Conditioned Medium From Bone Marrow-Derived Mesenchymal Stem Cells For Ex Vivo Expansion Of Cardiac Stem Cells
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Date
2016-08
Authors
Umar Fuaad, Mimi Zulaikha
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Abstract
Bone marrow-derived mesenchymal stem cells (MSCs) have been shown to
secrete paracrine factors which can stimulate activation of endogenous cardiac stem
cells (CSCs) and ameliorate infarcted heart function. These factors can be
harvested through conditioning of MSCs in vitro. This thesis aimed to optimise
MSC growth conditions and medium formulation for generating conditioned
medium (CdM) with CSC-cytoprotective properties. Murine MSCs were isolated
from tibia and femur bones of 3-5 weeks old C57BL6/N mice using flushed
marrow or crushed bone and was characterised by flow cytometry. Preconditioning
time and seeding density before initiation (Phase I), medium
formulation, oxygen, conditioning time, effects of repeated harvesting, and
treatment concentration (Phase II) were assessed and optimised based on the effects
of the produced CdM on CSCs survival in vitro. Then, the optimized CdM were
tested on CSC migration. To reduce metabolic waste, CdM were concentrated 8
times (Phase III) and tested on CSC survival, and compared to the crude CdM. All
data were analysed using ANOVA and t-test. Optimal MSC seeding density during
phase I was 20,000 cells /cm2 and no significant deterioration in CSC survival with
CdM generated from MSCs at lower density (p>0.05). Long MSC preconditioning
time (48 and 96 h) decreased CSC survival to 45.7% and 53.8%, respectively under
serum starved conditions. This effect could be reversed by adding 10% B27.
Combination of 24 h pre-conditioning time with high glucose supplemented (25
mM) DMEM produced CdM that offered significantly greatest cryoprotective
effects on CSCs (p<0.05). No change in CSC survival when ascorbic acid was
supplemented in the media. Similarly, hypoxic MSCs did not produce CdM with
cytoprotective effect superior to that of normoxic MSCs. Furthermore, repeated
harvesting of CdM, regardless of the conditioning time, shows reduced CSC
survival compared to CdM from the first harvest, and also the protein level in CdM
concentrates correlated with CSC survival. This thesis shows guided optimisation
could produce CSC-stimulating CdM from bone marrow-derived MSCs, and such
method can be translated onto standardisation of CdM production from human
MSCs at a larger scale for clinical therapy.
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Keywords
Bone Marrow-Derived Mesenchymal Stem Cells For Ex Vivo Expansion , Of Cardiac Stem Cells