Scientists have demonstrated that a combination of four transcription factors can prompt the direct conversion of fetal and mature human fibroblast cells into functional induced neurons (iN). The Stanford University School of Medicine researchers had previously shown that the forced expression of a combination of three transcription factors—Brn2 (also known as Pou3f2), Ascl1, and Myt1l (collectively termed BAM factors)—could efficiently prompt mouse fibroblasts to convert into functional neuronal cells.
The latest work has shown that by adding a fourth transcription factor gene into the mix, similar results can be obtained with human fibroblasts. Marius Wernig, Ph.D., and colleagues report their achievements in Nature. The paper is titled “Induction of human neuronal cells by defined transcription factors.”
The team initially tested whether forced expression of the BAM transcription factors could induce a neuronal fate in human pluripotent cells by using lentiviral vectors to transfect undifferentiated human embryonic stem cells (hESCs) with Brn2, Ascl1, and Myt1l. They surprisingly found the conversion was rapid. Within three days bipolar neuron-like cells surround the ESC colonies. By day six neuron-like cells could generate action potentials, and by day eight cells with more mature neuronal morphologies and gene expression were also evident. Similar results were observed when the transcription factors genes were expressed in induced pluripotent stem cells (iPSCs).
The researchers then moved on to see whether the approach could be used to prompt the conversion of human fibroblasts directly into neuronal cells. They tested the technique on three independent primary human fetal fibroblast lines. Interestingly, they found that while infecting the cells with the three BAM factors rapidly led to the generation of cells with immature neuronal morphologies, these cells remained functionally immature. “Thus, the BAM factors seemed to induce neuronal features but were insufficient to generate functional neurons from human fetal fibroblasts under these conditions,” the authors write.
They then looked for additional factors that might prompt further maturation of the immature fibroblast-derived neuronal cells when combined with the BAM pool. This search led to the finding that transfecting the fibroblasts with the BAM genes plus the gene for anther transcription factor, NeuroD1, triggered the production of more mature induced neurons.
In fact, two weeks after induction, the resulting BAMN-iN cells showed neuronal morphologies and produced nerve cell proteins. After 4–5 weeks of culture, there were also neuron-like cells with neurofilaments and neuronal processes that produced synaptic vesicle proteins.
Gene expression studies on individual iN cells demonstrated the expression of combinations of pan-neuronal , neuronal subtype-specific, and synaptic markers. Tests on a number of cells confirmed that they could also generate action potentials. Significantly, when expression of the exongenous BAMN transcripts was switched off in the iN cells, they started to express the endogenous versions of the genes at gradually increasing levels.
The vector-mediated approach was separately shown to prompt the production of induced neurons with active membrane properties from more mature fibroblasts, including dermal fibroblasts derived from an 11-year-old human subject. And in a final set of studies, the researchers demonstrated that after 4–5 weeks of co-culture with primary mouse cortical cells, human induced neurons expressed functional neurotransmitter receptors and formed functional synapses.
“Like mouse iN cells and neurons derived from ES cells iPS cells, the human iN cells seem relatively immature, as indicated by their slightly depolarized membrane potentials and the relatively low-amplitude synaptic responses,” the authors admit. In addition, in comparison with mouse iN cells, the human equivalents require longer culture periods to develop synaptic activity.
“Future studies will be necessary to thoroughly optimize conditions for human iN cell generation and maturation, which would facilitate applications of this method for the study of human neuronal development and disease.”
Nevertheless, they conclude, “our data demonstrate that non-neural human somatic cells as well as pluripotent stem cells can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modeling or future applications in regenerative medicine.”