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Nanog Breathes New Life into Old Cells

Original article from STEM CELLS

"Nanog Reverses the Effects of Organismal Aging on Mesenchymal Stem Cell Proliferation and Myogenic Differentiation Potential"

Autologous mesenchymal stem cells (MSCs) are an attractive source of cells for use in a regenerative capacity, however several problems have arisen which may limit their extensive clinical use. These problems include the decrease number and quality of cells with increasing donor age (Caplan 2007, Han et al 2010 and Sethe et al) and the loss of proliferation and differentiation potential upon expansion in vitro (Banfi et al, Baxter et al and Bonab et al). Previous studies have found that expression of the pluripotency-associated gene Nanog in bone marrow derived MSCs (BM-MSCs) leads to accelerated growth and the enhancement of chondrogenic and osteogenic differentiation (Go et al and Liu et al). Now in a study published in Stem Cells, researchers from the laboratory of Stelios T. Andreadis from the University at Buffalo, State University of New York, USA have analysed the effects of ectopic expression of Nanog on BM-MSCs from older donors, and their results show that this can reverse the aging-mediated loss of proliferation and myogenic differentiation potential, partly mediated through activation of the TGF-β pathway (Han et al).

They started by lentivirally infecting ovine neonatal BM-MSCs (Nanog. Successfully transfected cells expressed high levels of Nanog which was localized exclusively to the nucleus, but this did not lead to the induction of endogenous pluripotency factors (Oct4, Nanog and Sox2). Global gene expression analysis found that 5.3% of genes (623 transcripts; 243 up-regulated, 380 down-regulated) were differentially expressed between nBM-MSCs and aBM-MSCs and therefore may be affected by organismal aging. Expression of Nanog in nBM-MSCs caused changes in 5.7% (678 genes; 283 up-regulated and 395 down-regulated) and 8.2% (967 transcripts; 387 up-regulated, 580 down-regulated) in aBM-MSCs. Hierarchical analysis found that Nanog-expressing MSCs clustered together rather than with their non-transfected control counterparts, while further detailed analysis suggested that Nanog over-expression modified the gene expression of aBM-MSCs to that more akin to nBM-MSCs. Comparison of genes differentially expressed in response to Nanog over-expression that have known Nanog-bindings sites within regulatory regions (Chambers et al) identified 75 genes in aBM-MSC and 56 genes in nBM-MSC which may be directly regulated by Nanog. Pathway analysis found that the chemokine- and PPAR signalling pathways were altered in both nBM-MSCs and aBM-MSCs, although some pathways were differentially expressed in response to Nanog. In aBM-MSCs, pathways involved in the cell cycle, DNA synthesis/replication and nucleotide mismatch/repair were altered to a greater extent than in nBM-MSCs, while in nBM-MSCs genes involved in TGF-β and cancer signaling pathways were upregulated to a greater extent. Further cellular analysis found that both clonogenic potential and cell proliferation were increased in BM-MSCs overexpressing Nanog, an effect that was more pronounced in the aBM-MSCs, where the number of population doublings (PDs) until senescence increased from an estimated 14 PDs to 39 PDs upon Nanog over-expression. Importantly, for the potential of MSCs in therapeutic use, the increased proliferation rate was not accompanied by an increase in chromosomal abnormalities.

Previous studies have found that Nanog expression in MSCs can promote osteogenic and chondrogenic differentiation and inhibit adipogenesis (Go et al, Kochupurakkal et al, Lang et al and Liu et al) although any effects on myogenesis and aging properties had yet to be investigated. Smooth muscle cell (SMC) markers αSMA and calponin both increased after Nanog expression in both MSC cell types, but no differences were observed during myogenic differentiation. Q-PCR analysis of other smooth-muscle contractility-related genes found that SM22 and caldesmon were increased in Nanog-expressing aBM-MSCs only, while smoothelin expression was increased in both MSC types. These findings correlated to an increase in the contractility of SMC differentiated Nanog-expressing nBM-MSCs and aBM-MSCs as measured after imbedding in fibrin gels and being allowed to contract in the presence of TGF-β1. Vasoreactivity of vascular constructs in response to receptor (Endothelin-1, U46619) or non-receptor (KCl)-mediated pathways prepared from both MSC types fond that Nanog expression increased contractility significantly, with aBM-MSCs showing a 10-fold increase taking contractility levels above that of control nBM-MSCs. These experiments were then moved into human MSCs from three donors and two anatomical locations (BM and hair-follicle (HF)), which again found that Nanog over-expression led to enhanced vascular contractility.

Finally, the molecular pathways underlying Nanog's effects were studied; focusing on the role of TGF-β1. Q-PCR validated the increase in TGF-β1 expression upon Nanog over-expression in nBM-MSCs and aBM-MSCs, while Nanog expression also induced Smad2 phosphorylation, a TGF-β1 target. Addition of a TGF-β type I receptor kinase inhibitor (SB431542) also inhibited Smad2 phosphorylation in response to Nanog expression; overall suggesting that TGF-β1 mediated the Nanog induced enhanced myogenic potential of MSCs.

Altogether, these data suggest that the over-expression of Nanog can rejuvenate aged MSCs to give characteristics of neonatal MSCs with seemingly no ill effects, such as karyotypic abnormalities. As most patients who will require MSC-based therapeutics and tissue replacement are likely to be elderly, Nanog over-expression may allow the expansion of endogenous MSCs to a number and of a quality which would significantly enhance their therapeutic usefulness. This research, primarily ovine-based could now be easily investigated in human cells in detail; in part started within this study. Interestingly, the authors note that Oct4 over-expression in their system did not yield the same effects as Nanog over-expression, while Nanog over-expression additionally did not lead to the increase in endogenous expression of pluripotency-associated genes, together suggesting that Nanog plays a specialised role in MSCs that is perhaps aside from pluripotency associated effects.



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STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.