Brief exposure to small molecules allows induction of mouse embryonic fibroblasts into neural crest‐like precursors

Abstract

In this study, we propose a novel method for inducing neuronal cells by briefly exposing them to small‐molecule cocktails in a step‐by‐step manner. Global gene expression analysis with immunohistochemical staining and calcium flux assays reveal the generation of neurons from mouse embryonic fibroblasts. In addition, time‐lapse imaging of neural precursor‐specific enhancer expression and global gene expression analyses show that the neurons are generated by passing through a neural crest‐like precursor stage. Consistent with these results, the neural crest‐like cells are able to differentiate into neural crest lineage cells, such as sympathetic neurons, adipocytes, osteocytes, and smooth muscle cells. Therefore, these results indicate that brief exposure to chemical compounds could expand and induce a substantial multipotent cell population without viral transduction.

Exposure to small molecule cocktails allows induction of neural crest lineage cells from human adipose-derived mesenchymal stem cells

“…Based on our previous study, we selected the following four chemical patterns (cp1–4), and examined their effects on human ADSCs ( Fig 1A ); cp1: the same combination as we previously reported [ 14 ]; cp2: adding DZNep, a histone methyltransferase inhibitor [ 17 ], and EPZ, a selective inhibitor of DOT1L (a protein methyltransferase catalyzing the methylation of H3K4 [ 18 ]) to cp1; cp3: adding ISX9, inducer of neuronal differentiation [ 19 ], to cp1; and cp4: excluding VPA and Tranyl from cp3. Subsequently, NGFR expression was analyzed via immunofluorescence measurement.…”

“…Immunohistochemical staining analysis was performed as previously reported [ 5 , 14 ]. Briefly, the samples were fixed with 3.7% formaldehyde (FUJIFILM Wako Pure Chemical Industries) and permeabilized with PBS containing 0.2% Tween-20 (FUJIFILM Wako Pure Chemical Industries).…”

“…RT-qPCR analysis was performed as previously reported [ 14 ]. Briefly, total RNA was isolated from the samples using the ReliaPrep RNA Cell Miniprep System (Promega).…”

“…Viral delivery of transcriptional factors or small molecule-based signaling modulation have allowed direct conversion of somatic cells into other cell types without passing through pluripotent state [ 11 – 13 ]. Based on this concept, we previously reported that the combination of 7 small molecules (valproic acid (VPA), a histone deacetylase inhibitor; CHIR99021 (CHIR), a glycogen synthase kinase 3 inhibitor which activates WNT signaling; E-616452, an inhibitor of TGF-β type 1 activin-like kinase receptor (ALK) 5; Forskolin (FSK), a cAMP signaling activator; Tranylcypromine (Tranyl), a lysine-specific demethylase 1 inhibitor; Dorsomorphin (DM), a bone morphogenic protein (BMP) signaling inhibitor; SB431542 (SB), an inhibitor of TGF-β type1 ALK4, 5, 7) allowed induction of mouse embryonic fibroblasts (MEFs) into neural crest-like precursors [ 14 ]. This approach is useful for cell induction because of their cell permeability, non-immunogenicity, and ease of standardization of the small molecules.…”

Current progress in the derivation and therapeutic application of neural stem cells

“…These cells resemble primary NSCs in terms of their long-term self-renewal and tripotent differentiation abilities. Takayama et al 39 also developed a small-molecule cocktail composed of VPA, Forskolin (an adenylyl cyclase activator), Parnate, CHIR99021, Repsox, Dorsomorphin (a selective inhibitor of BMP signaling) and SB431542 (a selective inhibitor of TGF- β receptor I, such as ALK4 and ALK7) to induce neural crest-like precursors from mouse embryonic fibroblasts. Zheng et al 40 showed that a combination of A83-01, Purmorphamine (a smoothened receptor agonist), VPA and Thiazovivin (a selective Rho-associated protein kinase inhibitor) can directly lead to the generation of ciNSCs.…”