BMP and TGF-β pathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human pluripotent stem cells.
Although many laboratories currently use small molecule inhibitors of the BMP (Dorsomorphin/DM) and TGF-β (SB431542/SB) signaling pathways in protocols to generate midbrain dopamine (mDA) neurons from hES and hiPS cells, until now, these substances have not been thought to play a role in the mDA differentiation process. We report here that the transient inhibition of constitutive BMP (pSMADs 1, 5, 8) signaling, either alone or in combination with TGF-β inhibition (pSMADs 2, 3), is critically important in the upstream regulation of Wnt1-Lmx1a signaling in mDA progenitors. We postulate that the mechanism via which DM or DM/SB mediates these effects involves the up-regulation in SMAD-interacting protein 1 (SIP1), which results in greater repression of the Wnt antagonist, secreted frizzled related protein 1 (Sfrp1) in stem cells. Accordingly, knockdown of SIP1 reverses the inductive effects of DM/SB on mDA differentiation while Sfrp1 knockdown/inhibition mimics DM/SB. The rise in Wnt1-Lmx1a levels in SMAD-inhibited cultures is, however, accompanied by a reciprocal down-regulation in SHH-Foxa2 levels leading to the generation of few TH+ neurons that co-express Foxa2. If however, exogenous SHH/FGF8 is added along with SMAD inhibitors, equilibrium in these two important pathways is achieved such that authentic (Lmx1a+Foxa2+TH+) mDA neuron differentiation is promoted while alternate cell fates are suppressed in stem cell cultures. These data indicate that activators/inhibitors of BMP and TGF-β signaling play a critical upstream regulatory role in the mDA differentiation process in human pluripotent stem cells.
Department of Neuroscience, Farber Institute for Neurosciences, Thomas Jefferson University, 900 Walnut Street, Philadelphia, PA. 19107, USA.
Jingli
J
Cai
Stephanie
S
Schleidt
Joshua
J
Pelta-Heller
Danielle
D
Hutchings
Gregory
G
Cannarsa
Lorraine
L
Iacovitti
D015153:Blotting, Western; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D059290:Dopaminergic Neurons / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000187:drug effects / Q000502:physiology*; D000094742:Secreted Frizzled-Related Proteins
D019485:Bone Morphogenetic Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D016212:Transforming Growth Factor beta; C108381:GEMIN2 protein, human; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins; C516138:dorsomorphin