Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation.
The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells.


CONCLUSIONS
Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction.
Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065, Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10065.
Elizabeth L
EL
Calder
Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065, studerl@mskcc.org tchieuj@mskcc.org.
Jason
http://orcid.org/0000-0002-9793-9836
J
Tchieu
Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065.
Julius A
JA
Steinbeck
Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065.
Edmund
E
Tu
Pediatrics, Weill Cornell Medical College, New York, New York 10065, and.
Sotirios
S
Keros
Departments of Anesthesiology and.
Shui-Wang
SW
Ying
Departments of Anesthesiology and.
Manoj K
http://orcid.org/0000-0001-9198-7450
MK
Jaiswal
Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065.
Daniela
http://orcid.org/0000-0003-0897-8161
D
Cornacchia
Departments of Anesthesiology and.
Peter A
http://orcid.org/0000-0002-5973-9104
PA
Goldstein
Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065, Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10065.
Viviane
V
Tabar
Center for Stem Cell Biology and Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10065, Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10065 studerl@mskcc.org tchieuj@mskcc.org.
Lorenz
L
Studer
D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D053823:Hedgehog Proteins / Q000494:pharmacology*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D008297:Male; D009046:Motor Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology; D000074290:Zinc Finger Protein Gli3
C065683:GLI3 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; C509239:SHH protein, human; D000074290:Zinc Finger Protein Gli3; D014212:Tretinoin
Gli3; SHH signaling; directed differentiation; motoneurons; pluripotent stem cells; retinoic acid