A new role for microRNA-9 in human neural progenitor cells

A common feature of various age-dependent neurodegenerative disorders is the loss of specific types of neurons during disease progression.¹ One promising yet daunting approach is to replenish the affected brain regions with newly generated healthy neurons. This prospect is made possible by the controlled differentiation of human embryonic stem cells (hESCs) and patient-specific induced pluripotent stem cells (iPSCs) into human neural progenitor cells (hNPCs).² However, much needs to be learned about the molecular mechanisms that control the cellular behavior of hNPCs.

The transcriptional regulation of many aspects of neuronal development is well established,³ but posttranscriptional mechanisms remain to be further explored. MicroRNAs (miRNAs) are a class of small, noncoding RNAs (21–23 nucleotides) that regulate gene expression through base-pairing, mostly to 3′ untranslated regions (3′UTRs) of target mRNAs.^4,5 Among hundreds of human miRNAs, one of the most interesting is miR-9, which has been extensively studied due to its 100% conservation at the nucleotide level among different species and its specific expression in the mammalian brain.⁶ MiR-9 participates in neural cell fate determination and neuronal differentiation in flies, fish and rodents. However, its roles in the proliferation and migration of hNPCs are unknown.

hESCs can be differentiated into human postmitotic neurons through a series of steps in vitro.⁷ Embryoid bodies are derived first from hESCs grown in suspension, followed by neural induction and rosette formation, a hallmark of neuroepithelial cells in the neural tube.⁸ Rosette cells are isolated and expended in suspension to form neurospheres containing young hNPCs that can be heterogeneous in their developmental potential.⁸ After a month of maturation, these hNPCs can be differentiated into postmitotic neurons in the presence of certain growth factors. Interestingly, miR-9 expression is not detectable in SOX2- and PAX6positive rosette cells. Rather, it is specifically turned on in early-stage hNPCs and its level further increases during hNPC maturation, suggesting a unique function during this developmental process.

MiR-9 is processed from the same precursor miRNA as miR-9*. In young hNPCs, miR-9 can be knocked down with a locked nucleic acid (LNA) antisense probe without affecting miR-9* activity. Loss of miR-9 function led to smaller neurospheres and reductions in BrdU labeling and proliferation, as measured by the WST-1 assay, without affecting the survival of hNPCs. A similar phenotype was observed in rat NPCs. This effect was not due to precocious differentiation of hNPCs in the absence of miR-9 activity. In fact, as shown by detailed analyses with various molecular markers, miR-9 loss of function delayed the progression of hNPCs to a more mature neural progenitor fate.

Unexpectedly, loss of miR-9 activity also led to enhanced migration of young hNPCs away from neurospheres in a three-dimensional Matrigel cell migration assay, a system widely used to study cancer cells.⁹ Since these migrating cells retained their progenitor properties, the migratory phenotype was not a consequence of precocious differentiation. Again, this phenotype was confirmed in rat NPCs. Studies of the underlying mechanisms showed that the mRNA encoding stathmin, a developmentally regulated cytosolic phosphoprotein with a catastrophe-promoting microtubule-depolymerization acitivty,¹⁰ is a direct target of miR-9 regulation. During neuronal differentiation of hESCs, stathmin expression in hNPCs correlates inversely with that of miR-9, and partially loss of stathmin suppresses the effect of loss of miR-9 on hNPC migration and proliferation. Thus, stathmin is a key essential target of miR-9. This conclusion does not exclude the possibility that other targets are also important in hNPCs.

Do these findings in in vitro cell cultures have implications for improving in vivo therapeutic values of hESC-derived hNPCs? hNPCs without miR-9 activity show enhanced migration toward neocortex when transplanted into the medial ganglionic eminence of brain slices from embryonic day 14.5 mouse embryos. This finding confirms the developmental function of miR-9 in an in vivo setting, in which stathmin seems to be a key target as well. More importantly, when transplanted into adult brain of a mouse model of stroke,¹¹ hNPCs without miR-9 activity migrated more vigorously toward the site of injury. Thus, manipulation of the miR-9-stathmin interaction may limit unwanted proliferation and promote the migration of transplanted hNPCs. These functions may improve the efficacy of stem cell–based therapies for neurodegenerative diseases.

Figure 1.

A novel role for mir-9 in hNPCs. Mir-9 is not expressed in rosette cells derived from heSCs and its expression increases while its key target stathmin decreases during hNPC maturation. Mir-9 functions to maintain proliferation but limits the migration of young hNPCs.

Abbreviations

hESC

human embryonic stem cell

hNPC

human neural progenitor cell

iPSC

induced pluripotent stem cell

LNA

locked nucleic acid

miRNA

microRNA