The hallmark of all undifferentiated stem cells is their nearly unlimited self-renewal capacity and their potential to differentiate into a diverse range of specialised cell types. These unique properties of stem cells make them invaluable research tools and can potentially serve as a source of cells for regenerative therapies. Pluripotent stem cells such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPS cells) are capable of producing almost any cell type in the human body, whereas multipotent adult stem cells exhibit limited self-renewal and a differentiation capacity that is restricted to the cell types of a particular lineage or a closely related family of cells. Since the discovery of stem cells, diverse culture conditions have been evaluated for different types of stem cells. In current standard in vitro cell culture techniques, xenogeneic reagents are used in the establishment, cultivation, and differentiation procedures. For the clinical a! pplications of stem cells, however, xenogeneic reagents pose the risk of a severe immune response, and the transmission of viral or bacterial infections, prions, and unidentified zoonoses.
In this study, we assessed the applicability of an automated cell culturing, imaging, and analysis system to evaluate undifferentiated growth dynamics of hESCs maintained in different culture media. The molecular mechanisms regulating self-renewal and pluripotency of hESCs under hypoxic conditions were also elucidated. Furthermore, defined and xeno-free culture conditions for the expansion of stem cells were evaluated, developed, and optimised to meet the regulatory standards set by the directives of the European Union for the clinical application of stem cells. The applicability of a defined xeno-free medium for the derivation and maintenance of hESCs as well as for the expansion of iPS cells and adipose stem cells (ASCs) was evaluated.
The results indicated that the automated cell culturing, imaging, and analysis system enables reliable analysis of the undifferentiated growth dynamics of hESCs in different culture conditions, and revealed more information than does conventional microscopic observation. Exposure to hypoxic conditions prevented spontaneous differentiation, supported self-renewal, and significantly increased the hESC proliferation. Fundamental differences in genes that are central to hypoxia signalling, calcium and PKC pathway, and the retinoic acid pathway were detected in different culture conditions under hypoxia. A key transcription factor for self-renewal, Oct-4, was significantly upregulated under hypoxic conditions, indicating a possible mechanism for hypoxia-induced self-renewal and prevention of spontaneous differentiation.
The results of these studies suggest that although a population of hESCs was able to adapt to human serum-containing culture conditions, several of the xeno-free culture media evaluated were unable to maintain hESC self-renewal. Here we developed a xeno-free medium formulation, RegES, that allowed for the derivation of pluripotent hESC lines. Furthermore, hESCs, iPS cells, and ASCs can be propagated in the RegES medium for a prolonged period of time with a reasonable proliferation rate while maintaining their characteristics and differentiation potential.
Clinical stem cell therapy trials are ongoing, which calls for a strong focus on the safety and quality of in vitro expanded stem cell transplants. By replacing xenogeneic products with a defined xeno-free medium, the safety and quality of the cells with therapeutic potential may be enhanced significantly. The use of completely defined conditions will allow for a better understanding of stem cell regulation and differentiation as well as provide more reproducible and reliable results. Culture conditions may have significant impact on the cell characteristics, thus proper characterization of cells with specific analyzing methods is necessary. Future studies should focus on validating the xeno-free culture conditions to demonstrate the ability for long-term culture, maintenance of key features of self-renewal, differentiation potency, and genetic stability, as well as derivation, reprogramming, or isolation of new stem cell lines as a full proof-of-principle, and to provide for scale-up to a manufacturing level. Additional pre-clinical safety and efficacy studies are needed before the promise of the xeno-free products can be fully realised. The results of the present study indicate that the xeno-free RegES medium is applicable for further optimization of xeno-free establishment, culture, and differentiation of various stem cell types and can ultimately serve as a platform for the production of clinical-grade multi- and pluripotent stem cells and their derivatives for safer clinical cell-based therapy.
