In the latest round of published fibroblast-to-neuron research, scientists report on the generation of human induced neurons (hiNs) directly from skin fibroblasts taken from Alzheimer disease patients.They say that the viral transduction of fibroblasts with transcription regulator genes Ascl1, Brn2, Zic1, and Myt1 can trigger up to 100% of adult human fibroblasts cultured under appropriate conditions to convert directly into in vivo functional, glutamatergic forebrain-type neurons.
Asa Abeliovich, M.D., and colleagues from Columbia University and the Robert F. Furchgott Center for Neural Behavioral Science, found that hiNs generated from fibroblasts taken from AD patients exhibited altered processing, localization of amyloid precursor protein (APP), and increased production of Aβ relative both to the source patient fibroblasts and hiN cells generated from unaffected individuals.
The scientists report their technique and results in Cell in a paper titled “Directed Conversion of Alzheimer’s Disease Patient Skin Fibroblasts into Functional Neurons.”
As with other research reported in May, Dr. Abeliovich’s team’s starting point was the 2010 report in Nature by Vierbuchen, Wernig et al., demonstrating that mouse skin fibroblasts could be converted to neurons by the expression of three forebrain transcription regulators Ascl1, Brn2, and Myt1l.
The Columbia researchers found these three factors weren’t sufficient to transform human fibroblasts. By testing different mixes of transcription factors they found that a single polycistronic lentivirus vector harboring the genes Ascl1, Brn2, and Zic1 (ABZ vector) was sufficient for the conversion process and demonstrated up to 68% efficiency. Combining the ABZ vector with another vector carrying Myt1 was even more efficient and led to 85% +/- 15% of all transduced cells acquiring a MAP2-positive neuronal morphology phenotype.
The resulting hiN cells also expressed a range of other neuronal markers including those indicative of mature neurons. Significantly, the results of hierarchical clustering carried out to broadly compare hiN cell gene-expression profiles with those seen in human neurons and other cell types confirmed that hiN cell samples clustered most closely with CNS neurons than with fibroblasts, astrocytes, neural progenitors, or, indeed, pluripotent ES or iPS cells.
Patch claim recordings showed the majority of fibroblast hiN cells displayed typical neuronal Na+, K+, and Ca2+ channel properties days 21–28 of culture. Most were also able to fire at least one action potential in response to depolarizing current injections, and on termination of hyperpolarizing pulses the hiN cells displayed a typical rebound spike.
Passive membrane properties were also consistent with an in vitro neuronal phenotype, with resting membrane potentials ranging from -67 mV to -32 mV, the researchers note. Local calcium transients generated as a result of depolarization within the axon-like processes of hiN cells were indicative of putative synaptic release sites.
The functional connectivity of hiN cells was supported through in vitro tests with hiN cells that had been co-cultured with murine RFP-expressing glial cells and also through in vivo studies of brain samples from seven-day old mice that had received, while still in utero, brain implants of GFO-labelled hiNs.
To evaluate the potential utility of hiN cells for disease modeling, the researchers used their technique to generate hiN cells from a panel of human skin fibroblasts derived from patients with familial AD (FAD) due to mutations in PSEN-1 or -2, those with sporadic AD (SAD), and unaffected individuals. hiNs generated from both the FAD and control fibroblasts appeared similar with respect to neuronal reprogramming characteristics such as efficiency of MAP2-positive hiN cell generation and the percentage of neurons that expressed vGLUT1 and the mature neuron marker synaptophysin.
However, the FAD-derived hiNs displayed obvious AD-associated phenotypes, the team found. FAD patient brain is typified by an increased Aβ42/Aβ40 ratio. Consistent with this, the Aβ42/Aβ40 ratio was dramatically increased in FAD hiN cell cultures relative to the control hiN cells.
“Importantly, the Aβ42/Aβ40 ratio in FAD hiN cell cultures was also elevated relative to the originating FAD fibroblast cultures,” they state. FAD hiN cell conversion in addition led to an increase in the level of total Aβ (combined Aβ42 and Aβ40 polypeptides) relative to the originating FAD fibroblasts, indicating that hiN cell conversion appears to amplify an FAD-associated phenotype in the context of PSEN1 or PSEN2 mutations.
Immunocytochemical analysis of hiN cells with an antibody to the APP amino terminus domain revealed the presence of APP-positive puncta within soma, which were not present in the original fibroblasts. These APP-positive puncta were significantly increased in FAD-derived hiN cell cultures, both in terms of size and number.
To verify that the altered intracellular APP-positive puncta in FAD hiN cells were related to FAD mutations, the researchers carried out a rescue experiment designed to force the overexpression of wild-type PSEN1 in FAD PSEN1-mutant hiN cells by transfecting them with a PSEN1 plasmid vector. As expected, overexpression of wild-type PSEN1 rescued the endosomal APP-positive endocytic phenotype in the FAD-derived hiN cells but had no effect on puncta in the contol hIN cells.
“Our analysis of FAD patient-derived hiN cell cultures underscores the potential utility of such human neuronal disease models,” they conclude. “Surprisingly, the impact of FAD PSEN mutations on the Aβ42/Aβ40 ratio was amplified upon hiN cell conversion from fibroblasts. This suggests a model in which PSEN FAD mutants may alter APP processing at multiple levels, directly through modified γ-secretase activity as well as indirectly with altered cellular context.”
The availability of Alzheimer patient-derived hiNs will also facilitate the study of other AD-associated pathologies such as defective synaptic function, the researchers add. “To this end the ability of hiN cells to functionally integrate into neuronal circuitry will be particularly useful. It is also conceivable that such integration of hiN cells into murine AD disease models will test the therapeutic potential of hiN cells.”