MSACL 2019 EU Abstract

Intact-Cell Mass Spectrometry for Monitoring of Stem Cells Cultures

Petr Vaňhara (1,2), Lukáš Moráň (1,2), Vendula Pelková (2), Volodymyr Porokh (2), Hana Kotasová (2), Josef Havel (1,3), Aleš Hampl (1,2)
(1) International Clinical Research Center, St. Anne's University Hospital Brno, Czech Republic (2) Faculty of Medicine, Masaryk University, Brno, Czech Republic (3) Faculty of Science, Masaryk University, Brno, Czech Republic

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Petr Vanhara (Presenter) St. Anne's University Hospital Brno, Czech Republic >> POSTER (PDF)

Presenter Bio: Petr Vanhara focuses primarily on cancer and stem cell biology. He is involved in development of mass spectrometric tools for characterization of cancer and stem cells in various biomedical scenarios, aiming to reveal hidden phenotypes and diagnostic markers.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction
Stem cells are capable of self-renewal and differentiation to somatic cell types. Since the derivation of human embryonic stem cells (hESCs), enormous potential for biomedicine has been recognized. hESCs possess immense differentiation potential, and hence they are inherently prone to respond to various stimuli. This cellular plasticity can results in hazardous phenotypic instability and represents one of major biosafety obstacles in the development of bio-industrial or clinical-grade stem cell cultures. Therefore, there is an ongoing need for novel robust, feasible, and sensitive methods for determining status of otherwise identical cells, and for revealing hidden divergences from their optimal states. Here we modeled phenotypic changes in stem cells using different strategies and addressed changes in cell status by optimized intact-cell mass spectrometry (MS).
Methods
hESCs were cultured under identical conditions and processed immediately for intact-cell MS. Cells were washed in phosphate-buffered saline, counted, resuspended in the isotonic ammonium bicarbonate buffer, mixed with an acidified matrix containing sinapinic and 2,2,2-trifluoroacetic acid, and applied to a target plate. The mass spectra were recorded in linear positive ion mode over the 2,000–20,000 m/z range. Data sets were exported in ASCII. The final normalized spectral dataset containing selected m/z values with assigned peak intensities was subjected to statistical analyses.
Results
We compared mass spectra of hESCs cultured for varying time that developed distinct karyotypic or molecular traits. The final normalized spectral dataset was subjected to statistical analyses. Using PCA, the correctly clustered populations corresponding to short and long time of culture were clearly identified. Next, we monitored shifts in the hESC phenotypes after induction of differentiation towards early lung progenitors (ELEPs). Mass spectra recorded from stimulated and control cells reflecting the metabolomic profile between 2000-20000 Da allowed discrimination by cluster analysis, and monitoring of the differentiation process. In summary, the MS approach can therefore discriminate unapparent but potentially hazardous alterations that are not detected by other techniques, or track the proper differentiation route.
Conclusion
The quality of stem cell cultures is an essential prerequisite for clinical applications in which the product variability, batch consistency, or phenotype stability are significant. Here we show that intact-cell MS is capable to distinguish stem cell types in various scenarios. Analysis of spectral fingerprints allow discrimination of otherwise identical cells and provide an elegant tool for quality control of hESCs cultures.
Supported by Ministry of Health of the Czech Republic, grant numbers: NV18-08-00299 and 16-31501A (Ministry of Health of the Czech Republic, all rights reserved) and MUNI/A/1565/2018 (Faculty of Medicine, Masaryk University).