Optimization of protein quantification methods for analysis of FGF2 signaling during hypoblast differentiation
Hautala, Emilia (2025)
2025
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2025-10-20
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-2025102010005
https://urn.fi/URN:NBN:fi:tuni-2025102010005
Tiivistelmä
Fibroblast growth factor 2 (FGF2) and its receptors FGFR1 and FGFR2 are key regulators of hypoblast lineage specification during early human embryogenesis. Understanding the molecular mechanisms guiding hypoblast differentiation is essential for advancing knowledge of early human development. This thesis builds on transcriptomic data from Paloviita et al. (manuscript in preparation), which identified relevant gene expression patterns during hypoblast differentiation. While transcript-level data are available, protein-level regulation, especially isoform-specific dynamics, remains poorly understood. The aim of this thesis was to develop and optimize protein quantification methods for analyzing FGF2 signaling during hypoblast differentiation and in human embryonic stem cells (hESCs), with a particular focus on distinguishing isoforms and receptor usage in naïve and primed pluripotent state.
Hypoblast differentiation was carried out using the protocol described by Linneberg-Agerholm et al. (2019). To detect and quantify FGF2, FGFR1, and FGFR2 proteins, microscopy-based immunofluorescence staining and Western blot workflows were established. Two Western blot platforms were evaluated: Bio-Rad’s Stain-Free system and Jess automated capillary system. Experimental samples included naïve and primed H9 hESCs, as well as cells undergoing hypoblast differentiation with three treatments: a hsa-miR-92a-3p inhibitor, a non-targeting inhibitor NegA or culture medium-only control. Workflow optimization involved testing antibody dilutions, sample concentrations, and loading parameters to improve signal specificity and resolution.
FGF2, FGFR1, and FGFR2 proteins were successfully visualized in naïve and primed hESCs, as well as in naïve hESCs undergoing hypoblast differentiation. Western blot analysis identified two distinct FGF2 isoforms and confirmed the microscopy results on FGFR1 and FGFR2. FGFR1 protein levels were minimal in naïve hESCs despite moderate transcript expression, whereas FGFR2 was consistently detected in both naïve and primed hESCs. The Western blot workflows enabled protein quantification on hESCs but showed limitations in isoform resolution. Overall, immunofluorescence staining and blotting results were mostly consistent with transcriptomic data, although technical challenges limited direct quantitative comparisons.
This study established workflows for isoform-specific protein quantification in hESCs and revealed insights into FGF2 signaling naïve and primed pluripotent states. FGFR2 was consistently detected and may play a more prominent role in naïve state signaling than FGFR1. However, due to technical limitations, the study did not yield conclusive insights into FGF2 signaling during hypoblast differentiation. Future improvements could include refined sample preparation, isoform-specific antibodies, and imaging strategies such as higher magnification and focal plane adjustments to better exclude the inactivated mouse embryonic fibroblast (iMEF) cells and improve signal specificity.
Hypoblast differentiation was carried out using the protocol described by Linneberg-Agerholm et al. (2019). To detect and quantify FGF2, FGFR1, and FGFR2 proteins, microscopy-based immunofluorescence staining and Western blot workflows were established. Two Western blot platforms were evaluated: Bio-Rad’s Stain-Free system and Jess automated capillary system. Experimental samples included naïve and primed H9 hESCs, as well as cells undergoing hypoblast differentiation with three treatments: a hsa-miR-92a-3p inhibitor, a non-targeting inhibitor NegA or culture medium-only control. Workflow optimization involved testing antibody dilutions, sample concentrations, and loading parameters to improve signal specificity and resolution.
FGF2, FGFR1, and FGFR2 proteins were successfully visualized in naïve and primed hESCs, as well as in naïve hESCs undergoing hypoblast differentiation. Western blot analysis identified two distinct FGF2 isoforms and confirmed the microscopy results on FGFR1 and FGFR2. FGFR1 protein levels were minimal in naïve hESCs despite moderate transcript expression, whereas FGFR2 was consistently detected in both naïve and primed hESCs. The Western blot workflows enabled protein quantification on hESCs but showed limitations in isoform resolution. Overall, immunofluorescence staining and blotting results were mostly consistent with transcriptomic data, although technical challenges limited direct quantitative comparisons.
This study established workflows for isoform-specific protein quantification in hESCs and revealed insights into FGF2 signaling naïve and primed pluripotent states. FGFR2 was consistently detected and may play a more prominent role in naïve state signaling than FGFR1. However, due to technical limitations, the study did not yield conclusive insights into FGF2 signaling during hypoblast differentiation. Future improvements could include refined sample preparation, isoform-specific antibodies, and imaging strategies such as higher magnification and focal plane adjustments to better exclude the inactivated mouse embryonic fibroblast (iMEF) cells and improve signal specificity.