Nanog Reverses the Effects of Senescence on Proliferation and Myogenic Differentiation of Human Mesenchymal Stem Cells

Nanog reverses the effects of
senescence on proliferation and myogenic differentiation of human mesenchymal
stem cells

Panagiotis Mistriotis¹,
Maoshih Liang¹, Juhee Han¹, Stelios T. Andreadis^1,
2

1. Bioengineering Laboratory, 908
Furnas Hall, Department of Chemical and Biological Engineering, University at
Buffalo, State University of New York, Amherst, NY 14260-4200, USA

2. Center of Excellence in
Bioinformatics and Life Sciences, Buffalo, NY 14203, USA

Introduction: Human Mesenchymal Stem Cells (hMSCs)
show great promise as an autologous smooth muscle cell source for vascular
tissue engineering and regenerative medicine. However culture senescence and
organismal aging reduce the proliferative and myogenic differentiation capacity
of MSCs. To address this issue, we recently reported that ectopic expression of
Nanog can restore the lost contractile phenotype due to aging and enhance the
proliferative potential of ovine MSCs (Han et al., 2012, Stem Cells). In the
current study we aim in identifying the molecular mechanism that mediates the
rejuvenating effects of Nanog in MSCs.

Materials
and Methods: MSCs
isolated from both hair follicle or bone marrow and transduced with a
tetracycline regulatable vector that carries the Nanog gene. This system
enables Nanog expression upon Doxycycline (Dox) treatment. After serial
passaging, MSCs were induced to senescence and Dox was added to the culture
medium. Subsequently, the effect of Nanog on proliferative and myogenic
capacity of MSCs were evaluated and compared with early passage (EP) MSCs.

Results
and Discussion: Ectopic
expression of Nanog in late passage (LP) MSCs significantly increases the
proliferation (fig. 1A) and decreases the percentage of senescent cells as
indicated by SA-β-GAL staining (fig. 1B).
Furthermore, we show that Nanog controls the expression of key regulators of
senescence such as cyclin inhibitors and DNA methyltransferases. Next we
studied the myogenic differentiation upon Nanog expression. SMC-specific
markers such as ACTA2 and Calponin were highly upregulated in Nanog⁺LP
MSCs resembling EP MSCs (fig. 1C). In agreement, the contractility in response
to vasoactive agonists was also enhanced (fig.1D). Interestingly, we also
report that Nanog reverses the loss of the expression of key myogenic
transcription such as SRF, MRTF-b, SMAD2 and MYOCD due to senescence (fig. 1E,F).
Notably, we identified that Nanog co-immunoprecipitatets with SRF and enhances the
transcriptionaly activity of the SRF DNA binding site (CArG box) (fig. 1G). At
this framework, SRF knockdown abolished the effect of Nanog on myogenic markers
and contractility (fig. 1H). On the other hand, over-expression of SRF in LP
MSCs enhances the myogenic phenotype resembling the effect of Nanog.

Conclusion:
Taken together
these data suggest that Nanog reverses the effects of senescence on MSC proliferation
and myogenic differentiation capacity, thereby increasing the potential of MSC from aged
donors for cellular therapy and tissue regeneration.