DMH1, a Highly Selective Small Molecule BMP Inhibitor Promotes Neurogenesis of hiPSCs: Comparison of PAX6 and SOX1 Expression during Neural Induction

Abstract

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist, Noggin, and the small molecule SB431542, respectively, induces efficient neuralization of hiPSCs, a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost, consistent activity, and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1, a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration, we could selectively modulate the number of SOX1 expressing cells, whereas PAX6, another neural precursor marker, remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations, therefore, suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons, a subset of which also express tyrosine hydroxylase. Thus, the combined use of DMH1, a highly specific BMP-pathway inhibitor, and SB431542, a TGF-β1-pathway specific inhibitor, provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors.

Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification

“…6). YAP activity is decreased during mammalian neurogenesis (19,36,37) in conjunction with Sonic Hedgehog (36,37) and Smad (38) signaling pathways, which are ubiquitous targets of soluble factors used to promote neuronal differentiation of hES cells (39)(40)(41)(42)(43)(44)(45)(46).…”

Small molecule inhibitor of the bone morphogenetic protein pathway DMH1 reduces ovarian cancer cell growth

“…BMP small molecule inhibitors have radically progressed from toxic non-selective/specific inhibitors such as Dorsomorphin and LDN-193189 to more selective and specific inhibitors such as DMH1 [51; 52; 53; 54; 55]. Other groups have used Dorsomorphin and LDN-193189 for successful outcomes in many cancer types such as Lung, Prostate, Breast and acute lymphoblastic leukemia (ALL) [56; 57; 58; 59].…”

Dedifferentiation of patient‐derived glioblastoma multiforme cell lines results in a cancer stem cell‐like state with mitogen‐independent growth

“…Immunocytochemistry was performed as described previously 18. The following primary antibodies were used: Oct-4, GFAP (1:200, Cell Signaling), ALDH1A1 (1:200), smooth muscle actin (1:50), GATA4 (1:200; Sigma-Aldrich), CD44 (1:400, Abcam) and β-III tubulin (1:200, Millipore).…”

“…Reactions for each sample were performed in triplicate with negative controls. Primer sequences were as follows: EGFR forward, 5′-TCCTCTGGAGGCTGAGAAAA-3′, EGFR reverse, 5′-GGGCTCTGGAGGAAAAGAAA-3′ 19, Oct4 forward, 5′-AAAGCGAACCAGTATCGAGAAC-3′, Oct4 reverse, 5′-GCCGGTTACAGAACCACACT-3′ and GAPDH forward, 5′-GCTCTCTGCTCCTCCTGTTC-3′, GAPDH reverse, 5′-CGTTGACTCCGACCTTCAC-3′ 18.…”