Investigating the role of SOX9 in human neural stem cells

Abstract

Neural stem cells (NSCs) exist in both embryonic and adult neural tissues and are characterized by their self-renewal capacity and multipotency that contribute to the generation of three major cell types in the vertebrate central nervous system (CNS):neurons, oligodendrocytes and astrocytes. The tremendous therapeutic potential of NSCs to treat the neurodegenerative diseases and repair brain injuries has provoked intensive study in the molecular regulation of their induction, maintenance and differentiation.

Previous study reported that Sox9, a member of high-mobility-group(HMG) containing SoxE transcription factors family, plays important roles in regulating the formation and maintenance of NSCs in both mouse and chick CNS, as well as the cell fate switch between neuronal and glial. Whether it plays similar roles in human NSCs (hNSCs)is still unknown. My RT-qPCR analysis showed that SOX9is expressed at a basal level in human embryonic stem cells (hESCs) and up-regulated upon commitment into neural lineage and maintained at a high level in hESCs-derived hNSCs. I therefore hypothesized that SOX9 might also be involved in the induction, maintenance and differentiation of hNSCs.

To test this, two stable hESC lines(HES2)were generated with each constitutively expressing short hairpin RNA (shRNA) against SOX9andGL2 Luciferase (Luc, as control) respectively. Upon neural induction, SOX9-knock-down(KD) hESCs were able to commit neural lineage and differentiate into NSCs/neurospheres (NSPs), however, these NSCs exhibited reduced multipotency and glial marker (GALC, CD44) expressions but enhanced self-renewal compared to the shLuc NSCs. Hence, SOX9 is required for both the induction and maintenance of multipotent hNSCs. Strikingly, extensive TUJ1+ neurites and advance groupings of these neurites into bundles were observed in SOX9-KD NSPs after three days and seven days neuronal differentiation respectively, suggesting premature neurogenesis as a result of SOX9 ablation.

In addition, RT-qPCR analysis revealed down-regulated expression of NSC marker HES1but induced proneural basic helix-loop-helix transcription factor MASH1in shSOX9-1208 NSCs. The inhibitory role of HES1 on the expression and functions of MASH1 has been reported to be essential for the timely generation of neurons. Hence, ablation of SOX9 is likely to relieve the inhibition on MASH1activity via down-regulated HES1expression and leads to early neuronal differentiation. Expression of the potent neurite blocker NG2 was also found to be reduced in SOX9-KD NSCs which may explain the extensive neurite network observed. Altogether, similar to previous studies in mouse NSCs, SOX9 is also required for the induction and maintenance of hNSCs. However, this study further reveals a putative novel role of SOX9 in preventing premature neuronal differentiation by regulating the expressions of HES1 to counteract MASH1 function and NG2 to control neurite outgrowth.