Abstract
The ability to generate human neurons of specific subtypes of clinical importance offers experimental platforms that may be instrumental for disease modeling. We recently published a study demonstrating the use of neuronal microRNAs (miRNAs) and transcription factors to directly convert human fibroblasts to a highly enriched population of striatal medium spiny neurons (MSNs), a neuronal subpopulation that has a crucial role in motor control and harbors selective susceptibility to cell death in Huntington's disease. Here we describe a stepwise protocol for the generation of MSNs by direct neuronal conversion of human fibroblasts in 30 d. We provide descriptions of cellular behaviors during reprogramming and crucial steps involved in gene delivery, cell adhesion and culturing conditions that promote cell survival. Our protocol offers a unique approach to combine microRNAs and transcription factors to guide the neuronal conversion of human fibroblasts toward a specific neuronal subtype.
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Acknowledgements
We thank all members of the Yoo laboratory for helpful suggestions. M.B.V. is supported by a National Science Foundation Graduate Research Fellowship (DGE-1143954). A.S.Y. is supported by a US National Institutes of Health (NIH) Director's Innovator Award (DP2), and awards from the Mallinckrodt, Jr. Foundation and the Ellison Medical Foundation, as well as a Presidential Early Career Award for Scientists and Engineers (PECASE).
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M.R., M.B.V. and A.S.Y. designed and performed experiments, and wrote the manuscript. Y.L. contributed to Table 1. D.A. contributed to Supplementary Information.
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Integrated supplementary information
Supplementary Figure 1 Vector maps of all lentiviral plasmids used to generate MSNs.
Plasmids maps and their full sequences can be obtained directly from Addgene at addgene.org/Andrew_Yoo. All plasmids are Ampicillin resistant. Mammalian selection resistance is denominated as follows; N144 – Hygromycin, N174 – Neomycin, N106 – Blasticidin.
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Supplementary Text and Figures
Supplementary Figure 1 (PDF 294 kb)
Live imaging of neonatal fibroblasts during microRNA-mediated reprogramming.
Cells were re-plated at PID 3 and imaged every three hours from PID 7 to PID 21. Time stamp of the video is indicative of time since re-plating, and not post-transduction days. The black arrow indicates the cell shown in Figure 5 undergoing reprogramming. Other colored arrows were added to aid the tracking of additional cells during reprogramming. Due to a small degree of movement of the coverslip, the arrows are intended to help viewers follow single cells over time. (MOV 24535 kb)
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Richner, M., Victor, M., Liu, Y. et al. MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons. Nat Protoc 10, 1543–1555 (2015). https://doi.org/10.1038/nprot.2015.102
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DOI: https://doi.org/10.1038/nprot.2015.102
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