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CD49f enhances multipotency and maintains stemness through the direct regulation of OCT4 and SOX2

From Stem Cells 
By Stuart P. Atkinson

Heterogeneity of stem cells and their progeny has been rightly identified as being a major problem in taking stem cell therapies toward the clinic. Many important studies have attempted to address this problem with the ultimate aim of devising strategies to segregate interesting or potentially useful cell types. A example of this was a recent report on the StemCellsPortal, which described a study in which researchers have started to identify cohorts of cell surface markers for mouse stem cells and embryo’s which may ultimately allow high purity cell sorting (Rugg-Gunn, Cox and Lanner et al). Towards this goal in human stem cells, researchers from the group of Kyung-Sun Kang, while working of cultivation techniques for human mesenchymal stem cells (hMSC) which allow better proliferation and higher osteogenic and adipogenic capabilities, discovered the cell surface marker CD49f to be an important mediator of stemness through PI3K/AKT/p53 activity and went on to show a further role for CD49f in pluripotency of human ESCs (hESCs) through direct regulation of OCT4 and SOX2 (Yu and Yang et al).

Under normal culture conditions, hMSCS are grown as a monolayer, however in this study, hMSC grown under non-adherent conditions as spheroid colonies of about 10,000 cells were examined. Cells grown under both adherent and non-adherent conditions were positive for the hMSC markers CD44 and CD90 and negative for the human haematopoietic stem cells (hHSC) markers CD34 and CD117 and could both differentiate adequately to the adipogenic and osteogenic lineage. Indeed, spheroid-hMSCs showed higher levels of induction for both lineages, exhibiting higher levels of genes indicative of adipogenic (C/EBP-b, aP2, PPAR-g and LEP) and osteogenic (RUNX2 and BGLAP (osteocalcin)) genes which was coupled with an increased proliferative rate. Levels of phosphorylated PI3K and AKT were also elevated while the levels of endogenous p53, p21 and p16 were reduced in spheroid-hMSCs as compared to monolayer-hMSCs. Inhibition of GSK3 (with small molecules BIO or CHIR99021) led to an increase in spheroid formation while inhibition of PI3K (with LY294002 or wortmannin) decreased spheroid formation suggesting a critical role for the PI3K/AKT/GSK3b signaling pathway. As Integrins are understood to activate growth-associated signal transduction pathways such as PI3K/AKT via local protein kinases, they were analysed to understand any potential role in hMSC-spheroid formation.

Analysis of cell surface markers through flow cytometric and immunohistochemcial analysis of the two hMSC populations found that both CD49f (or ITGA6) and CD104, which form a heterodimer, were up-regulated in spheroid-hMSCs, with a greater proportion of cells each expressing higher levels of CD49f compared to monolayer-hMSCs.   CD49f is known to be associated with both tumourigenic potential (Lawson et al), therapeutic potential in bone marrow-derived multipotent stem cells (Lee et al) and in long-term multi-lineage grafts in HSCs (Notta et al) and is prominently expressed in hESCs, with its ligand, laminin, used as a growth substrate for hESCs (Meng et al, Miyazaki et al, Rodin et al and Rowland et al). Subsequent purification of CD49f positive and negative hMSCs demonstrated that tyrosine kinases downstream of the integrins (PXN, FAK and ILK) were increased in the positive fraction, and these cells were more efficient at spheroid formation. Spheroids formed from these cells also expressed higher protein levels of activated FAK, PI3K and AKT and lower levels of p53, p21 and p16 than spheroids formed from CD49f negative hMSCs or monolayer-hMSCs. Additionally siRNA of CD49f or p53 led to a respective decrease and increase in the number and size of hMSC spheroids formed. CD49f-overexpressing hMSCs also had a significantly enhanced osteogenic differentiation potential when compared to wild-type hMSCs, which was further boosted in the presence of BIO and inhibited by LY294002, while adipogenic differentiation was also enhanced in the CD49f-overexpressing hMSCs, but BIO and LY294002 both inhibited this enhancement.

This enhancement in multi-potentiality led the authors to study a potential link of CD49f with pluripotency. Spheroid-hMSCs expressed higher levels of pluripotency associated mRNAs (OCT4, SOX2, NANOG and LIN28) than monolayer-hMSCs, while reduction of either OCT4 or SOX2 through siRNA significantly repressed CD49f expression, resulting in decreased sphere formation. Overexpression of OCT4, SOX2, OCT4/SOX2 or OCT4/SOX2/LIN28/NANOG in hMSCs all led to an increase in CD49f expression, while ChIP assays found increased SOX2 and OCT4 binding at specific regions of the CD49f promoter. A potential role for CD49f in pluripotency was then studied in hESCs and during differentiation as embryoid bodies. hESCs had higher levels of CD49f, OCT4, SOX2 and NANOG than hMSCs, which all decreased upon differentiation, while a reduction of CD49f in hMSCs led to a gradual increase in a differentiated morphology, a reduction in AP-positive colonies and a decrease in OCT4 and SOX2 protein and RNA expression. Further, an inhibition of the PI3K/AKT/GSK3b pathway was also noted along with an increase in the p53, p21 and p16 expression, while treatment of hESCs with LY294002 also led to a dose-dependent decrease in NANOG and CD49f expression overall suggesting that CD49f plays a pivotal role in maintaining cellular pluripotency through the PI3K/AKT/p53 pathway.

Overall this paper represents another step forward in our understanding of the complex interplay between cell surface interactions, signalling pathways and direct DNA/chromatin interaction that mediates multipotentiality in human stem cell populations and further represents an important step in the right direction for stem cells potential in the clinic. The necessity for high quality and high purity stem cells is paramount for any stem cell strategy for human disease treatment, and hMSCs represent one of the more important stem cell populations for therapeutics in an aging society.

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