In this study, Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS, or IRMS), coupled with multivariate analysis is shown to be an effective tool to further investigate the differentiation of human pluripotent stem cells and monitor the alternative affects different retinoid compounds have on the induction of differentiation. Stem cells and differentiated phenotypes being distinguished based on the spectral fingerprint recorded and not through the addition of fluorescent or magnetic biomarkers. IRMS detected differences between cell populations as early as 3 days of retinoid compound treatment. Populations of stem cells treated with different retinoid compounds could easily be distinguished from one another during the early stages of cell differentiation, demonstrating the potential of that IRMS technology as a sensitive screening technique to monitor the status of the stem cell phenotype and progression of differentiation along alternative pathways in response to different compounds. The study also investigates some of the problems with intrinsic resonant Mie scattering (RMieS), produced when recording IR spectra from single cells in air. Some single cell spectra recorded containing RMieS were not corrected properly when using the RMieS-EMSC scattering correction and typical correction parameter options; correction of such spectra using the RMieS-EMSC algorithm resulted in the production of cell spectra with odd absorption band structures after correction. This study demonstrates that alternative correction parameter options for the RMieS-EMSC correction can successfully overcome this problem. Early experimental results show that that IRMS is a sensitive technique capable of capturing discrete molecular changes of pluripotent stem cells as they are induced to differentiate, without the need for expensive and potentially damaging exogenous labels. Studies in the literature predominately use IRMS to investigate chemically fixed cells, however, this is a form of cell death and once cells are fixed they become un-viable. Critically, cell phenotype should be understood from living cells with the biochemistry being maintained after information is recorded so that single cells can retain their biological function. IR spectroscopy has the potential to record spectra from living cells when coupled with the micro-devices to provide a suitable water barrier for IR spectroscopic investigations. This study demonstrates that cell spectra can be recorded from cells in an aqueous environment, with a new processing method being described to accurately remove the water buffer contributions contained within raw single cell spectra recorded in an aqueous environment. Therefore, highlighting the potential of IRMS for the screening of living cells, potentially identifying stem cells from other cell phenotypes based on their biochemical make-up and without cellular destruction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:632242 |
| Date | January 2014 |
| Creators | Clemens, Graeme |
| Contributors | Gardner, Peter |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-development-and-use-of-infrared-microspectroscopic-techniques-to-monitor-the-differentiation-of-pluripotent-stem-cells-induced-by-retinoic-acid-and-synthetic-retinoid-analogues(07f40c35-6aa9-4204-b037-b41574b850be).html |
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