Motor neurons created by direct lineage reprogramming of FUS-ALS fibroblasts model several important aspects of ALS, according to a new study in the January 5 online issue of Cell Reports. Unlike induced pluripotent stem cells (iPSCs), the directly reprogrammed cells bypass the embryonic-like state, and thus retain some of the molecular characteristics of the aging cells from which they are derived (see Jan 2010 news). This may offer a cellular model of ALS in which the effects of aging, as well as disease-linked mutations, can be explored.
Recapitulation of the cell donor phenotype is the sine qua non of cell-based models of disease, and questions have arisen over how faithfully iPSC-derived cells can reproduce that phenotype, since the fibroblasts they arise from are wiped clean of the epigenetic markers that define the differentiated cell. “The neurons created from iPSCs are actually embryonic neurons, because all the aging markers have been removed,” noted senior author of the new study Chun-Li Zhang, of UT Southwestern Medical Center in Dallas, Texas.
Recently, for instance, Mertens et al. showed that induced neurons (iNs) generated by direct conversion from fibroblasts had an age-related reduction in the level of a nuclear transport receptor, which decreased the degree of nucleocytoplasmic compartmentalization. This characteristic that was lost in iPSC-derived neurons.
To explore the use of human induced motor neurons (hiMNs) for modeling ALS, Zhang developed a protocol for direct lineage reprogramming of human skin fibroblasts from both healthy and FUS-ALS donors. Zhang had previously shown that cholinergic neurons could be created by direct lineage reprogramming. Here, he went further, adding the transcription factors ISL1 and LHX3, delivered via lentivirus, along with other factors delivered in the growth media, in order to induce a motor neuron phenotype.
Working with co-first authors Meng-Lu Liu and Tong Zang, Zhang found that about 90% of healthy adult fibroblasts were converted to motor neuron-like cells, characterized by development of axons and dendrites and expression of multiple motor neuron markers, including HB9 and ChAT. Discrete puncta of the presynaptic marker synaptotagmin suggested the development of synaptic terminals. Further analysis indicated the neurons were a mix of cervical and thoracic motor neurons. When cocultured with astrocytes, the cells lived for 49 days, and continued to strongly express ChAT, indicating continued synthesis of acetylcholine neurotransmitter. The neurons fired repetitive action potentials when current was applied, and formed functional neuromuscular junctions when allowed to contact skeletal myotubes.
The team then performed direct conversion on fibroblast samples from three different FUS-ALS patients, and observed similar differentiation into HB9- and ChAT-expressing hiMNs by 21 days. While FUS in patient-derived fibroblasts localized in a similar pattern to that in healthy controls, in the hiMNs derived from ALS patients, an abnormal fraction of the protein was cytoplasmic. The neurons displayed a shrunken soma and an increased death rate compared to non-ALS hiMNs neurons. FUS neurons fired infrequently and abnormally, and while they did form synapses, they rarely formed neuromuscular junctions.
Steven Finkbeiner of the Gladstone Institute of Neurodegenerative Disease in San Francisco, who was not involved in the study, commented that these results “add importantly to the body of work demonstrating the value of reprogrammed cells for disease modeling. Significantly, these motor neurons exhibit phenotypic deficits reminiscent of neurodegeneration seen in ALS.”
Direct lineage reprogramming has “enormous potential” to complement existing models of ALS, added Dhruv Sareen of Cedars-Sinai Medical Center in Los Angeles, especially since the cells retain aging-related epigenetic modifications, which may be critical for understanding adult neurodegenerative diseases. However, he noted, the random integration of lentivirus into the genome may affect the disease phenotype in ways that may vary from cell to cell.
It is encouraging, Finkbeiner said, that the conversion process appears to be relatively fast and efficient. However, he cautioned, the method will need to be replicated elsewhere, as there have been reports of improved methods for producing specific types of brain cells which have been difficult to match in terms of speed and efficiency when tested in other labs.
“The appeal to me of direct reprogramming is actually not the speed or efficiency but the prospect that cells may retain some of the epigenetic marks of the starting cell,” Finkbeiner added, “which could reflect contributions of aging or the environment that might be very relevant for ALS.” Thus, direct reprogramming could be a valuable complementary approach to the use of patient-derived iPSCs.
The potential advantages of direct reprogramming as a research tool are twinned with some severe limitations for use as a drug screening platform. Unlike iPSCs, which can proliferate in culture to supply the raw materials for an unlimited supply of motor neurons, each fibroblast is directly converted into one and only one motor neuron.
“This will not be a high-throughput system because of the availability of the cells,” Zhang said. “We can do thousands of compounds, perhaps, but not hundreds of thousands,” making the cells perhaps most useful for validation rather than discovery. There may be batch-to-batch variations in purity and quality that are also harder to control than with iPSC-derived motor neurons.
As a first exploration of their utility for drug evaluation, Zhang tested several compounds known to promote neuronal survival: valproic acid, isoxazole, lithium chloride, and kenpaullone. Kenpaullone (see April 2013 News) was the most effective in an induced-stress screen, enhancing outgrowth of neuronal processes, improving electrical activity, and extending survival. Kenpaullone itself is unlikely to be a good drug candidate, Zhang said, not least because it quickly induces a form of dependence in the neurons exposed to it, which die rapidly when it is withdrawn.
The fact that the disease phenotype can be rescued by pharmacological treatment, however, “further establishes the potential utility of the system to evaluate drug candidates,” according to Finkbeiner.
The biggest value of the direct-induction system in the immediate future is likely to come from its ability to shed light on the effects of aging on neurons in culture. “We haven’t had the tools before to study this,” Zhang said.
1. Liu ML, Zang T, Zhang CL. Direct Lineage Reprogramming Reveals Disease-Specific Phenotypes of Motor Neurons from Human ALS Patients. Cell Rep. 2016 Jan 5;14(1):115-28. Epub 2015 Dec 24.[Pubmed]
2. Mertens J, Paquola AC, Ku M, Hatch E, Böhnke L, Ladjevardi S, McGrath S, Campbell B, Lee H, Herdy JR, Gonçalves JT, Toda T, Kim Y, Winkler J, Yao J, Hetzer MW, Gage FH. Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects. Cell Stem Cell. 2015 Dec 3;17(6):705-18. Epub 2015 Oct 8.[Pubmed]