RRC ID 69254
Author Nefzger CM, Su CT, Fabb SA, Hartley BJ, Beh SJ, Zeng WR, Haynes JM, Pouton CW.
Title Lmx1a allows context-specific isolation of progenitors of GABAergic or dopaminergic neurons during neural differentiation of embryonic stem cells.
Journal Stem Cells
Abstract LIM homeobox transcription factor 1 alpha (Lmx1a) is required for the development of midbrain dopaminergic neurons, roof plate formation, and cortical hem development. We generated a reporter embryonic stem cell (ESC) line for Lmx1a and used it to track differentiation and extract neural progenitors from differentiating mouse ESCs. Lmx1a(+) cells gave rise to functional cortical upper layer GABAergic neurons or dopaminergic neurons depending on the culture conditions used for differentiation. Under chemically defined neurobasal conditions, ESC differentiation resulted in widespread and transient expression of Lmx1a, without the addition of exogenous factors such as sonic hedgehog (Shh), Wnts, and/or bone morphogenic proteins (BMPs). Under neutral conditions, Lmx1a(+) cells express genes known to be downstream of Lmx1a and cortical hem markers Wnt3a and p73. The majority of these cells did not express the ventral midbrain dopaminergic marker Foxa2 or dorsal roof plate marker BMP-2. Lmx1a(+) -Foxa2(-) cells were primed to become SatB2(+) GABAergic neurons and appeared to be resistant to dopaminergic patterning cues. PA6 coculture produced a substantial population of Lmx1a(+) progenitors that also expressed Foxa2 and on further differentiation gave rise to dopaminergic neurons at high frequency. We conclude that Lmx1a is a useful marker for the extraction of progenitors of GABAergic or dopaminergic neurons. We caution against the assumption that it indicates dopaminergic commitment during in vitro differentiation of ESCs. Indeed, in monolayer culture under neurobasal conditions, with or without the addition of Shh and fibroblast growth factor 8 (FGF8), Lmx1a(+) cells were predominantly progenitors of forebrain GABAergic neurons. We obtained dopaminergic cells in large numbers only by coculture with PA6 cells.
Volume 30(7)
Pages 1349-61
Published 2012-7-1
DOI 10.1002/stem.1105
PMID 22495882
MeSH Animals Cell Differentiation / physiology Cell Line Dopaminergic Neurons / cytology* Dopaminergic Neurons / metabolism* Embryonic Stem Cells / cytology* Embryonic Stem Cells / metabolism* Flow Cytometry GABAergic Neurons / cytology* GABAergic Neurons / metabolism* Immunohistochemistry LIM-Homeodomain Proteins / genetics LIM-Homeodomain Proteins / metabolism* MSX1 Transcription Factor / genetics MSX1 Transcription Factor / metabolism* Mice Polymerase Chain Reaction Transcription Factors / genetics Transcription Factors / metabolism*
IF 6.022
Human and Animal Cells MC3T3-G2/PA6(RCB1127)