SOX2 is required for adult human muller stem cell survival and maintenance of progenicity in vitro.
OBJECTIVE
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.
Bhatia
Bhairavi
B
UCL Institute of Ophthalmology, UK.
Singhal
Shweta
S
Tadman
Daniel N
DN
Khaw
Peng T
PT
Limb
G Astrid
GA
D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D015536:Down-Regulation; D018274:Electroporation; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing / Q000502:physiology; D006801:Humans; D020287:In Situ Nick-End Labeling; D019394:Ki-67 Antigen / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D055351:Retinal Neurons / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000502:physiology*; D014162:Transfection
D015415:Biomarkers; D019394:Ki-67 Antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D020169:Caspases