THE ROLE OF SUBSTRATE STIFFNESS IN MAINTAINING PLURIPOTENCY OF EMBRYONIC STEM CELLS IN VITRO CULTURE
D.I. Bilko1, Y.B. Chaikovsky2
- National University of Kyiv-Mohyla Acadaemy, Kyiv, Ukraine
- Bogomolets National Medical University, Kyiv, Ukraine
Murine embryonic stem cells (ESCm) cultured in vitro in the
presence of LIF (leukemia inhibitory factor) maintain pluripotency. However, when LIF is removed from the media, an
active differentiation into various specialized somatic cells is
observed. The aim of the study was to determine the role of
substrate stiffness in maintaining of pluripotency of embryonic
stem cells in vitro culture. To this aim, we used the method
of culturing pluripotent stem cells in vitro, the method of
“hanging drop”, the determination of the Young’s modulus
for polyacrylamide gel of different hardness, the immunocytochemical alkaline phosphatase (AP) streptavidin-biotin
method, microscopy. By culturing ESCm on a soft, medium
and hard density polyacrylamide gel as a substrate (0.8, 4.0,
8.0 кPа), we found that on a soft gel ESCm differentiation
does not occur even in the absence of LIF. ESCm cultured on a
soft substrate continue to show signs of pluripotency, namely,
create round compact colonies with high alkaline phosphatase
activity and form embryoid bodies (EB), the efficiency of
which (87.5 ± 3.2 per 100 cells seeded) did not decrease even
after LIF withdrawal. In the absence of LIF, ESCs cultured on a hard base showed a low level of EB formation (23.5 ± 2.24).
The results of our observations demonstrate that the process of
EB formation may be influenced not only by a composition of
nutrient medium, but also by complex interaction between the
physical forces of the matrix and the mechanical properties of
3D cell aggregates. The model is considered as a tool to study
early events in embryogenesis in the search of conditions for
effective culture of progenitor cells and differentiated cells
pluripotency, embryonic stem cells, embryoid body, differentiation, culture in vitro
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