Fourier transform infrared microspectroscopy for the characterisation of human embryonic and human induced pluripotent stem cells
thesisposted on 28.02.2017, 04:10 authored by Cao, Julie Oanh
Fourier transform infrared (FTIR) microspectroscopy shows potential as a benign, objective and rapid tool to screen clinically destined pluripotent and multipotent stem cells. In this work, we explored the utility of this technique to distinguish between several human embryonic and human induced pluripotent stem cell lines and their differentiated progeny, based on their macromolecular chemistry. We observed both intra-class and inter-class biochemical variation within the undifferentiated stem cell cohorts, in addition to spectroscopic differences between both classes during their differentiation towards mesendodermal and ectodermal lineages. Chemometric comparisons of the progeny cells alone, indicated that they were phenotypically distinct, suggesting that current reprogramming methods do not yield human induced pluripotent stem cells that are equivalent to human embryonic stem cells, in spite of immunohistochemistry results suggesting the antithesis. However, whether these phenotypical changes are indicative of the inadequacies of the reprogrammed cells to form therapeutically useful lineage committed progeny needs to be established. To elucidate the basis of the phenotypic differences observed between these pluripotent stem cells, we decided to investigate the role of environmental factors influencing these spectral 'signatures', by varying their growth environments. Accordingly, we employed Fourier transform infrared (FTIR) microspectroscopy to acquire spectra from several hESC lines and their derived cell types maintained in both feeder dependent and feeder independent conditions, in KOSR based or mTESR based hESC medium, and either mechanically or enzymatically dissociated. It was found that hESC lines grown under different conditions possessed unique FTIR spectroscopic ‘signatures’ and that these spectroscopic differences persisted even upon differentiation towards mesendodermal lineages. The results from this study illustrates the power of this modality for defining macromolecular phenotypic differences between several lines of human embryonic stem cells and human induced pluripotent stem cells and their lineage committed progeny. The technique has been shown to be highly sensitive, as indicated by its ability to detect biochemical differences even between stem cell lines of the same class. Further, the results indicate that the spectroscopic phenotypes are sensitive to a combination of genetics and environmental factors.