft production for different neurological disorders, such as spinal cord injuries (SCI). Multipotent human neural stem/precursor cells (NSCs/NPCs) can also be derived from human fetal central nervous system (CNS) forebrain and spinal cord tissues. In the case of SCI, neural cell transplantation therapies could be one option for regeneration of the damaged tissue and restoring loss of locomotor function. However, currently existing differentiation protocols for hESC-derived neural cell production often result in heterogeneous populations, which contain undifferentiated or partly differentiated hESCs or NSCs/NPCs proliferating in an uncontrolled manner and are tumorigenic upon grafting. Also, although it has been shown that fetal CNS-derived NSCs/NPCs are effective for the treatment of SCI in animal models some studies have suggested that these cells may be tumorigenic after transplantation. Thus, it is important to evaluate the safety of these human neural cell grafts properly and in reliable animal models to avoid grafting of unsafe cells for the patients in the future. Related to this it has been shown that in SCI animal models more specialized cell populations, such as oligodendrocyte precursors (OPCs), are safer upon grafting and can have more beneficial effects in the regeneration of damaged tissue compared to multipotent NSC/NPC transplantations. However, the oligodendrocyte differentiation protocols for pluripotent stem cells are not so effective and contain undefined and animal-derived products, which are not to be recommended for clinical applications due to the safety risks of possible immunological reactions or pathogen cross-transfers.
In this thesis work the first aim was to characterize hESC surface proteins to find novel markers related to these cells that could be used for the purification of hESC-derived neural cell populations from tumorigenic pluripotent stem cells. In addition, the aim was to characterize the surface protein expression profiles of hESC-derived neural cell populations during the neuronal differentiation process and select appropriate markers for the sorting of pure neuronal populations with fluorescence activated cell sorter (FACS). The second aim was to compare the differences between hESC- and fetal CNS-derived NPCs to ascertain their specific characteristics of pluripotency and neural marker expression levels. In addition, different immunodeficient rodent host tissues were evaluated for reliability in the determination of hESC- and fetal CNS-derived NPCs tumorigenicity and safety. Furthermore, the hESC-derived NPCs were grafted into SCI-model rats to evaluate their effects for regeneration after injury. In the third project the aim was to develop a novel differentiation protocol for hESC-derived OPCs and oligodendrocytes, including optimization of purification step for disposing of pluripotent stem cells. Finally, in the fourth project the protocol for hESC-derived OPC production was optimized into xeno-free conditions aiming at clinical grade cell production.
According to the results from the first study a novel marker related to pluripotent stem cells, namely epithelial cell adhesion molecule EpCAM/CD326 was found. This marker was successfully used for the purification of hESC-derived neural cell populations from pluripotent stem cells with FACS. The results from the second study showed that in contrast to the fetal NPCs, the hESC-derived NPC populations expressed pluripotency related markers at protein level, which indicated the presence of undifferentiated cells in the graft and made the cell population tumorigenic upon grafting. Also, the animal studies showed that there are remarkable differences between different tissues’ permissiveness for tumor formations caused by undifferentiated hESCs. After grafting of hESC-derived NPCs in immunodeficient mice testicles and subcutaneous tissues, no tumors or teratomas were observed. By contrast, grafting of the same cells in immunodeficient rats’ spinal cords resulted in tumor formations, and significant decline in the animals’ locomotor function was detected. According to the third study, a novel differentiation and purification protocol for hESC-derived OPC production was developed using only human recombinant growth factors and extracellular matrix proteins to induce the differentiation. Also, for purification of produced hESC-derived OPCs a gentle sorting method for FACS with NG2-antibody was developed. In addition, the myelination capacity of hESC-derived OPCs was demonstrated in co-cultures with neurons. Finally, the results from the fourth study showed that it is possible to efficiently differentiate OPCs from pluripotent stem cells in totally xeno-free conditions, and the xeno-free medium also supported subculturing and differentiation of sorted NG2+ OPCs.
These studies suggest that hESCs are a promising cell source for neuronal and oligodendroglial differentiation. These results also showed that it is very important to purify the differentiated populations of pluripotent stem cells prior grafting them to avoid tumor formations. Importantly, since remarkable differences were detected between different tissues’ and animal species’ propensity for tumor formations caused by hESCs, we concluded that there may be similar or even bigger differences in graft tumorigenicity between commonly used animal models and human patients. In conclusion, for the future treatment of SCI it will be important that pluripotent - or multipotent - stem cell-derived neural grafts are properly produced and characterized using reproducible and traceable manufacturing and characterization methods.
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