MSACL 2019 EU Abstract

Expression of Cellular and Neurodevelopmental Markers in Cerebral Organoids

Markéta Nezvedová (1), Eliška Stuchlíková (1), Veronika Vidová (1), Gabriela Přibyl Dovrtělová (1), Tereza Váňová (2), Dáša Bohačiaková (2), Zdeněk Spáčil (1)
(1) The RECETOX Centre, Faculty of Science, Masaryk University, Brno, Czech Republic, (2) Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

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Marketa Nezvedova (Presenter) Masaryk University >> POSTER (PDF)

Presenter Bio: Marketa Nezvedova is a doctoral research fellow in the Metabolomics and targeted proteomics group headed by Dr. Zdenek Spacil. She holds a PharmD degree in analytical chemistry from Charles University in Prag.

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

INTRODUCTION:
The dementia epidemic affects 47 million people globally, with increasing incidence [1]. Cerebral organoids, derived from induced pluripotent stem cells, represents an emerging model system to study biological processes leading to the neurological diseases and to an exploration of novel hypotheses [2]. Prior to that, suitable tools are required to properly characterize cerebral organoids in terms of cellular composition and neuronal maturation [3,4].

OBJECTIVES:
Our aim in this study was to develop an assay based on selected reaction monitoring tandem mass spectrometry (SRM-MS/MS) to identify cellular and neurodevelopmental markers and applied the assay to cerebral organoids of various age. The multiplex assay determines expression levels of cellular markers attributable to a specific cell type, e.g. neuroepithelial cells (SOX2), radial glia (BLBP), immature neurons (DCX, TUJ), mature neurons (MAP2), serotoninergic neurons (SERT), postsynaptic proteins (GRIN1) and oligodendrocytes (OLIG2).

METHODS:
Cerebral organoids cultivated at Dept. of Histology and Embryology following the protocol published by Lancaster et al. [5] were collected on the day 47, 97, 120 and 145. Each organoid was individually lyophilized. Dried pellets were extracted into 50mM ammonium bicarbonate buffer. Protein mixtures were reduced, alkylated and spiked with isotopically labeled peptides used as retention indices for a prediction model. Samples were enzymatically digested by trypsin, purified by solid phase extraction, dried down and injected on C18 Peptide CSH column. Ultra-high performance liquid chromatography mass spectrometry with triple quadrupole mass analyzer (6495B series, Agilent technologies, CA, USA) was used to investigate cellular markers in positive ion mode. Measured data were normalized to a response of housekeeping protein Glyceraldehyde 3 phosphate dehydrogenase (GAPDH).

RESULTS:
We have identified cell markers represented by 2-4 proteotypic peptides with 3 5 transitions. We used Skyline software for peptide identification based on predicted retention time and product ions. The iRT Calculator was recalibrated using retention indices covering the chromatography gradient. We normalized the response of specific cellular markers to the response of the GAPDH. This workflow was used for identification and relative quantification of eight cellular markers characterizing various developmental stages in the organoids and for grouped comparative analysis in organoids of different age.

CONLCUSION:
In this study, we present multiplex SRM assay for identification of cellular markers, associated with specific cell type composition and dependent on the maturity of organoids. We discovered age-dependent variable levels of certain cell markers in organoids of different age.