(486f) Restoration of Cellular Homeostasis of Human Mesenchymal Stem Cells during in Vitro Culture Expansion

text-align:center;line-height:normal;text-autospace:none">Restoration
of Cellular Homeostasis of Human Mesenchymal Stem Cells during In Vitro Culture
Expansion

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text-align:center;line-height:normal;text-autospace:none">Xuegang
Yuan¹, Yijun Liu¹, Brent M. Bijonowski¹, Qin Fu¹
and Teng Ma¹

text-align:center;line-height:normal;text-autospace:none"> ¹Department
of Chemical and Biomedical Engineering, Florida State University

text-align:center;line-height:normal;text-autospace:none">2525
Pottsdamer St, Tallahassee, US

text-align:center;line-height:normal;text-autospace:none">1-330-734-7036,
font-family:" arial>xy13b@my.fsu.edu

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normal;text-autospace:none">Key Words " arial>:  Human mesenchymal stem cell, Replicative
senescence, Metabolic alteration, Redox cycle, Sirt-1, Rejuvenation.

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justify;text-justify:inter-ideograph;line-height:normal">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 regeneration. 
However, immediately after isolation and upon in vitro culture
expansion, hMSCs lose their in vivo quiescent state and start to
accumulate genetic and phenotypic changes that significantly reduce their stemness
and therapeutic potential [1].  hMSCs has limited proliferation potential and
in vitro expansion leads to replicative senescence and a breakdown of
cellular homeostasis. Since clinical application requires large-scale
production of hMSCs with defined cellular properties, preserving cellular
homeostasis during in vitro culture expansion has become a major barrier for
hMSCs based therapy.

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justify;text-justify:inter-ideograph;line-height:normal">In this
study, we report the breakdown of cellular homeostasis during in vitro
expansion of bone marrow-derived hMSCs. Cellular homeostasis, characterized by
proliferation potential, maintenance of basal autophagy and metabolic activity,
and absence of cellular senescence, was disrupted during culture expansion of
hMSCs. Basal autophagy and mitophagy decreases in hMSCs with a corresponding
increase in replicative senescence and impairment of stemness [2].  Metabolomics
and proteomic analysis suggested the loss of metabolic homeostasis during
expansion, which reconfigured hMSC metabolism from glycolysis towards OXPHOS. Increased
energy demand during in vitro expansion of hMSCs alters mitochondrial fitness and
dynamics, leading to reduced mitochondrial biogenesis and mitophagy. Proteomic
analysis showed that activity EIF2 signaling pathway increased in high passage
hMSC, suggesting accumulating cellular stress induced during expansion. To
understand the role of energy metabolism and redox cycle in regulating cellular
homeostasis, we investigated intracellular NAD+/NADH level and Sirt-1
expression during hMSC expansion. Our results show that NAD+/NADH redox cycle
was significantly altered during expansion of hMSCs, potentially due to changes
in NAD+ biosynthesis and metabolism. Sirt-1, a NAD+-dependent enzyme which regulates
aging-related cellular events, including mitochondrial biogenesis, electron
transport activity, DNA damage repair and oxidative stress protection, was also
found to decrease with increasing passages of hMSCs. By restoring intracellular
NAD+ level in senescent hMSCs, Sirt-1 activity, along with multiple stem cell functions
have been recovered. Autophagic (and mitophagic) activity and mitochondrial fitness
were also improved to counter culture stress and maintain cellular homeostasis during
culture expansion of hMSC.  Besides, we also found hMSCs are more
sensitive to artificial environment compare to human fibroblast, indicating a
potential specific response for stem cells. Together, current study provides
metabolic and biochemical insight of replicative senescence of hMSCs and
indicated the regulatory role of cellular homeostasis. It also provides a
potential strategy to maintain stem cell quality for biomanufacturing.

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justify;text-justify:inter-ideograph;line-height:normal">Figure 1. Culture
expansion induces hMSC senescence and a breakdown of cellular
homeostasis.  (A-D): Culture expansion induced hMSC morphological
alteration with increase senescence and DNA damage; (E): mitochondrial
biogenesis was down-regulated; (F) and (G) hMSC metabolic reconfiguration and
signaling pathway changes characterized by proteomics; (H) and (I): Culture
induced decrease of NAD+ and Sirt-1 activity in hMSCs; (J): NAD+/NADH ratio in
hFBs following culture expansion.

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justify;text-justify:inter-ideograph;line-height:normal">References

margin-left:.25in;margin-bottom:.0001pt;text-align:justify;text-justify:inter-ideograph;
text-indent:-.25in;line-height:normal"> " arial>[1]. Liu, Y., N. Munoz, B.A. Bunnell, T.M.
Logan, and T. Ma, Density-Dependent Metabolic Heterogeneity in Human
Mesenchymal Stem Cells. Stem Cells, 2015. 33(11): p. 3368-3381.

margin-left:.25in;margin-bottom:.0001pt;text-align:justify;text-justify:inter-ideograph;
text-indent:-.25in;line-height:normal"> " arial>[2]. Liu, Y., N. Munoz, A.C. Tsai, B.A.
Bunnell, T.M. Logan, and T. Ma, Metabolic Reconfiguration Supports
Reacquisition of Primitive Phenotype in Human Mesenchymal Stem Cell Aggregates.
Stem Cells, 2017. 35(2): p398-410 8.0pt;font-family:" arial>.