Researchers say induced neurons exhibit neuronal morphology and marker gene expression and produce functional synapses between each other.
Scientists claim human adult fibroblasts can be reprogrammed to transform into functional neurons by the expression of miR-124 and two transcription factors, Myt1l and Brn2. The researchers say the resulting human induced neurons (hiNs) exhibit typical neuronal morphology and marker gene expression, fire action potentials, and produce functional synapses between each other.
The team points out that other reports published this year have demonstrated that different combinations of transcription factors can trigger the generation of neuronal-like cells or dopaminergic neurons from human fibroblasts. However, they stress, the combined miRNA/transcription factor approach generates neurons with mature functional synapses between adult fibroblast-derived hiNs in the absence of other cell types.
Scientists from Scripps Research Institute, Sanford-Burnham Medical Research Institute, and the University of California’s Gladstone Institute of Cardiovascular Disease collaborated on this work. Findings are reported in Cell Stem Cell in a paper titled “Direct Reprogramming of Adult Human Fibroblasts to Functional Neurons under Defined Conditions.”
The publication follows just a couple of weeks after a Stanford University team reported that human fibroblasts can be triggered to transform into neurons by the expression of just miR-124 and miR-9/9*. miR-124 is the most abundant microRNA in the mammalian central nervous system and is significantly upregulated in differentiating and mature neurons, in which it modulates the activity of major antineuronal differentiation factors, the researchers note.
To evaluate whether miR-124 in combination with transcription factor expression could prompt fibroblasts to convert into neurons, the Scripps-led researchers first transduced human primary postnatal human fibroblast cells with lentiviruses separately carrying 11 different transcription factors and miR-124 tagged with a red fluorescent protein (RFP) marker. Eighteen days after infection a number of the RFP-positive cells also expressed the early neuronal marker βIII-tubulin (Tuj1).
The team then moved on to test whether any combination of one, two, or three of the transcription factors (rather than all 11) plus miR-124 was sufficient to trigger neuron-like transformation of the fibroblasts. They found that within just three days of transduction with miR-124, Brn2, and Myt1l (designated the miBM combination), many of the RFP-positive cells exhibited small, compact cell bodies with monopolar or bipolar projections and weak βIII-tubulin expression.
By 15 days the cells developed a characteristic neuronal morphology consisting of multiple neurite extensions and elaborate branching. Also, about half of these cells were positive for the mature neuronal markers MAP2 and NeuN.
By comparing the number of Tuj1-positive hiN cells present on day 18 with the total number of cells in the starting fibroblast population, the researchers estimated an efficiency of 4–8% for hiN generation. Further evaluation suggested that fibroblasts destined to become hiN cells were most likely postmitotic within 24 hour of transgene induction, “and thus it is likely that hiN conversion occurred in the absence of a mitotic progenitor cell stage,” they note.
Interestingly, studies in a doxycycline or cumate-inducible system suggested that that expression of miBM for seven days was sufficient to produce hiN cells at a frequency comparable to that of the earlier experiments.
The researchers carried out electrophysiological characterization of the hIN cells at 25 days, when the majority displayed synapsin immunoreactivity, a marker associated with functional maturation of neuronal synapses. About 60% of hiN cells tested exhibited rapidly inactivating inward current with a rise time of 2–3 ms followed by outward currents “most likely corresponding to opening of voltage-dependent Na+ and K+ channels, respectively,” the authors suggest.
The resting membrane potential averaged about -45mV, and as the cells’ time in culture increased, over 80% fired action potentials. About 15% of the hIN cells exhibited spontaneous action potentials, and about 20% repetitive trains of evoked action potentials.
Importantly some of the hIN cells produced functional neurotransmitters: About 8% were positive for the inhibitory neurotransmitter GABA, and 12% stained positive for VGAT, a protein involved in vesicular transport of GABA.
Sixty percent of the hiN cells responded to exogenous GABA by producing whole-cell currents. Nearly 45% of cells displayed presynaptic properties of excitatory glutamatergic neurons, indicated by positive VGLUT1 staining, the researchers add. Very few hiN cells, meanwhile, exhibited markers indicative of dopaminergic phenotype, but none stained positive for peripherin, choline acetyltransferase, or serotonin.
By 30 days post-infection, patch-clamp recordings demonstrated miniature excitatory postsynaptic currents (mEPSCs) indicative of functional synapses in 25% hiN cells. The currents were sensitive to a glutamate receptor antagonist but not a competitive inhibitor of GABAA receptors, “thus further confirming their excitatory nature.”
As a final test of their reprogramming approach, the researchers transduced adult human dermal fioblasts taken from two different Caucasian females, with the miBM transgenes. The efficiency of conversion to hiN cells was 1.5–2.9% for one cell population and 9.5–11.2% for the other. When tested on day 25 postinfection, 47% of the hiN cells derived from adult fibroblasts also exhibited rapidly inactivating Na+ currents, and 12% fired action potentials.
Twenty eight percent of cells also exhibited VGLUT1 immunoreactivity, and 60% exhibited NMDA-evoked responses indicative of excitatory neuronal properties. When plated at high density, nearly half the relevant cells displayed excitatory synaptic currents that reflected functional contacts with neighboring cells.
