Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors

Abstract

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models.

Synergistic Effects of Hypoxia and Morphogenetic Factors on Early Chondrogenic Commitment of Human Embryonic Stem Cells in Embryoid Body Culture

“…The cells underwent self-selection to form aggregates and initiate the first step of chondrogenic differentiation - the cell condensation. Formation of pellets also helped the formation of more homogenous cell aggregates compared to the heterogeneous EBs, which are characterized by gastrula-like tissues with varying local concentrations of morphogenic factors [43–45]. At 6 weeks, chondrogenic pellets derived from transient hypoxia EBs (Group 4) exhibited an excellent homogeneity, which could partially be attributed to the loss of unspecified cells remaining in cell suspension and washed away during medium changes in pellet cultures.…”

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

“…We have previously shown that this 3D bioreactor system supports stable culture of PHH under serum-free conditions 24,25 and is suitable for differentiation of human embryonic stem cells (hESC). 26,27 …”

The 3R principle: advancing clinical application of human pluripotent stem cells

“…To address this, animal-free in vitro methods of teratoma formation are being explored. One recent study examined the spontaneous differentiation of human ES cells in a perfused three-dimensional multi-compartment bioreactor [24]. This system reported generation of embryoid bodies with histological and transcriptional features similar to those that develop in the mouse teratoma assay.…”