Abstract
The purification of pluripotent stem cell-derived cortico-fugal projection neurons (PSC-CFuPNs) is useful for disease modeling and cell therapies related to the dysfunction of cortical motor neurons, such as amyotrophic lateral sclerosis (ALS) or stroke. However, no CFuPN-specific surface markers for the purification are known. Recently, microRNAs (miRNAs) have been reported as alternatives to surface markers. Here, we investigated this possibility by applying the miRNA switch, an mRNA technology, to enrich PSC-CFuPNs. An array study of miRNAs in mouse fetal brain tissue revealed that CFuPNs highly express miRNA-124-3p at E14.5 and E16.5. In response, we designed a miRNA switched that responds to miRNA-124-3p and applied it to mouse embryonic stem cell (ESC)-derived cortical neurons. Flow cytometry and quantitative polymerase chain reaction (qPCR) analyses showed the miRNA-124-3p switch enriched CFuPN-like cells from this population. Immunocytechemical analysis confirmed vGlut1/Emx1/Bcl11b triple positive CFuPN-like cells were increased from 6.5 to 42%. Thus, our miRNA-124-3p switch can uniquely enrich live CFuPN-like cells from mouse ESC-derived cortical neurons.
Highlights
Corticofugal projection neurons (CFuPNs) are projection neurons that connect the cerebral cortex and subcortex and transmit excitatory input to the subcerebral nucleus
We found that miRNA 124-3p is a specific marker of CFuPNs
Scientists have theorized that the transplantation of CFuPNs is a promising regenerative medicine related to these ailments
Summary
Corticofugal projection neurons (CFuPNs) are projection neurons that connect the cerebral cortex and subcortex and transmit excitatory input to the subcerebral nucleus. In some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS), corticospinal motor neurons (CSMNs), which are a subset of CFuPNs, degenerate. Several groups have used CFuPNs to model these disorders in vitro (Sances et al, 2016) and to develop cell therapies to treat them (Gaillard et al, 2007; Steinbeck et al, 2012; EspunyCamacho et al, 2013; Motono et al, 2016; Sano et al, 2017). Cell sorting is done after the differentiation to purify the desired cell population (Okano et al, 2013; Doi et al, 2014; Takeda et al, 2018), but this is not an option for CFuPNs, because no specific cell surface markers or antibodies are known. The insertion of a reporter gene is one option for the purification, but this approach prohibits cell therapies
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