Abstract
Undifferentiated human embryonic stem cells have a distinct morphology (hESC). Changes in cell morphology during culture can be indicative of differentiation. hESC, maintained in diverse medias, demonstrated alterations in morphological parameters and subsequent alterations in underlying transcript expression and lineage differentiation. Analysis of morphological parameters showed distinct and significant differences between the undefined, less defined and Xeno-free medias while still maintaining pluripotency markers. This suggested that the less defined media may be creating dynamic instability in the cytoskeleton, with the cytoskeleton becoming more stabilised in the Xeno-free media as demonstrated by smaller and rounder cells. Examination of early lineage markers during undirected differentiation using d5 embryoid bodies demonstrated increased mesodermal lineage preference as compared to endodermal or ectoderm in cells originally cultured in Xeno-free media. Undefined media showed preference for mesoderm and ectoderm lineages, while less defined media (BSA present) demonstrated no preference. These data reveal that culture media may produce fundamental changes in cell morphology which are reflected in early lineage differentiation choice.
Highlights
Human embryonic stem cells are commonly defined by their ability to self renew and maintain their undifferentiated state
Cultures performed in conditioned on MEFs (CM) and SP, were observed to have a looser colony structure and flatter appearance when compared to mT and the Xeno-free medias E8 and SM (Fig 1A, S1A and S2A Figs)
Analysis of our data established that the N:C ratio decreased in MEL1 but increased in both WA09 and ESI-hES3 as media became more defined
Summary
Human embryonic stem cells (hESC) are commonly defined by their ability to self renew and maintain their undifferentiated state. Investigations into individual hESC lines have demonstrated that substantial variability occurs between cell lines in their differentiation efficiency [1, 2]. As human pluripotent stem cells (hPSC) progress towards use in clinical applications and drug development [3,4,5] it becomes imperative to understand how exogenous factors, such as media composition, may influence cellular differentiation through affecting changes in morphological parameters. Reports have demonstrated that altering the physical microenvironment of PSC resulted in different cytoskeletal organisation and behaviour of selfrenewal and lineage specification [6, 7].
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