Abstract
Purpose.:
SOX2, a high-mobility group transcription factor, is expressed by retinal progenitors during development. It has been associated with the ability of progenitor cells to differentiate into retinal neurons and is highly expressed by human Müller stem cells (hMSCs) in culture. The authors investigated the role of this factor in the maintenance of progenicity and neural differentiation of hMSCs in vitro.
Methods.:
SOX2 silencing was induced by transfection of hMSCs in culture with two pGSU6-GFP SOX2 silencing constructs and a scrambled control vector. Silencing was confirmed by examination of gene and protein expression coding for SOX2. Effects of SOX2 downregulation were investigated by expression of proliferation (Ki67) and apoptotic (TUNEL, caspase) cell markers and by the expression of markers of retinal neurons (HuD, βIII tubulin, rhodopsin, BRN3B, ISL1), glia (vimentin), and the progenitor marker PAX6.
Results.:
SOX2 silencing caused hMSCs to rapidly adopt a neural-like morphology and was accompanied by the upregulation of specific markers of retinal neurons, including βIII tubulin, rhodopsin, BRN3B, and ISL1, and by the downregulation of the neural progenitor marker PAX6 and the glial cell marker vimentin. Interestingly, SOX2 silencing induced apoptosis, suggesting a crucial role of this factor on hMSC survival in vitro.
Conclusions.:
These in vitro results parallel that seen when Sox2 is silenced in neural stem cells of lower species during development, and they suggest that Sox2 may have an important role in adult hMSC differentiation into retinal neurons in vitro.
During development, Müller glia and retinal neurons share a common progenitor that is multipotent at all stages of retinal histogenesis.
1 Müller cells constitute a source of neural retinal progenitors in the adult zebrafish
2 and the postnatal and damaged adult chick
3 , rat
4,5 and mouse retinas.
6,7 Recent studies have demonstrated that the adult human retina also harbors a population of Müller glia with stem cell characteristics.
8,9 Human Müller stem cells (hMSCs) express various markers of neural stem cells in vitro and in situ, including the transcription factor SOX2.
8,9 This gene encodes a member of the SRY-related high-mobility group (HMG) family of transcription factors involved in regulation of embryonic development and determination of cell fate.
10 SOX2 plays a role in many developmental processes and is essential for the development of the CNS,
11 sensory taste bud cells,
12 sensory cells of the inner ear and eye,
13 and branching morphogenesis and epithelial cell differentiation in the lung.
14 Sox2 is also expressed in the adult brain where neural stem cells divide at a low rate, both renewing themselves and generating neural precursors.
15 In culture, SOX2-positive hMSCs show functional properties of neural stem cells, including self-renewal and multipotency.
8 Sox2 has also been shown, in combination with the expression of the genes
Oct 3/4,
cMyc, and
Klf to induce pluripotent stem cells from various somatic cells, including fibroblasts and epithelial cells.
16
Inhibition of SOX2 by injection of antisense morpholinos or dominant-negative forms of SOX2 into
Xenopus embryos results in reduced or missing eyes.
17 Within the retina Sox2 is expressed in proliferating cells and is downregulated by all cell types as they start to migrate to the different retinal areas.
18 Sox2 is also required for the development of amacrine cells and Müller glia,
19 and the inhibition of Sox2 signaling results in neural progenitor cells delaminating from the ventricular zone and exiting the cell cycle prematurely. This is associated with general loss of pan-neural and regional progenitor markers and the onset of expression of early neuronal differentiation markers.
15,16 In the adult Sox2 mutant mouse, precursor cell proliferation and generation of new neurons in neurogenic areas are decreased, whereas GFAP/Nestin-positive hippocampal cells are greatly diminished.
20 Given that hMSCs can be made to differentiate in vitro into cells that exhibit characteristics of retinal neurons
8 and given the importance of this gene during neural retinal development and its well-recognized function in the maintenance of “stemness” on neural stem cells, we investigated whether SOX2 may also play a role in the maintenance of progenicity of adult hMSCs in culture and whether downregulation of this gene may modify the neural differentiation ability of these cells in vitro.
Effect of SOX2 Silencing on Progenitor, Glial, and Neuronal Cell Marker Expression