Reprogramming of Rat Fibroblasts into Induced Neurons by Small-Molecule Compounds In Vitro and In Vivo

Abstract

Cell replacement is a promising approach for neurodegenerative disease treatment. Somatic cells such as fibroblasts can be induced to differentiate into neurons by specific transcription factors; however, the potential of viral vectors used for reprogramming to integrate into the genome raises concerns about the potential clinical applications of this approach. Here, we directly reprogrammed rat embryonic skin fibroblasts into induced neurons (iNs) via six small-molecule compounds (SMs) (VPA, CHIR99021, forskolin, Y-27632, Repsox, and P7C3-A20). iNs exhibit typical neuronal morphology, and immunofluorescence showed that more than 96% of the iNs expressed the early neuronal marker class III beta-tubulin (TUJ1) and that more than 91% of iNs expressed the mature neuronal marker neuron-specific enolase (NSE) after 10 days of reprogramming. Quantitative real-time polymerase chain reaction also showed that most iNs expressed the dopaminergic neuron marker tyrosine hydroxylase, the neural marker Nur correlation factor 1, the (γ-aminobutyric acid, GABA) GABAergic neuronal marker GABA, and the cholinergic neuron marker choline acetyltransferase. In addition, we found that cell proliferation decreased during reprogramming and that protein synthesis increased initially and then decreased. SMs were mixed with hydrogels, and the hydrogels were implanted subcutaneously into the backs of rats. After 7 days, the TUJ1 and NSE proteins were expressed in surrounding tissues, indicating that SMs caused reprogramming in vivo. In summary, rat skin fibroblasts can be efficiently reprogrammed into iNs by SMs in vitro and in vivo.

Tissue engineering RPE sheet derived from hiPSC-RPE cell spheroids supplemented with Y-27632 and RepSox

“…The TGF-β signaling pathway is known to regulate many biological functions including cell proliferation, differentiation, apoptosis, adhesion, and migration through the Smad and non-Smad pathways [ 36 ]. The TGF-β signaling pathway also promotes EMT and the inhibition of it can reduce the passage-dependent loss of epithelial potential [ 37 ]. Based on its effect on the TGF-β signaling pathway, Repsox is used in cellular reprogramming and EMT intervention [ 18 , 37 ].…”

“…The TGF-β signaling pathway also promotes EMT and the inhibition of it can reduce the passage-dependent loss of epithelial potential [ 37 ]. Based on its effect on the TGF-β signaling pathway, Repsox is used in cellular reprogramming and EMT intervention [ 18 , 37 ]. It has also been reported to help maintain epithelial-like morphology in primary mouse RPE cells [ 18 , 38 ].…”

Innovative Approaches and Advances for Hair Follicle Regeneration

“…Previous reports have revealed that treatment of fibroblasts with small molecules can produce papilla-like cells and that mouse fibroblasts produce hair papilla-like cells with greater hair induction capacity . Recent studies use small-molecule compounds (VCFYRP) efficiently reprogrammed rat skin fibroblasts into induced neurons in vitro and in vivo . There was also research that used small molecules such as SB431542, LDN-193189, CHIR99021, and SU5402 to inhibit fibroblast growth factor receptor, vascular endothelial growth factor receptor, and DAPT (inhibits γ-secretase) to replace transcription factors to improve reprogramming efficiency. , Lee et al reported that the formation of pigmented HF organoids can be obtained through the aggregation of PSCs to form follicular-like skin-like organs and progressive regulation of TGF-β, BMP, and FGF signaling pathways.…”

Reprogramming Glioblastoma Cells into Non-Cancerous Neuronal Cells as a Novel Anti-Cancer Strategy

“…Non-surgical genetic approaches that can alter the inherited cancerous properties of GBM cells have become possible thanks to recent conceptual and technical breakthroughs. An attractive strategy is cell transdifferentiation using transcriptional factors [ 22 , 23 , 24 ]. Soon after the groundbreaking generation of induced pluripotent stem cells (iPSCs) from somatic cells such as skin fibroblasts [ 25 , 26 ], Kim et al and others reported and developed direct reprogramming of fibroblasts.…”

“…Converted iNPCs possess the potential to give rise to all three major subtypes of neural cells: astrocytes, oligodendrocytes, and neurons, all of which are presumably useful for disease modeling and basic studies of early neural fate induction [ 29 , 30 ]. Moreover, transdifferentiation provides a novel and more efficient genetic method of manipulating cell lineages for the treatment of brain injuries and neurodegenerative diseases [ 22 , 23 , 24 ].…”