Abstract
MicroRNAs are key regulators of neural cell proliferation, differentiation and fate choice. Due to the limited access to human primary neural tissue, the role of microRNAs in human neuronal differentiation remains largely unknown. Here, we use a population of long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from human embryonic stem cells to study the expression and function of microRNAs at early stages of human neural stem cell differentiation and neuronal lineage decision. Based on microRNA expression profiling followed by gain- and loss-of-function analyses in lt-NES cells and their neuronal progeny, we demonstrate that miR-153, miR-324-5p/3p and miR-181a/a* contribute to the shift of lt-NES cells from self-renewal to neuronal differentiation. We further show that miR-125b and miR-181a specifically promote the generation of neurons of dopaminergic fate, whereas miR-181a* inhibits the development of this neurotransmitter subtype. Our data demonstrate that time-controlled modulation of specific microRNA activities not only regulates human neural stem cell self-renewal and differentiation but also contributes to the development of defined neuronal subtypes.
Highlights
Based on their ability to self-renew and differentiate into any type of somatic cells, human embryonic and induced pluripotent stem cells represent an unrestricted source of specialized cells for basic and applied research
To identify miRNAs associated with early stages of human neuronal differentiation, we analyzed miRNA expression profiles of human embryonic stem cells, hES cell-derived neuroepithelial-like stem cells and their differentiated neuronal progeny (Figure 1C, D)
We selected two different time points during the in vitro differentiation of lt-NES cells: 15 days (ND15, Figure 1C), when about 20% of the cells express the pan-neuronal marker b-III tubulin, and 30 days (ND30, Figure 1D), when the number of b-III tubulin-positive neurons reaches more than 50% (Figure S1)
Summary
Based on their ability to self-renew and differentiate into any type of somatic cells, human embryonic and induced pluripotent stem (hES and iPS) cells represent an unrestricted source of specialized cells for basic and applied research. Different methods have been developed to derive neural stem cells and differentiated neural cell types from human pluripotent stem cells (hPSC) We have established a protocol to obtain homogeneous long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) from hPSC. Lt-NES cells have been successfully used to model human neurodegenerative diseases [4,5] and represent a reductionist model for studying early stages of human neural stem cell differentiation in vitro [6]. During development miRNAs contribute to the establishment and maintenance of specific cell fates [8]. MiR-9/9* and miR-124 were shown to significantly contribute to the direct conversion of fibroblasts into neurons [12,13]
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