Stem cell–based transplantation therapies for brain injuries and degenerative diseases hold great promise but also face major challenges, including the risk of uncontrolled proliferation and tumor formation and the failure to migrate to injured brain regions and properly incorporate into the damaged neuronal circuitry. Our findings here demonstrate an unexpected role for miR-9 in coordinating the proliferation and migration of young hNPCs derived from hESCs. Loss of miR-9 activity enhanced the migration of hNPCs transplanted into embryonic or adult mouse brains in a stroke model. Since so little is known about the cellular behavior of human neurons and their progenitor cells, our results may help improve the clinical efficacy of cell therapies in the nervous system using hESCs or iPS cells.
There are at least 400 miRNAs in humans and many of them are not evolutionarily conserved, suggesting human-specific functions. However, few have been studied with loss-of-function approaches (Bartel, 2009
). hESCs are an excellent model system for studying the physiological functions and specific targets of each endogenous human miRNA in different human cell types, such as in neural progenitors during neuronal specification from hESCs. To this end, the loss-of-function studies are critically important. Such approaches have been widely used in other model organisms. For instance, genetic knockout of miR-9 in Drosophila
revealed an essential role in ensuring the precision of SOP specification (Li et al., 2006
). Morpholino knockdown in zebrafish indicated a role for miR-9 in regulating the organizing activity and neurogenesis at the midbrain-hindbrain boundary (Leucht et al., 2008
). Interestingly, although miR-9 is 100% conserved at the sequence level, its expression patterns and functions differ significantly in different organisms (Delaloy and Gao, 2008
). In neuronal differentiation of hESCs, miR-9 expression is specifically turned on in hNPCs and maintained in postmitotic neurons. Our cell transplantation experiments further demonstrate a novel cell-autonomous function for miR-9 in regulating the migratory behavior of hNPCs.
Each miRNA may regulate hundreds of target mRNAs (Bartel, 2009
). It is possible many miRNAs exert their functions in a specific biological process mainly through a few target mRNAs. For instance, Senseless seems to be the key target in miR-9a regulation of SOP specification in Drosophila
(Li et al., 2006
). At the midbrain-hindbrain boundary in zebrafish, several components of the FGF signaling pathway is targeted by miR-9 (Leucht et al., 2008
). In fly wing development, dLMO is another key target of miR-9a (Biryukova et al., 2009
). It was reported that miR-9 suppressed the expression of the transcription factor Foxg1 but not the nuclear receptor Tlx (also known as Nr2e1) during Cajal-Retzius cell differentiation (Shibata et al., 2008
). However, another report suggested that overexpression of miR-9 suppressed the expression of Tlx during the terminal differentiation of adult mouse neural progenitor cells, resulting in increased differentiation and migration of newborn neurons (Zhao et al., 2009
). The reasons for this discrepancy are unknown. In our current study, stathmin seems to be a novel key target that is essential for miR-9 to promote proliferation and limit migration of multipotent hNPCs in the early neurosphere stage. Thus, this specific miR–target interaction is essential to maintain the hNPC pool at this particular stage. This conclusion, based on extensive loss-of-function studies both in cell culture and in transplantation experiments in mouse brains, does not exclude the likely possibility that some other miR-9 targets are also essential in this process.
There are several significant differences between the study by Zhao et al. and ours. First, we used hESC-derived neural progenitors instead of adult mouse neural progenitors. Second, we performed our studies using loss-of-function approaches to examine the physiologically relevant functions of endogenous miR-9 in newly formed hNPCs. After miR-9 knockdown, hESC-derived hNPCs maintained their neural progenitor properties and exhibited enhanced migration, both in vitro and after transplantation into mouse embryonic or adult brains. In contrast, Zhao et al. reported that migrating cells after miR-9 overexpression expressed neuronal but not progenitor markers, and they failed to detect a change in cell differentiation in NPCs treated with miR-9 antisense RNA. Third, the miR-9–Tlx
mRNA interaction was thought to be relevant only in adult neural progenitors shortly before terminal differentiation was induced with retinoic acid or forskolin (Zhao et al., 2009
). In contrast, we studied newly formed hNPCs before the expansion of the progenitor pool. Thus, the same miRNA may have distinct functions at different developmental stages of various NPCs, likely by regulating different spatially and temporally expressed mRNA targets (Figure S7D
The molecular interaction between miR-9 and stathmin may have important implications for other biological processes as well. For instance, miR-9 is overexpressed in primary but not metastatic brain tumors (Nass et al., 2008
), and stathmin enhances cancer cell migration and the metastatic phenotype (Baldassarre et al., 2005
; Rosenfeld et al., 2008
). Thus, manipulation of the miR-9–stathmin interaction may suppress tumor formation and enhance the migration of transplanted hNPCs and is therefore a potential target for improving the efficacy of stem cell–based therapies.