RESUMEN
Maintaining the homogeneity of a stem cell population is one of the challenges in bioprocessing prior to therapeutic applications of stem cells. Concerning human induced pluripotent stem cell (hiPSC) colonies cultured on feeder cells, cells at the peripheral region of the colony were found to have a higher average movement rate than cells at the central region of the colony. This spatial difference in average movement rate might lead to spatial heterogeneity of cell fate decision in the colony. We have developed a kinetic model to clarify the origin of this phenomenon which was difficult to understand by in vitro studies alone. Using a kinetic model based on a cellular automaton, we described fundamental cell behaviors including cell division, contact inhibition, cell migration, cell-cell connections, and cell-substrate connections. With all parameter values estimated from experimental data, the appropriateness of our kinetic model was indicated by good agreement between simulated and experimental data. Using the kinetic model, the average cell movement rate in a colony became homogenous after cell division stopped, implying that cell division was the main cause of the observed spatial heterogeneity. The result also showed a directly proportional relationship between the frequency of cell pushing and cell movement rate in the colony, confirming the role of cell division. Our kinetic model is expected to be useful for studying behaviors of hiPSCs and proposing good strategies to improve hiPSC bioprocessing.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Nutrientes/química , Células Nutrientes/citología , Humanos , Células Madre Pluripotentes Inducidas/química , Cinética , Modelos BiológicosRESUMEN
BACKGROUND: Human embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos. METHODS: A comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines. RESULTS: We developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement. CONCLUSIONS: The present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies.
Asunto(s)
Técnicas de Cultivo de Célula , Embrión de Mamíferos/citología , Células Madre Embrionarias Humanas/citología , Medicina Regenerativa/normas , Animales , Biomarcadores/análisis , Masa Celular Interna del Blastocisto/química , Masa Celular Interna del Blastocisto/citología , Diferenciación Celular , Línea Celular , Proliferación Celular , Separación Celular , Medios de Cultivo/química , Células Nutrientes/química , Haplotipos/genética , Células Madre Embrionarias Humanas/química , Humanos , Células Madre Pluripotentes/químicaRESUMEN
Until recently, human embryonic stem cells (hESCs) were shown to exist in a state of primed pluripotency, while mouse embryonic stem cells (mESCs) display a naive or primed pluripotent state. Here we show the rapid conversion of in-house-derived primed hESCs on mouse embryonic feeder layer (MEF) to a naive state within 5-6 days in naive conversion media (NCM-MEF), 6-10 days in naive human stem cell media (NHSM-MEF) and 14-20 days using the reverse-toggle protocol (RT-MEF). We further observe enhanced unbiased lineage-specific differentiation potential of naive hESCs converted in NCM-MEF, however, all naive hESCs fail to differentiate towards functional cell types. RNA-seq analysis reveals a divergent role of PI3K/AKT/mTORC signalling, specifically of the mTORC2 subunit, in the different naive hESCs. Overall, we demonstrate a direct evaluation of several naive culture conditions performed in the same laboratory, thereby contributing to an unbiased, more in-depth understanding of different naive hESCs.