Optimized technology generates inhibitory as well as stimulatory mature neurons.
The expression of two miRNAS, miR-9/9* and miR-124, is sufficient to prompt the conversion of human fibroblasts directly into neurons, researchers claim. The team at Stanford University that reports the discovery admits that the findings are particularly surprising, as miRNAs normally play a role in suppressing genes.
Nevertheless a series of experiments by Gerald R. Crabtree, M.D., and colleagues, has shown that infecting both adult and neonatal fibroblasts with a vector that expresses precursors of miR-9/9* and miR-124 triggers some of the cells to stop proliferating, take on neuron-like morphologies, and express MAP2, a marker of post-mitotic neurons. The researchers report their work in Nature in a paper titled “MicroRNA-mediated conversion of human fibroblasts to neurons.”
The Stanford team’s research is founded on previous studies by Andrew Yoo, Ph.D. (now at Washington University in St. Louis), that implicated miR-9/9* and miR-124 in the maturation of neurons from neural stem cells. Dr. Yoo went on to investigate the function of the two miRNAs further by examining the effects of their expression in non-neuronal cells.
What the researchers didn’t expect to find was that expression of the two miRNAs in fibroblasts turned the cells into neurons. “It was very weird. We were astounded,” remarks Dr. Crabtree. In fact, the miR-9/9*-124-induced neurons expressed SCN1a, a key contributor to neuronal excitability, as well as synapsin 1 and NMDA. The cells also generated electrical signals and were able to form functional presynatic terminals.
Although the basic two-miRNA technique was very inefficient, the researchers found that adding the neurogenic transcription factor Neurod2 to the vector further increased the transformation rate. Importantly, both miR-9/9* and miR-124 were necessary: fibroblasts expressing either miR-9/9* or miR-124 on its own, even in combination with Neurod2, failed to transform.
Because the miR-9/9*-124-Neurod2-induced cells appeared relatively immature and only occasionally demonstrated repetitive action potentials, the researchers went on to see if adding in other neurogenic factors could prompt further maturation of the induced neurons. Previous research had shown that Ascl1 and Myt1l are important factors in the conversion of mouse embryonic fibroblasts into functionally mature neurons, so Dr. Crabtree’s team generated a vector for the expression of miR-9/9*-124 together with Neurod2, Ascl1, and Myt1l (collectively designated DAM).
Expressing this combination in fibroblasts further boosted the number of induced neurons that expressed MAP2, and the resulting cells demonstrated extensive neurite outgrowth. About 80% of the miR-9/9*-124-DAM-transformed cells were able to fire repetitive action potentials and showed typical sodium and potassium currents during voltage clamp depolarizations.
About 10% of the recorded cells were spontaneously active, and spontaneous excitatory postsynaptic currents (EPSCs) were observed in 10/14 induced cells without co-culturing with primary neurons. Furthermore, the induced neurons exhibited evoked EPSCs and inhibitory postsynaptic currents (IPSCs) in response to local stimulation.
“Importantly, neuronal identity was stable after the removal of exogenous expression of miR-9/9*-124 and DAM after three weeks of induction,” the authors add.
The next stage was to try and identify the types of neurons generated by miR-9/9*-124-DAM transformation. Gene-expression studies showed that most of the induced cells were positive for genes expressed in cortical layers. While striatal markers, serotonergic markers, and cerebellar genes were expressed in a small number of cells, none displayed a peripheral nervous system marker or dopaminergic/noradrenergic markers. Interestingly, the cell population appeared to contain inhibitory cells as well as excitatory cells.
Work thus far had focused on converting human neonatal foreskin fibroblasts, but the team subsequently found that the miR-9/9*-124-DAM technique also worked with adult human dermal fibroblasts, just more slowly. The converted adult cells were also able to generate action potentials, demonstrated typical voltage-gated sodium and potassium currents, spontaneous EPSCs, and evoked EPSCs and IPSCs without co-cultured primary neurons.
The Stanford researchers claim their work is unique in demonstrating that miRNAs can promote the conversion of fibroblasts into neurons. It follows two sets of studies published this year that suggest at least two different combinations of proteins can also trigger fibroblasts to transform into neurons.
Reporting in Nature in May, Stanford professor of pathology, Marius Wernig, M.D., and colleagues, demonstrated for the first time that fetal and mature human fibroblasts could be prompted to convert into functional (albeit relatively immature) neurons by the expression of Brn2, Ascl1, and Myt1l, and NeuroD1. It was Dr. Werning’s initial work with mouse fibroblasts a year prior that helped Dr. Crabtree’s team decide which factors to add to their miRNA combination.
While both the optimized miRNA approach and Dr. Wernig’s technique generate neurons characteristic of the frontal cortex, only the miRNA-based technique has resulted in the production of inhibitory neurons as well.
Earlier this month, Vania Broccoli, Ph.D., and colleagues at the San Raffaele Scientific Institute, separately reported in Nature that expression of three neurodevelopmental factors, Mash1, Nurr1, and Lmx1a, could transform prenatal and adult fibroblasts into functional dopaminergic neurons that showed spontaneous electrical activity. The Italian team hopes their achievement will pave the way for the development of cell therapies against Parksinson disease and related disorders.
Dr. Crabtree suggests the three studies will represent just the tip of the iceberg in this field. “Its been a long time in coming to this,” he remarks. “But science often progresses in leaps and starts, and then all of a sudden many scientists come to the same position at the same time. Now these studies have come out, and more will be coming, all of which are going to say that not only can you make in neurons different ways, but also you can make neurons of different types.”