Complex analysis of metabolic status, intracellular pH, viscosity and cytoskeleton of human

Metabolism
pH
Cytoskeleton
Functional-structural changes of MSCs during differentiation
Viscosity
Mesenchymal
Stem
Cells

Effective control of MSCs differentiation - great challenge
Complex analysis is required!!!

Methods of the stem cells morphology and physiology investigation
Feature
Method
Cell markers
• Flow cytometry
• Immunocytochemistry
• Magnetic-activated cell sorting
Genotype
• Polymerase chain reaction (PCR)
Differentiation potency
• Immunocytochemistry
• Fluorescence Microscopy + fluorescence dyes
/protein
• Fluorescence Lifetime Imaging Microscopy
(FLIM) +exso/endogenous markers
• Stochastic Optical Reconstruction Microscopy
(STORM) +fluorescence dyes/protein

Outline of the experiment
• MSCs – human mesenchymal stem cells bone marrow
Metabolism: fluorescence microscopy and FLIM of NAD(P)H and FAD
pH: fluorescence microscopy and SypHer–2
• YFP, monomer
• two peaks of fluorescence excitation
(420 nm and 500 nm), peak emission 516 nm
• at alkaline pH values, the excitation peak at 420 nm
decreases, and at 500 nm - increases,
while for acidic - on the contrary
LSM 710 laser scanning confocal
microscope (Carl Zeiss, Germany)
FLIM system based on
Simple Tau 152 TCSPC system
(Becker & Hickl GmbH)
Nicotinamide adenine dinucleotide, NADH: excitation - 750 nm ,detection - 455-500 nm
Flavine adenine dinucleotide, FAD: excitation - 900 nm , detection – 500-550 nm
redox ratio FAD/NAD(P)H
Lifetimes
λ, nm

• MSCs – human mesenchymal stem cells bone marrow
LSM 710 laser scanning confocal
microscope (Carl Zeiss, Germany)
FLIM system based on
Simple Tau 152 TCSPC system
(Becker & Hickl GmbH)
Viscosity: FLIM and Bodipy 2
Cytoskeleton: STORM and TagRFP
TagRFP
em=550nm
detection= 584nm
EclipseTi (Nikon, Japan),
module N-STORM, system PSF
ex = 800 nm,
detection range = 409-660 nm
Outline of the experiment

Metabolism
Functional-structural changes of MSCs during differentiation
Mesenchymal
Stem
Cells

Optical redox ratio of FAD/NAD(P)H changes during chondrogenic differentiation
NADH:
excitation -750 nm (5 mW)
detection - 455-500 nm
FAD:
excitation - 900 nm (5mW) ,
detection - 500-550 nm
image size is 213 × 213 μm
(1024 × 1024 pixels)
[Meleshina et al. Stem Cell Research & Therapy (2017) 8:15]

Dynamic of bound NAD(P)H in MSCs during chondrogenic differentiation
[Meleshina et al. Stem Cell Research & Therapy (2017) 8:15]
Pseudocolor-coded FLIM images of the free (t1) and protein-bound (t2) forms of NAD(P)H.
For NAD(P)H: excitation - 750 nm, detection - 455–500 nm. Field of view 213*213μm (512*512 pixels)

pH
Functional-structural changes of MSCs during differentiation
Mesenchymal
Stem
Cells

Intracellular pH analysis in MSCs during differentiation
by fluorescence microscopy and SypHer–2
days of differentiation
pH, a.u.
bias to acidic pH values
ex = 405 nm and 488 nm, detection range = 500-550 nm
[unpublished data]

Analysis of collagen formation during chondrogenic differentiation using SHG
green –
cell autofluorescence
red- collagen fiber
Alcian blue staining
on acidic polysaccharides
Hematoxylin
staining
[Meleshina et al. Stem Cell Research & Therapy (2017) 8:15]
SHG of collagen was excited at wavelength of 750 nm and detected in the range 373-387 nm
the image size is 130×130 μm (512 × 512 pixels)

Cytoskeleton
Functional-structural changes of MSCs during differentiation
Viscosity
Mesenchymal
Stem
Cells

MSCs viscosity analysis during differentiation
using FLIM and Bodipy 2
chondrogenic differentiation
undifferentiated MSCs
viscosity increase – cholesterol accumulation
viscosity, cP
days of differentiation
ex of Bodipy 2 = 800 nm, detection range = 409-660 nm
[unpublished data]

Analysis of cytoskeleton organization in MSCs during differentiation
by STORM and TagRFP
Undifferentiated MSCs
7 day
14 day
21 day
Increase of actin fibers thickness
ex of TagRFP = 555 nm, em=584 nm
[unpublished data]

take home message
Metabolic plasticity of MSCs during chondrogenic differentiation: glycolysis – more glycolytic state
Intracellular pH
bias of pH values towards a more acidic pH
3. Membrane viscosity
viscosity increase – cholesterol accumulation
4. Cytoskeleton organization
undifferentiated MSCs having a fibroblast-like morphology, the actin fibers are represented by long, parallel fibrils extending through the cytoplasm of the cells. Chondrocytes have increased the thickness of end parts of actin fibers. In addition, chondrocytes have changed their orientation: actin fibrils crossed cells in different directions

Acknowledgements
This work has been financially supported by Russian Science Foundation (grants No. 14-15-00536)
M.V. Shirmanova
M.K. Kuimova
N.V. Klementieva
O. Furman
F.A. Kulagin
V.V. Dudenkova
A.S. Bystrova
E.V. Zagaynova