Scientists have managed to reprogram non-neuronal pericytes found in the human brain directly into action potential-firing immature neurons, using just two transcription factors. The achievement, carried out in cultures of human brain tissue in vitro, could feasibly pave the way to the development of new regenerative treatments for traumatic brain injury or neurodegenerative disorders such as Alzheimer’s disease or Parkinson’s disease, claim researchers at Ludwig-Maximilians University Munich, who headed the studies.
Prompting non-neuronal cells to convert into neurons isn’t a new concept, Benedikt Berninger, Ph.D., and colleagues point out. Prior studies have demonstrated that rodent and human fibroblasts can be induced to convert into neurons by the forced expression of three or four transcription factors, and postnatal astroglia from the mouse cerebral cortex can be reprogrammed directly into functional neuronal cells in vitro by the forced expression of just one transcription factor.
However, the investigators stress, one of the major challenges to translating neuronal reprogramming into clinical therapy is getting somatic cells residing in the brain itself to convert directly into neurons. To attempt this in an in vitro setting, the researchers first needed a population of cells, so they generated adherent cell cultures from surgically removed, nonmalignant human brain samples. Initial analyses showed that the majority of the cells supported in the cultures expressed platelet-derived growth factor receptor-β (PDGFRβ), indicating that they were pericytes, a type of brain cell involved in establishing and maintaining the blood-brain barrier and regulating local blood flow.
When the team transduced the cultured cells with retroviral vectors carrying Sox2 and Mash1, they found that those cells that co-expressed both the transcription factors lost their pericyte identity and acquired a neuronal phenotype. In some of the cultures that had contained up to 97% pericytes before transduction, up to 46% of the cells that co-expressed both Sox2 and Mash1 lost their pericyte markers and starting to express the neuronal marker βIII-tubulin, and up to 26% also exhibited neuronal morphology.
Intriguingly, the researchers note, conversion of the adult non-neuronal pericytes into pericyte-derived induced neuronal cells (hPdiNs) didn’t require cell division. Rather, the process appeared to involve a direct switch from one cell type to the other. In fact, only a small percentage of fluorescence-tagged Sox2- and Mash1-coexpressing cells that the team followed over time underwent cell division, “indicating that Sox2- and Mash1-induced reprogramming does not require cell division, but is accompanied by immediate cell cycle exit,” the team writes.
And encouragingly, the hPdiNs displayed the functional membrane properties of neurons. Up to 71% of neuronal cells derived from five different patient samples generated an action potential in response to a step-current injection, and exhibited clearly discernible sodium and potassium currents in voltage-clamp studies.
The authors admit that the cells’ neuronal properties were more akin to those of immature neurons than mature neurons, “as reflected by the relatively high-input resistances, low-action potential, and peak sodium current amplitudes, even after prolonged time in culture, consistent with the slow maturation of human neurons,” they write. However, when the the pericyte-derived neuronal cells were cocultured with embryonic mouse brain neurons they took on a more complex morphology, were capable of repetitive action potential firing and, importantly, could receive functional glutamatergic input from the cocultured neurons, “demonstrating that they express functional transmitter receptors, are capable of assembling a postsynaptic compartment, and can be recognized by other neurons as functional targets.” Moreover, the hPdiN dendrites displayed multiple presynaptic terminals that contained vesicular glutamate transporters.
Dr. Beringer admits that much work will be needed to optimize the reprogramming process and adapt it for application in vivo. However, he claims, “our data provide strong support for the notion that neuronal reprogramming of cells of pericytic origin within the damaged brain may become a viable approach to replace degenerated neurons.”
The investigators describe their technology and results in Cell Stem Cell, in a paper titled “Reprogramming of Pericyte-Derived Cells of the Adult Human Brain into Induced Neuronal Cells.”