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(190e) Preservation of Therapeutic Potential of Culture Expanded Human Mesenchymal Stem Cells By Preventing a Breakdown of Cellular Homeostasis

Yuan, X., FAMU-FSU College of Engineering
Liu, Y., Florida State University
Tsai, A. C., Florida State University
Ma, T., FAMU-FSU College of Engineering

Preservation of Therapeutic Potential of
Culture Expanded Human Mesenchymal Stem Cells by Preventing a Breakdown of
Cellular Homeostasis

Xuegang Yuan, Yijun Liu, Ang-Chen Tsai and Teng Ma

 Department of Chemical
and Biomedical Engineering, Florida State University

2525 Pottsdamer St, Tallahassee, US

1-330-734-7036, xy13b@my.fsu.edu

Key Words:   Human mesenchymal stem cell, Metabolism, Culture-induced
senescence, NAD+/Sirtuin.


Human mesenchymal stem cells (hMSCs)
isolated from various adult tissues are primary candidates in cell therapy and
being tested in clinical trials for a wide range of diseases.  The pro-regenerative and therapeutic
properties of hMSCs are largely attributed to their
trophic effects that coordinately modulate the progression of inflammation and
enhance the endogenous tissue repair by host progenitor cells.  However, immediately after isolation and
upon culture expansion, hMSCs lose their in vivo quiescent state and start to
accumulate genetic and phenotypic changes that significantly alter their
phenotypic properties with reduced clonogenic
population and therapeutic potential [1]. 
The proliferation of hMSCs is limited and
long-term culture-induced changes lead to cellular senescence and metabolic
alteration, resulting in reduced therapeutic outcome. Since clinical
application requires large-scale production of hMSCs
with defined cellular properties, preserving innate hMSC
properties during hMSCs in vitro expansion is a major
challenge in large scale hMSC production.

Figure 1. Culture expansion induces hMSC
senescence and breakdown of cellular homeostasis.  (A) Culture expansion induced cellular
and mitochondrial morphology alteration with decreased Sirt
expression; (B) stem cell gene and (C) colony forming ability decreased in
culture-induced senescent hMSC; (D) metabolic shift
from glycolysis towards OXPHOS in culture-induced senescent hMSCs;
and (E) NAD+ and NADH level in different passage of hMSCs.

Here, we reported that the reduced therapeutic properties of
culture expanded hMSC are a result of replicative
senescence and the breakdown of cellular homeostasis.  Our results show long-term culture expansion
alters hMSC mitochondrial function and reconfigures
energy metabolism towards oxidative phosphorylation (OXPHOS).  We also found a progressive reduction of
basal autophagy, which plays a key role in maintaining hMSC
clonogenicity and preventing senescence. 
Nicotinamide adenine
dinucleotide (NAD+), a key redox cofactor and co-substrate for many enzymatic
processes including Sirtuins, plays a key regulatory role
in cellular aging and longevity [2, 3].  NAD+/Sirtuin
axis plays a crucial role in restoring mitochondrial function, including
mitochondrial biogenesis, membrane potential and electron transport
ability.  Our results show a
passage-dependent decline of NAD+ level during hMSC culture
expansion and a corresponding reduction in Sirtuin
activity.  However, restore NAD+
level via supplementation in culture medium increases Sirtuin
activity and re-balance culture-induced senescent hMSC
cellular homeostasis by increasing mitochondrial membrane potential and
biogenesis.  Basal autophagy level
was also recovered after supplementation.  Together, our results reveal the key role
of NAD+/Sirtuin in regulating hMSC
replicative senescence and cellular homeostasis during culture expansion and
demonstrate the effectiveness of NAD supplementation in restoring hMSC regenerative capacity and therapeutic potentials. 


[1].  Liu, Y., et al. Density-Dependent
Metabolic Heterogeneity in Human Mesenchymal Stem Cells. Stem Cells, 2015. 33(11):
p. 3368-3381.

[2].  Imai, S., et al. NAD+ and Sirtuins in Aging and Disease. Trends. Cell. Biol., 2014. 24
(8): 464-471.

[3].  Zhang H., et al. NAD⁺ repletion improves
mitochondrial and stem cell function and enhances life span in mice. Science. 2016. 352(6292): 1436-1443