Ph.D Project: an overview

1. Control and Regulation of Stem Cell Differentiation
Triggering using Nanoparticles
Prof. Hugh J. Byrne
Dr Alan Casey
Dr Esen Efeoglu
Francesca Ravera, MSc., Ph.D Fiosraigh Scholar
City Campus, School of Physics and Clinical & Optometric Sciences
Nanolab Group
FOCAS Research Institute,
TU Dublin, Dublin 8, Ireland
Annual Evaluation 2020

3. Project Overview

4. Bone marrow derived adult
rodent Mesenchymal Stem
Cells (MSCs)
20 𝜇m
Chondrogenic differentiation
Raman
Microspectroscopy Nanoparticle exposure
Osteogenic differentiation
20 𝜇m
produce and
maintain the
cartilaginous matrix
star-shaped type
of bone cell

5.  in vitro monitoring of stem cells
differentiation process
Bone marrow derived adult
rodent Mesenchymal Stem Cells
20 𝜇m
14 day Chondrocytes 24 hour 40 nm Carboxylate-modified
Polystyrene exposed Chondrocyte
20 𝜇m 20 𝜇m
 Identifying the biochemical and
spectral markers of cellular events
governing the differentiation
MSCs differentiation
 Nanoparticle exposure, trafficking and
localisation
 Explore the interaction mechanisms,
changes and cellular responses

6. Research Objectives
3. Demonstrate the
dependence of control of
stem-cell triggering on the
physico-chemical
properties of the
nanoparticles for a
regulation of cells
commitment and
differentiation.
2. Provide an understanding of
structure-activity
relationships governing the
influence of the
nanoparticles on
differentiation, suppression
and enhancement of the
differentiation ratio.
1. Demonstrate that Raman
microspectroscopy can be
used to monitor and
better understand the
processes of
differentiation of stem
cells at a subcellular level
in vitro.
Next 12 months objectives:
Characterisation of the identified chondrogenic differentiation markers using
mass spectrometry imaging.
Identification and characterisation of commercially available nanoparticles and
in vitro investigation of toxicological properties of the nanomaterials.
Monitoring of nanoparticle uptake and localisation, and systematic
characterisation of spectroscopic signatures of differentiation without NP
exposure.

7. Methodology

8. Raman Microspectroscopy
 Simultaneous detection of
macromolecules
 Non-destructive, non-invasive
 Minimal sample preparation
 Label free, no dyes or toxic waste
products
 High specificity suitability for biological
sample in native state
 Water can be used as solvent
 Suitable for chemical analysis,
quantification, classification and
imaging of biological samples
Scattering of light: interacts
without causing electronic
transitions
The Raman Effect

9. 400 600 800 1000 1200 1400 1600 1800
0
0.05
0.1
Wavenumbers cm-1
Intensity
Determination of spectral features of Mesenchymal Stem Cells
Nucleolus
Nucleus
Cytoplasm
7dayMeanSpectra
DNA/RNA LipidsProteins

10. Principle Components Analysis for comparison of cytoplasm
region, nucleus and nucleolus region of rMsc
Nucleolus
Cytoplasm
Nucleus
Loading 1

11. Nucleolus
Cytoplasm
Nucleus
Principle Components Analysis for comparison of cytoplasm
region, nucleus and nucleolus region of rMsc

12. Results

13. The study of the substrates
In vitro cell culture
environment investigation:
Comparison of 2D and 3D
culture models.

14. CaF2
Collagen
CaF2
Collagen
CaF2
Collagen
PCA scatter plot and loading of PC1 of comparison of cytoplasm, nucleus and
nucleolus region of rMSC grown on CaF2 and Collagen substrates
Cytoplasm Nucleus Nucleolus
Comparison of Raman spectra of two substrates.
Mean spectrum of Collagen and CaF2
M. Gargotti, E. Efeoglu, H. J. Byrne and A. Casey, Analytical and Bioanalytical
Chemistry, 2018, 410, 7537–7550.

15. 0 100 200 300 400 500 600 700 800 900 1000
0
2000
4000
6000
8000
10000
12000
RAW SPECTRUM
BACKGROUND
(CaF2)
REFERENCE
SPECTRUM
CORRECTED
SPECTRUM
Pre-processing prior multivariate analysis:
Development of Improved Extended Multiplicative Scatter Correction
Raw data EMSC effectively removes both the background and
adjusts the baseline
 Explore the effect of these on subsequent multivariate
analysis for the purpose of cell classification

16. In vitro monitoring of Mesenchymal stem cell
differentiation to Chondrocytes using Raman
Microspectroscopy
(Amarilio et al., 2007)
Extracellular Matrix
Chondrogenic pellet development

17. 7 Days Chondrogenic Pellet Development 14 Days Chondrogenic Pellet Development
21 Days Chondrogenic Pellet Development
A) B)
C)A
B
C
Centre Region
Intermediate Region
Edge Region
Monitoring of Chondrogenic Pellet formation using Raman Microspectroscopy

18. Achievements and Future
Work

19. Overall Progresses
Team Work and Collaboration
‘’ Being able to determine
the best approach to a
question, find relevant data,
design a way to analyze it,
understand a large amount
of data, and then synthesize
your findings. ‘’
Leadership and Project
Management
Mentoring and teaching
other students as a teacher
or mentor, motivating
someone and help them
accomplish a goal. You also
get experience evaluating
someone’s performance
(grading) and giving
constructive feedback.
Critical Thinking
To approach problems
systematically, see the links
between ideas, evaluate
arguments, and analyze
information to come up with
your own conclusions.
Building your dissertation is
a solo project, but on a day
to day basis you work with
other people
knowing how to divide up a
task, get along with others,
communicate effectively,
and resolve conflict.
• Range of data
acquisition and analysis
software has been used
to extract information.
• Cell culture and cellular
assays bio-imaging
techniques.
• Spectroscopy techniques
Multidisciplinary
Skills
Lab Work and Research

20. Modules accomplished so far
ENEH 1004 Multivariate Analysis &
Data Mining for Biomedical
Application 1
Lecturer: Dr Aidan Meade
GradCAM XXXX Philosophy of Science
and Technology
Lecturer: Prof Noel Fitzpatrick
GRSO 1012 Research Integrity
Lecturer: Dr Steve Meaney ; Prof
Mary McNamara
GRSO 1011 Research Information
Retrieval, Literature Review
Skills and Academic Profile for
STEM
Lecturer: Dr Marek Rebow
9th Annual Graduate Research Symposium
Poster presentation: “Raman
Microspectroscopy for in vitro monitoring of
Mesenchymal Stem Cell differentiation into
different lineages and the effect of
Nanoparticles on differentiation process”
Microscopy Society of Ireland Winter
Symposium 2019 University College Dublin
Oral presentation “Control and Regulation of
Stem Cell Differentiation Triggering using
Nanoparticles”
Raman4clinics-Raman-based applications for
clinical diagnostics COST Training Summer
School 2018 University of Coimbra Interactive
Workshop
Conferences and academic events
The International Society for Clinical Spectroscopy
Summer School workshop Lake Windermare 2019
Techniques & Strategies in Molecular Medicine
Workshop 2019 Trinity College Dublin
Introduction to Molecolar
Spectroscopy University of
Manchester
Introduction to Programming with
MATLAB Vanderbilt University
From Diseases to Genes and Back
Novosibirsk University

21. FUTURE
WORK
- Characterization of
chondrogenic differentiation
markers using mass
spectrometry imaging
- Publication on Chondrogenic
and Osteogenic differentiation,
conducted by sub-culturing the
cell lines on different coated
and non-coated surfaces and
substrates
- Planned Conferences:
Before COVID: Spring SciX
2020.
XXVII International Conference
on Raman Spectroscopy
(ICORS) Rome 2020
- To perform the nanoparticle
exposure and an in vitro
investigation of
toxicological properties of the
nanomaterials, monitoring the
uptake and
localisation, and systematic
characterisation of
spectroscopic signatures
of differentiation without NP
exposure;
- Publication on nanoparticle
internalisation and trafficking in
stem cells by bio-imaging
techniques and vibrational
spectroscopy
THANK YOU!