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MicroRNA-based conversion of human fibroblasts into striatal medium spiny neurons

Nature Protocols volume 10, pages 1543–1555 (2015)Cite this article

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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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Figure 1: Direct conversion of human fibroblasts to striatal medium spiny neurons.
Figure 2: The expression level of CTIP2 is crucial for the production of DARPP-32, but at high levels it can be toxic.
Figure 3: Re-plating transduced cells ensures long-term survival in vitro.
Figure 4: Drop-plating at high-density levels forms cell clusters.
Figure 5: The acquisition of a striatal neuronal fate is gradual.
Figure 6: Acquisition of neuronal morphology is visible within 2 weeks, and it increases in complexity over time.

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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).

Author information

Author notes

  1. Michelle Richner and Matheus B Victor: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, USA

    Michelle Richner, Matheus B Victor, Yangjian Liu, Daniel Abernathy & Andrew S Yoo

  2. Division of Biology and Biomedical Sciences, Program in Neuroscience, Washington University School of Medicine, St. Louis, Missouri, USA

    Matheus B Victor

  3. Division of Biology and Biomedical Sciences, Program in Developmental, Regenerative and Stem Cell Biology, Washington University, St. Louis, Missouri, USA

    Daniel Abernathy

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Contributions

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.

Corresponding author

Correspondence to Andrew S Yoo.

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The authors declare no competing financial interests.

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.

Supplementary information

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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