Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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


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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others


  1. International Basal Ganglia Society. The Basal Ganglia VI (eds. Graybiel, A.M., DeLong, M.R. & Kitai, S.T.) (Kluwer Academic/Plenum Pub., 2003).

  2. Albin, R.L., Young, A.B. & Penney, J.B. The functional anatomy of basal ganglia disorders. Trends Neurosci. 12, 366–375 (1989).

    Article  CAS  Google Scholar 

  3. Gerfen, C.R. The neostriatal mosaic: multiple levels of compartmental organization in the basal ganglia. Annu. Rev. Neurosci. 15, 285–320 (1992).

    Article  CAS  Google Scholar 

  4. Yoo, A.S. et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231 (2011).

    Article  CAS  Google Scholar 

  5. Victor, M.B. et al. Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts. Neuron 84, 311–323 (2014).

    Article  CAS  Google Scholar 

  6. Zhang, N., An, M.C., Montoro, D. & Ellerby, L.M. Characterization of human Huntington's disease cell model from induced pluripotent stem cells. PLoS Curr. 2, RRN1193 (2010).

    Article  Google Scholar 

  7. Ma, L. et al. Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice. Cell Stem Cell 10, 455–464 (2012).

    Article  CAS  Google Scholar 

  8. Delli Carri, A. et al. Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons. Development 140, 301–312 (2013).

    Article  Google Scholar 

  9. HD iPSC Consortium. Induced pluripotent stem cells from patients with Huntington's disease show CAG-repeat-expansion-associated phenotypes. Cell Stem Cell 11, 264–278 (2012).

    Article  Google Scholar 

  10. Arber, C. et al. Activin A directs striatal projection neuron differentiation of human pluripotent stem cells. Development 142, 1375–1386 (2015).

    Article  CAS  Google Scholar 

  11. Aubry, L. et al. Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc. Natl. Acad. Sci. USA 105, 16707–16712 (2008).

    Article  CAS  Google Scholar 

  12. Pang, Z.P. et al. Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223 (2011).

    Article  CAS  Google Scholar 

  13. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).

    Article  CAS  Google Scholar 

  14. Caiazzo, M. et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476, 224–227 (2011).

    Article  CAS  Google Scholar 

  15. Xue, Y. et al. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82–96 (2013).

    Article  CAS  Google Scholar 

  16. Son, E.Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).

    Article  CAS  Google Scholar 

  17. Wainger, B.J. et al. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts. Nat. Neurosci. 18, 17–24 (2015).

    Article  CAS  Google Scholar 

  18. Nightingale, S.J. et al. Transient gene expression by nonintegrating lentiviral vectors. Mol. Ther. 13, 1121–1132 (2006).

    Article  CAS  Google Scholar 

  19. Yanez-Munoz, R.J. et al. Effective gene therapy with nonintegrating lentiviral vectors. Nat. Med. 12, 348–353 (2006).

    Article  CAS  Google Scholar 

  20. Chambers, S.M. & Studer, L. Cell fate plug and play: direct reprogramming and induced pluripotency. Cell 145, 827–830 (2011).

    Article  CAS  Google Scholar 

  21. Makeyev, E.V., Zhang, J., Carrasco, M.A. & Maniatis, T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol. Cell 27, 435–448 (2007).

    Article  CAS  Google Scholar 

  22. Packer, A.N., Xing, Y., Harper, S.Q., Jones, L. & Davidson, B.L. The bifunctional microRNA miR-9/miR-9* regulates REST and CoREST and is downregulated in Huntington's disease. J. Neurosci. 28, 14341–14346 (2008).

    Article  CAS  Google Scholar 

  23. Visvanathan, J., Lee, S., Lee, B., Lee, J.W. & Lee, S.K. The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development. Genes Dev. 21, 744–749 (2007).

    Article  CAS  Google Scholar 

  24. Yoo, A.S., Staahl, B.T., Chen, L. & Crabtree, G.R. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. Nature 460, 642–646 (2009).

    Article  CAS  Google Scholar 

  25. Conaco, C., Otto, S., Han, J.J. & Mandel, G. Reciprocal actions of REST and a microRNA promote neuronal identity. Proc. Natl. Acad. Sci. USA 103, 2422–2427 (2006).

    Article  CAS  Google Scholar 

  26. Kutner, R.H., Zhang, X.Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495–505 (2009).

    Article  CAS  Google Scholar 

  27. Arlotta, P., Molyneaux, B.J., Jabaudon, D., Yoshida, Y. & Macklis, J.D. Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum. J. Neurosci. 28, 622–632 (2008).

    Article  CAS  Google Scholar 

  28. Herigstad, B., Hamilton, M. & Heersink, J. How to optimize the drop plate method for enumerating bacteria. J. Microbiol. Methods 44, 121–129 (2001).

    Article  CAS  Google Scholar 

  29. Fedoroff, S. & Richardson, A. Protocols for Neural Cell Culture 3rd edn. (Humana Press, 2001).

  30. Lobo, M.K., Karsten, S.L., Gray, M., Geschwind, D.H. & Yang, X.W. FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains. Nat. Neurosci. 9, 443–452 (2006).

    Article  CAS  Google Scholar 

  31. Ouimet, C.C. & Greengard, P. Distribution of DARPP-32 in the basal ganglia: an electron microscopic study. J. Neurocytol. 19, 39–52 (1990).

    Article  CAS  Google Scholar 

  32. Graybiel, A.M., Aosaki, T., Flaherty, A.W. & Kimura, M. The basal ganglia and adaptive motor control. Science 265, 1826–1831 (1994).

    Article  CAS  Google Scholar 

  33. Deacon, T.W., Pakzaban, P. & Isacson, O. The lateral ganglionic eminence is the origin of cells committed to striatal phenotypes: neural transplantation and developmental evidence. Brain Res. 668, 211–219 (1994).

    Article  Google Scholar 

Download references


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

Authors and Affiliations



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.

Ethics declarations

Competing interests

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 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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing