Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons

Abstract

Recently, direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors. This approach may provide alternative cell resources for drug discovery and regenerative medicine, but applications could be limited by the genetic manipulation involved. Here, we show that mouse fibroblasts can be directly converted into neuronal cells using only a cocktail of small molecules, with a yield of up to >90% being TUJ1-positive after 16 days of induction. After a further maturation stage, these chemically induced neurons (CiNs) possessed neuron-specific expression patterns, generated action potentials, and formed functional synapses. Mechanistically, we found that a BET family bromodomain inhibitor, I-BET151, disrupted the fibroblast-specific program, while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes. Overall, our findings provide a "proof of principle" for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation, through disruption of cell-specific programs and induction of an alternative fate.

A molecular roadmap for induced multi-lineage trans-differentiation of fibroblasts by chemical combinations

“…It is possible that the iMT priming state is an important transitional state that lies upstream of chemical induced pluripotency. Our model is also supported by other published works on chemical induced trans-differentiation [24,25]. The iMT priming state is a synthetic, transient and unstable cellular state in which many unrelated lineage markers are coexpressed (Supplementary information, Table S5).…”

“…Remarkably, a combination of small molecules can replace all Yamanaka factors during the generation of induced pluripotent cells [20,21]. Enlightened by the idea, chemical cocktails that initiate neuronal trans-differentiation have been reported [22][23][24][25]. Similar approaches have been used to generate functional cardiac myocytes from both mouse and human fibroblasts [26][27].…”

Chemical reprogramming of mouse embryonic and adult fibroblast into endoderm lineage

“…With high-throughput screening and chemical biology, the role of transcription factors may be gradually replaced by chemicals (small chemicals and growth factors). To this end, several groups have succeeded in converting somatic cells to iPSCs and neural stem cells/neurons by small molecules (19,(21)(22)(23)38). In this report, we show that a similar approach can be used to convert fibroblasts into self-renewing ciEPCs and then eventually functional ciHeps.…”

Mitotic phosphorylation of SOX2 mediated by Aurora kinase A is critical for the stem-cell like cell maintenance in PA-1 cells

“…24 Moreover, differentiated cells can be reprogrammed by combinations of SOX2 with small molecules or even merely with small compounds. [24][25][26] However, the underlying mechanisms by which SOX2 contributes to selfrenewal or reprogramming processes remain unknown. The post-translational modifications are considered as important events during ESCs early differentiation.…”