Skip to main content

Advertisement

Log in

An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons

  • Original Paper
  • Published:
The Cerebellum Aims and scope Submit manuscript

Abstract

While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure–function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLCβ4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Marr D. A theory of cerebellar cortex. J Physiol. 1969;202:437–70. https://doi.org/10.1113/jphysiol.1969.sp008820.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ito M. Cerebellar circuitry as a neuronal machine. Prog Neurobiol. 2006;78:272–303. https://doi.org/10.1016/J.PNEUROBIO.2006.02.006.

    Article  PubMed  Google Scholar 

  3. Heintz TG, Eva R, Fawcett JW. Regional regulation of Purkinje cell dendritic spines by integrins and Eph/ephrins. PLoS One. 2016;11:e0158558. https://doi.org/10.1371/journal.pone.0158558.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bezprozvanny I. Calcium signaling and neurodegenerative diseases. Trends Mol Med. 2009;15:89–100. https://doi.org/10.1016/J.MOLMED.2009.01.001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Paulson HL. The spinocerebellar ataxias. J Neuroophthalmol. 2009;29:227–37. https://doi.org/10.1097/WNO0b013e3181b416de.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Sun Y-M, Lu C, Wu Z-Y. Spinocerebellar ataxia: relationship between phenotype and genotype—a review. Clin Genet. 2016;90:305–14. https://doi.org/10.1111/cge.12808.

    Article  CAS  PubMed  Google Scholar 

  7. Orr HT, Chung M, Banfi S, Kwiatkowski TJ, Servadio A, Beaudet AL, et al. Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat Genet. 1993;4:221–6. https://doi.org/10.1038/ng0793-221.

    Article  CAS  Google Scholar 

  8. Trott A, Houenou LJ. Mini-review: spinocerebellar ataxias: an update of SCA genes. Recent Pat DNA Gene Seq. 2012;6:115–21.

    Article  CAS  Google Scholar 

  9. Soong B-W, Morrison PJ. Spinocerebellar ataxias. Handb Clin Neurol. 2018:143–74. https://doi.org/10.1016/B978-0-444-64189-2.00010-X.

  10. Wagner W, Brenowitz SD, Hammer JA. Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons. Nat Cell Biol. 2011;13:40–8. https://doi.org/10.1038/ncb2132.

    Article  CAS  PubMed  Google Scholar 

  11. Wagner W, McCroskery S, Hammer JA. An efficient method for the long-term and specific expression of exogenous cDNAs in cultured Purkinje neurons. J Neurosci Methods. 2011;200:95–105. https://doi.org/10.1016/j.jneumeth.2011.06.006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Alexander CJ, Hammer JA. Optimization of cerebellar purkinje neuron cultures and development of a plasmid-based method for purkinje neuron-specific, miRNA-mediated protein knockdown. Methods Cell Biol. 2016;131:177–97. https://doi.org/10.1016/bs.mcb.2015.06.004.

    Article  CAS  PubMed  Google Scholar 

  13. Kim J-H, Auerbach JM, Rodríguez-Gómez JA, Velasco I, Gavin D, Lumelsky N, et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature. 2002;418:50–6. https://doi.org/10.1038/nature00900.

    Article  CAS  PubMed  Google Scholar 

  14. Wichterle H, Lieberam I, Porter JA, Jessell TM. Directed differentiation of embryonic stem cells into motor neurons. Cell. 2002;110:385–97. https://doi.org/10.1016/S0092-8674(02)00835-8.

    Article  CAS  PubMed  Google Scholar 

  15. Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neuronal properties in vitro. Dev Biol. 1995;168:342–57. https://doi.org/10.1006/DBIO.1995.1085.

    Article  CAS  PubMed  Google Scholar 

  16. Zhang S-C, Wernig M, Duncan ID, Brüstle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol. 2001;19:1129–33. https://doi.org/10.1038/nbt1201-1129.

    Article  CAS  PubMed  Google Scholar 

  17. Lee S-H, Lumelsky N, Studer L, Auerbach JM, McKay RD. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol. 2000;18:675–9. https://doi.org/10.1038/76536.

    Article  CAS  PubMed  Google Scholar 

  18. O’Shea KS. Embryonic stem cell models of development. Anat Rec. 1999;257:32–41. https://doi.org/10.1002/(SICI)1097-0185(19990215)257:1<32::AID-AR6>3.0.CO;2-2.

    Article  PubMed  Google Scholar 

  19. Sylvester KG, Longaker MT. Stem cells. Arch Surg. 2004;139:93–9. https://doi.org/10.1001/archsurg.139.1.93.

    Article  PubMed  Google Scholar 

  20. Temple S. The development of neural stem cells. Nat. 2001;4146859:2001.

    Google Scholar 

  21. Gage FH, Coates PW, Palmer TD, Kuhn HG, Fisher LJ, Suhonen JO, et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci. 1995;92:11879–83. https://doi.org/10.1073/pnas.92.25.11879.

    Article  CAS  PubMed  Google Scholar 

  22. Gage FH. Mammalian neural stem cells. Science. 2000;287(80):1433–8. https://doi.org/10.1126/science.287.5457.1433.

    Article  CAS  PubMed  Google Scholar 

  23. Reynolds B, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255(80):1707–10. https://doi.org/10.1126/science.1553558.

    Article  CAS  PubMed  Google Scholar 

  24. Temple S. Stem cell plasticity—building the brain of our dreams. Nat Rev Neurosci. 2001;2:513–20. https://doi.org/10.1038/35081577.

    Article  CAS  PubMed  Google Scholar 

  25. Qian X, Shen Q, Goderie SK, He W, Capela A, Davis AA, et al. Timing of CNS cell generation. Neuron. 2000;28:69–80. https://doi.org/10.1016/S0896-6273(00)00086-6.

    Article  CAS  PubMed  Google Scholar 

  26. Li M, Pevny L, Lovell-Badge R, Smith A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol. 1998;8:971–S2. https://doi.org/10.1016/S0960-9822(98)70399-9.

    Article  CAS  PubMed  Google Scholar 

  27. Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovitz-Eldor J, Goldstein RS, et al. Induced neuronal differentiation of human embryonic stem cells. Brain Res. 2001;913:201–5. https://doi.org/10.1016/S0006-8993(01)02776-7.

    Article  CAS  PubMed  Google Scholar 

  28. Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med. 2000;6:88–95.

    Article  CAS  Google Scholar 

  29. Desbaillets I, Ziegler U, Groscurth P, Gassmann M. Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol. 2000;85:645–51.

    Article  CAS  Google Scholar 

  30. Tao O, Shimazaki T, Okada Y, Naka H, Kohda K, Yuzaki M, et al. Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells. J Neurosci Res. 2010;88:234–47. https://doi.org/10.1002/jnr.22208.

    Article  CAS  PubMed  Google Scholar 

  31. Muguruma K, Nishiyama A, Ono Y, Miyawaki H, Mizuhara E, Hori S, et al. Ontogeny-recapitulating generation and tissue integration of ES cell–derived Purkinje cells. Nat Neurosci. 2010;13:1171–80. https://doi.org/10.1038/nn.2638.

    Article  CAS  PubMed  Google Scholar 

  32. Wang S, Wang B, Pan N, Fu L, Wang C, Song G, et al. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Sci Rep. 2015;5:9232. https://doi.org/10.1038/srep09232.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Brickman JM, Serup P. Properties of embryoid bodies. WIREs Dev Biol. 2017;6. https://doi.org/10.1002/wdev.259.

  34. Su H-L, Muguruma K, Matsuo-Takasaki M, Kengaku M, Watanabe K, Sasai Y. Generation of cerebellar neuron precursors from embryonic stem cells. Dev Biol. 2006;290:287–96. https://doi.org/10.1016/J.YDBIO.2005.11.010.

    Article  CAS  PubMed  Google Scholar 

  35. Srivastava R, Kumar M, Peineau S, Csaba Z, Mani S, Gressens P, et al. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons. Stem Cells. 2013;31:652–65. https://doi.org/10.1002/stem.1295.

    Article  CAS  PubMed  Google Scholar 

  36. Lindholm D, Castrén E, Tsoulfas P, Kolbeck R, da Berzaghi MP, Leingärtner A, et al. Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation. J Cell Biol. 1993;122:443–50. https://doi.org/10.1083/JCB.122.2.443.

    Article  CAS  PubMed  Google Scholar 

  37. Salero E, Hatten ME. Differentiation of ES cells into cerebellar neurons. Proc Natl Acad Sci. 2007;104:2997–3002. https://doi.org/10.1073/pnas.0610879104.

    Article  CAS  PubMed  Google Scholar 

  38. Crossley PH, Martinez S, Martin GR. Midbrain development induced by FGF8 in the chick embryo. Nature. 1996;380:66–8. https://doi.org/10.1038/380066a0.

    Article  CAS  PubMed  Google Scholar 

  39. Millen KJ, Steshina EY, Iskusnykh IY, Chizhikov VV. Transformation of the cerebellum into more ventral brainstem fates causes cerebellar agenesis in the absence of Ptf1a function. Proc Natl Acad Sci. 2014;111:E1777–86. https://doi.org/10.1073/pnas.1315024111.

    Article  CAS  PubMed  Google Scholar 

  40. Alder J, Cho NK, Hatten ME. Embryonic precursor cells from the rhombic lip are specified to a cerebellar granule neuron identity. Neuron. 1996;17:389–99. https://doi.org/10.1016/S0896-6273(00)80172-5.

    Article  CAS  PubMed  Google Scholar 

  41. Machold R, Fishell G. Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors. Neuron. 2005;48:17–24. https://doi.org/10.1016/J.NEURON.2005.08.028.

    Article  CAS  PubMed  Google Scholar 

  42. Inman GJ, Nicolás FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002;62:65–74.

    Article  CAS  Google Scholar 

  43. Ma X, Kawamoto S, Uribe J, Adelstein RS. Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development. Mol Biol Cell. 2006;17:2138–49. https://doi.org/10.1091/mbc.E05-10-0997.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Leitges M, Kovac J, Plomann M, Linden DJ. A unique PDZ ligand in PKCα confers induction of cerebellar long-term synaptic depression. Neuron. 2004;44:585–94. https://doi.org/10.1016/J.NEURON.2004.10.024.

    Article  CAS  PubMed  Google Scholar 

  45. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82. https://doi.org/10.1038/nmeth.2019.

    Article  CAS  PubMed  Google Scholar 

  46. Theodorou E, Dalembert G, Heffelfinger C, White E, Weissman S, Corcoran L, et al. A high throughput embryonic stem cell screen identifies Oct-2 as a bifunctional regulator of neuronal differentiation. Genes Dev. 2009;23:575–88. https://doi.org/10.1101/gad.1772509.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Katoh Y, Katoh M. Conserved POU-binding site linked to SP1-binding site within FZD5 promoter: transcriptional mechanisms of FZD5 in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer. Int J Oncol. 2007;30:751–5. https://doi.org/10.3892/ijo.30.3.751.

    Article  CAS  PubMed  Google Scholar 

  48. Zhang X, Huang CT, Chen J, Pankratz MT, Xi J, Li J, et al. Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell. 2010;7:90–100. https://doi.org/10.1016/j.stem.2010.04.017.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Zhang J, Jiao J. Molecular biomarkers for embryonic and adult neural stem cell and neurogenesis. Biomed Res Int. 2015;2015:1–14. https://doi.org/10.1155/2015/727542.

    Article  CAS  Google Scholar 

  50. Wen J, Hu Q, Li M, Wang S, Zhang L, Chen Y, et al. Pax6 directly modulate Sox2 expression in the neural progenitor cells. Neuroreport. 2008;19:413–17. https://doi.org/10.1097/WNR.0b013e3282f64377

  51. Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2004;101:12543–8. https://doi.org/10.1073/pnas.0404700101.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Li X-J, Du Z-W, Zarnowska ED, Pankratz M, Hansen LO, Pearce RA, et al. Specification of motoneurons from human embryonic stem cells. Nat Biotechnol. 2005;23:215–21. https://doi.org/10.1038/nbt1063.

    Article  CAS  PubMed  Google Scholar 

  53. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27:275–80. https://doi.org/10.1038/nbt.1529.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Dou D, Zhao H, Li Z, Xu L, Xiong X, Wu X, et al. CHD1L promotes neuronal differentiation in human embryonic stem cells by upregulating PAX6. Stem Cells Dev. 2017;26:1626–36. https://doi.org/10.1089/scd.2017.0110.

    Article  CAS  PubMed  Google Scholar 

  55. Hu B-Y, Weick JP, Yu J, Ma L-X, Zhang X-Q, Thomson JA, et al. Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc Natl Acad Sci. 2010;107:4335–40. https://doi.org/10.1073/pnas.0910012107.

    Article  PubMed  Google Scholar 

  56. Ericson J, Rashbass P, Schedl A, Brenner-Morton S, Kawakami A, Van Heyningen V, et al. Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell. 1997;90:169–80. https://doi.org/10.1016/S0092-8674(00)80323-2.

    Article  CAS  PubMed  Google Scholar 

  57. Sansom SN, Griffiths DS, Faedo A, Kleinjan DJ, Ruan Y, Smith J, et al. The level of the transcription factor Pax6 is essential for controlling the balance between neural stem cell self-renewal and neurogenesis. PLoS Genet. 2009;5:e1000511. https://doi.org/10.1371/journal.pgen.1000511.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Don S, Verrills NM, Liaw TYE, Liu MLM, Norris MD, Haber M, et al. Neuronal-associated microtubule proteins class III beta-tubulin and MAP2c in neuroblastoma: role in resistance to microtubule-targeted drugs. Mol Cancer Ther. 2004;3:1137–46.

    Article  CAS  Google Scholar 

  59. Morrison ME, Mason CA. Granule neuron regulation of Purkinje cell development: striking a balance between neurotrophin and glutamate signaling. J Neurosci. 1998;18:3563–73. https://doi.org/10.1523/JNEUROSCI.18-10-03563.1998.

    Article  CAS  PubMed  Google Scholar 

  60. Kuhar SG, Feng L, Vidan S, Ross ME, Hatten ME, Heintz N. Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation. Neurobiology. 1993;117(1):97–104.

    CAS  Google Scholar 

  61. Nayler S, Vanichkina D, Kanjhan R, Taft R. Transcriptome of the developing ataxia-telangiectasia cerebellum: RNA sequencing of human iPSC-derived cerebellar progenitors. n.d. https://doi.org/10.13140/RG.2.1.5064.8400.

  62. Ben-Arie N, Bellen HJ, Armstrong DL, McCall AE, Gordadze PR, Guo Q, et al. Math1 is essential for genesis of cerebellar granule neurons. Nature. 1997;390:169–72. https://doi.org/10.1038/36579.

    Article  CAS  PubMed  Google Scholar 

  63. Gazit R, Krizhanovsky V, Ben-Arie N, Samper E, Brown S, Aguilera RJ, et al. Math1 controls cerebellar granule cell differentiation by regulating multiple components of the Notch signaling pathway. Development. 2004;131:903–13. https://doi.org/10.1242/dev.00982.

    Article  CAS  PubMed  Google Scholar 

  64. Schüller U, Heine VM, Mao J, Kho AT, Dillon AK, Han Y-G, et al. Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma. Cancer Cell. 2008;14:123–34. https://doi.org/10.1016/J.CCR.2008.07.005.

    Article  PubMed  PubMed Central  Google Scholar 

  65. Watson LM, Wong MMK, Vowles J, Cowley SA, Becker EBE. A simplified method for generating Purkinje cells from human-induced pluripotent stem cells. Cerebellum. 2018;17:419–27. https://doi.org/10.1007/s12311-017-0913-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Ryan KE, Kim PS, Fleming JT, Brignola E, Cheng FY, Litingtung Y, et al. Lkb1 regulates granule cell migration and cortical folding of the cerebellar cortex. Dev Biol. 2017;432:165–77. https://doi.org/10.1016/J.YDBIO.2017.09.036.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Wefers AK, Lindner S, Schulte JH, Schüller U. Overexpression of Lin28b in neural stem cells is insufficient for brain tumor formation, but induces pathological lobulation of the developing cerebellum. Cerebellum. 2017;16:122–31. https://doi.org/10.1007/s12311-016-0774-0.

    Article  CAS  PubMed  Google Scholar 

  68. McDougall ARA, Hale N, Rees S, Harding R, De Matteo R, Hooper SB, et al. Erythropoietin protects against lipopolysaccharide-induced microgliosis and abnormal granule cell development in the ovine fetal cerebellum. Front Cell Neurosci. 2017;11:224. https://doi.org/10.3389/fncel.2017.00224.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Wechsler-Reya RJ, Scott MP. Control of neuronal precursor proliferation in the cerebellum by sonic hedgehog. Neuron. 1999;22:103–14. https://doi.org/10.1016/S0896-6273(00)80682-0.

    Article  CAS  PubMed  Google Scholar 

  70. Falsig J, Sonati T, Herrmann US, Saban D, Li B, Arroyo K, et al. Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures. PLoS Pathog. 2012;8:e1002985. https://doi.org/10.1371/journal.ppat.1002985.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Levin SI, Khaliq ZM, Aman TK, Grieco TM, Kearney JA, Raman IM, et al. Impaired motor function in mice with cell-specific knockout of sodium channel Scn8a (Na V 1.6) in cerebellar Purkinje neurons and granule cells. J Neurophysiol. 2006;96:785–93. https://doi.org/10.1152/jn.01193.2005.

    Article  CAS  PubMed  Google Scholar 

  72. Martenson JS, Yamasaki T, Chaudhury NH, Albrecht D, Tomita S. Assembly rules for GABAA receptor complexes in the brain. Elife. 2017;6. https://doi.org/10.7554/eLife.27443.

  73. Aller MI, Jones A, Merlo D, Paterlini M, Meyer AH, Amtmann U, et al. Cerebellar granule cell Cre recombinase expression. Genesis. 2003;36:97–103. https://doi.org/10.1002/gene.10204.

    Article  CAS  PubMed  Google Scholar 

  74. Wang B, Harrison W, Overbeek PA, Zheng H. Transposon mutagenesis with coat color genotyping identifies an essential role for Skor2 in sonic hedgehog signaling and cerebellum development. Development. 2011;138:4487–97. https://doi.org/10.1242/dev.067264.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Nakatani T, Minaki Y, Kumai M, Nitta C, Ono Y. The c-Ski family member and transcriptional regulator Corl2/Skor2 promotes early differentiation of cerebellar Purkinje cells. Dev Biol. 2014;388:68–80. https://doi.org/10.1016/J.YDBIO.2014.01.016.

    Article  CAS  PubMed  Google Scholar 

  76. Skinner PJ, Vierra-Green CA, Clark HB, Zoghbi HY, Orr HT. Altered trafficking of membrane proteins in Purkinje cells of SCA1 transgenic mice. Am J Pathol. 2001;159:905–13. https://doi.org/10.1016/S0002-9440(10)61766-X.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Laure-Kamionowska M, Maślińska D. Calbindin positive Purkinje cells in the pathology of human cerebellum occurring at the time of its development. Folia Neuropathol. 2009;47:300–5.

    CAS  PubMed  Google Scholar 

  78. Liu Y, Lee JW, Ackerman SL. Mutations in the microtubule-associated protein 1A (Map1a) gene cause Purkinje cell degeneration. J Neurosci. 2015;35:4587–98. https://doi.org/10.1523/JNEUROSCI.2757-14.2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Sillitoe RV, Stephen D, Lao Z, Joyner AL. Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum. J Neurosci. 2008;28:12150–62. https://doi.org/10.1523/JNEUROSCI.2059-08.2008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Sarna JR, Marzban H, Watanabe M, Hawkes R. Complementary stripes of phospholipase Cβ3 and Cβ4 expression by Purkinje cell subsets in the mouse cerebellum. J Comp Neurol. 2006;496:303–13. https://doi.org/10.1002/cne.20912.

    Article  CAS  PubMed  Google Scholar 

  81. Oue M, Handa H, Matsuzaki Y, Suzue K, Murakami H, Hirai H. The murine stem cell virus promoter drives correlated transgene expression in the leukocytes and cerebellar Purkinje cells of transgenic mice. PLoS One. 2012;7:e51015. https://doi.org/10.1371/journal.pone.0051015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Miller TE, Wang J, Sukhdeo K, Horbinski C, Tesar PJ, Wechsler-Reya RJ, et al. Lgr5 marks post-mitotic, lineage restricted cerebellar granule neurons during postnatal development. PLoS One. 2014;61:288. https://doi.org/10.3311/PPch.10689.

    Article  Google Scholar 

  83. Zhang J, D’Ercole AJ. Expression of Mcl-1 in cerebellar granule neurons is regulated by IGF-I in a developmentally specific fashion. Dev Brain Res. 2004;152:255–63. https://doi.org/10.1016/j.devbrainres.2004.07.008.

    Article  CAS  Google Scholar 

  84. O’Hearn E, Molliver ME. Degeneration of Purkinje cells in parasagittal zones of the cerebellar vermis after treatment with ibogaine or harmaline. Neuroscience. 1993;55:303–10. https://doi.org/10.1016/0306-4522(93)90500-F.

    Article  PubMed  Google Scholar 

  85. Luo L, Hensch TK, Ackerman L, Barbel S, Jan LY, Jan YN. Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature. 1996;379:837–40. https://doi.org/10.1038/379837a0.

    Article  CAS  PubMed  Google Scholar 

  86. Yang Q, Hashizume Y, Yoshida M, Wang Y, Goto Y, Mitsuma N, et al. Morphological Purkinje cell changes in spinocerebellar ataxia type 6. Acta Neuropathol. 2000;100:371–6. https://doi.org/10.1007/s004010000201.

    Article  CAS  PubMed  Google Scholar 

  87. Alexander CJ, Wagner W, Copeland NG, Jenkins NA, Hammer JA. Creation of a myosin Va-TAP tagged mouse and identification of potential myosin Va-interacting proteins in the cerebellum. Cytoskeleton. 2018;75:395–409. https://doi.org/10.1002/cm.21474.

    Article  CAS  PubMed  Google Scholar 

  88. McGee AW, Topinka JR, Hashimoto K, Petralia RS, Kakizawa S, Kauer F, et al. PSD-93 knock-out mice reveal that neuronal MAGUKs are not required for development or function of parallel fiber synapses in cerebellum. J Neurosci. 2001;21:3085–91.

  89. Miyazaki T, Watanabe M, Yamagishi A, Takahashi M. B2 exon splicing of nonmuscle myosin heavy chain IIB is differently regulated in developing and adult rat brain. Neurosci Res. 2000;37:299–306. https://doi.org/10.1016/S0168-0102(00)00130-9.

    Article  CAS  PubMed  Google Scholar 

  90. Miyata M, Miyata H, Mikoshiba K, Ohama E. Development of Purkinje cells in humans: an immunohistochemical study using a monoclonal antibody against the inositol 1,4,5-triphosphate type 1 receptor (IP3R1). Acta Neuropathol. 1999;98:226–32.

    Article  CAS  Google Scholar 

  91. Hisatsune C, Miyamoto H, Hirono M, Yamaguchi N, Sugawara T, Ogawa N, et al. IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice. Front Neural Circuits. 2013;7:156. https://doi.org/10.3389/fncir.2013.00156.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Kawaai K, Mizutani A, Shoji H, Ogawa N, Ebisui E, Kuroda Y, et al. IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation. Proc Natl Acad Sci U S A. 2015;112:5515–20. https://doi.org/10.1073/pnas.1503310112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Yang D, Shcheynikov N, Muallem S. IRBIT: it is everywhere. Neurochem Res. 2011;36:1166–74. https://doi.org/10.1007/s11064-010-0353-6.

    Article  CAS  PubMed  Google Scholar 

  94. Takagishi Y, Oda S, Hayasaka S, Dekker-Ohno K, Shikata T, Inouye M, et al. The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice. Neurosci Lett. 1996;215:169–72. https://doi.org/10.1016/0304-3940(96)12967-0.

    Article  CAS  PubMed  Google Scholar 

  95. Dekker-Ohno K, Hayasaka S, Takagishi Y, Oda S, Wakasugi N, Mikoshiba K, et al. Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat. Brain Res. 1996;714:226–30.

    Article  CAS  Google Scholar 

  96. Richter K, Langnaese K, Kreutz MR, Olias G, Zhai R, Scheich H, et al. Presynaptic cytomatrix protein Bassoon is localized at both excitatory and inhibitory synapses of rat brain. J Comp Neurol. 1999;408:437–48. https://doi.org/10.1002/(SICI)1096-9861(19990607)408:3<437::AID-CNE9>3.0.CO;2-5.

    Article  CAS  PubMed  Google Scholar 

  97. Wang W, Stock RE, Gronostajski RM, Wong YW, Schachner M, Kilpatrick DL. A role for nuclear factor I in the intrinsic control of cerebellar granule neuron gene expression. J Biol Chem. 2004;279:53491–7. https://doi.org/10.1074/jbc.M410370200.

    Article  CAS  PubMed  Google Scholar 

  98. Yuzaki M, Forrest D, Verselis LM, Sun SC, Curran T, Connor JA. Functional NMDA receptors are transiently active and support the survival of Purkinje cells in culture. J Neurosci. 1996;16:4651–61. https://doi.org/10.1523/JNEUROSCI.16-15-04651.1996.

    Article  CAS  PubMed  Google Scholar 

  99. Linden DJ, Connor JA. Participation of postsynaptic PKC in cerebellar long-term depression in culture. Science. 1991;254:1656–9.

    Article  CAS  Google Scholar 

  100. Ahn S, Ginty DD, Linden DJ. A late phase of cerebellar long-term depression requires activation of CaMKIV and CREB. Neuron. 1999;23:559–68. https://doi.org/10.1016/S0896-6273(00)80808-9.

    Article  CAS  PubMed  Google Scholar 

  101. Südhof TC. The presynaptic active zone. Neuron. 2012;75:11–25. https://doi.org/10.1016/J.NEURON.2012.06.012.

    Article  PubMed  PubMed Central  Google Scholar 

  102. Dieck S, Sanmartí-Vila L, Langnaese K, Richter K, Kindler S, Soyke A, et al. Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J Cell Biol. 1998;142:499–509. https://doi.org/10.1083/JCB.142.2.499.

    Article  PubMed Central  Google Scholar 

  103. Nomura T, Kakegawa W, Matsuda S, Kohda K, Nishiyama J, Takahashi T, et al. Cerebellar long-term depression requires dephosphorylation of TARP in Purkinje cells. Eur J Neurosci. 2012;35:402–10. https://doi.org/10.1111/j.1460-9568.2011.07963.x.

    Article  PubMed  Google Scholar 

  104. Seki T, Yoshino K, Tanaka S, Dohi E, Onji T, Yamamoto K, et al. Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron. PLoS One. 2012;7:e31232. https://doi.org/10.1371/journal.pone.0031232.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Shimobayashi E, Kapfhammer JP. Increased biological activity of protein kinase C gamma is not required in spinocerebellar ataxia 14. Mol Brain. 2017;10:34. https://doi.org/10.1186/s13041-017-0313-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Miyata M, Finch EA, Khiroug L, Hashimoto K, Hayasaka S, Oda S-I, et al. Local calcium release in dendritic spines required for long-term synaptic depression. Neuron. 2000;28:233–44. https://doi.org/10.1016/S0896-6273(00)00099-4.

    Article  CAS  PubMed  Google Scholar 

  107. Takagishi Y, Hashimoto K, Kayahara T, Watanabe M, Otsuka H, Mizoguchi A, et al. Diminished climbing fiber innervation of Purkinje cells in the cerebellum of myosin Va mutant mice and rats. Dev Neurobiol. 2007;67:909–23. https://doi.org/10.1002/dneu.20375.

    Article  CAS  PubMed  Google Scholar 

  108. Takagishi Y, Murata Y. Myosin Va mutation in rats is an animal model for the human hereditary neurological disease, Griscelli syndrome type 1. Ann N Y Acad Sci. 2006;1086:66–80. https://doi.org/10.1196/annals.1377.006.

    Article  CAS  PubMed  Google Scholar 

  109. Kim Y, Kim T, Rhee JK, Lee D, Tanaka-Yamamoto K, Yamamoto Y. Selective transgene expression in cerebellar Purkinje cells and granule cells using adeno-associated viruses together with specific promoters. Brain Res. 1620;2015:1–16. https://doi.org/10.1016/j.brainres.2015.05.015.

    Article  CAS  Google Scholar 

  110. Hall B, Limaye A, Kulkarni AB. Overview: generation of gene knockout mice. Curr Protoc Cell Biol 2009;Chapter 19:Unit 19.12 19.12.1–17. https://doi.org/10.1002/0471143030.cb1912s44.

  111. Longenecker G, Kulkarni AB. Generation of gene knockout mice by ES cell microinjection. Curr Protoc Cell Biol 2009;Chapter 19:Unit 19.14 19.14.1–36. https://doi.org/10.1002/0471143030.cb1914s44.

Download references

Acknowledgements

We thank Dr. Chengyu Lui (NHLBI Transgenic Core) for providing the mouse embryonic stem cells.

Funding

This study was funded by the National Heart, Lung, and Blood Institute Intramural Research Program at the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to John A. Hammer.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

All animal care and experiments performed in this study were approved by the Institutional Animal Care and Use Committee of the National Heart, Lung, and Blood Institute in accordance with the National Institute of Health guidelines. This article does not contain any studies with human participants performed by any of the authors.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material

Figure S1

Generation of PN precursors and GN precursors. Shown is a representative example of a precursor PN culture 10 days after initiation from NP cells that was stained for TubJ to mark all neurons (A1) and Neph3 to mark PN precursors (A2). The overlaid image in shown in A3. Also shown is a representative example of a precursor GN culture 12 days after initiation from NP cells that was stained for TubJ to mark all neurons (B1) and NeuN to mark GN precursors (B2). The overlaid image in shown in B3. Mag bars: 100 μm (A3 and B3). (PNG 3039 kb)

High Resolution Image (TIF 4082 kb)

Figure S2

A small number of GN precursors develop into Calbindin D28K-positive cells. Shown is a representative example of a Calbindin D28K-positive cell present when GN precursor cells are cultured for 14 days in PN Basal Growth Medium. Mag bar: 20 μm (PNG 143 kb)

High Resolution Image (TIF 192 kb)

Figure S3

An optimized extracellular substrate creates thinner cultures suitable for high-resolution microscopy. Shown is a representative example of a GN/PN precursor co-culture grown for 21 days on gelatin (the standard surface) (A), or on a combination of Geltrex, poly-l-lysine (PLL) and poly-l-ornithine (PLO) (B). The latter substrate results in thinner cultures more suitable for high-resolution microscopy. Mag bars: 100 μm (A and B). (PNG 642 kb)

High Resolution Image (TIF 836 kb)

Figure S4

Yield of developed PNs per dish. The co-culture of GN/PN precursors yields a similar number of developed PNs per dish as primary culture. Cells were cultured on Cellvis 35 mm #1.5 glass-bottomed dishes having a total growth area of 154 mm2. A total of 3 independent experiments for each condition were analyzed. Error bars represent the standard deviation; n.s., not significant. (PNG 74 kb)

High Resolution Image (TIF 237 kb)

Figure S5

Primary PNs express multiple proteins used previously to mark PNs. Shown are representative Day-21 primary PNs that were double-stained for the PN-specific protein Calbindin D28K in red and the following proteins known to be expressed by PNs in green: PSD93 (A1-A4), PLCβ4 (B1-B4), myosin IIB-B2 (C1-C4), IP3R1 (D1-D4), and IRBIT (E1-E4). Each sample is presented as a lower magnification, overlaid image in the first panel, and higher magnification split and overlaid images of the boxed region in the second, third and fourth panels, respectively. Mag bars: 20 μm (A1-E1) and 5 μm (A4-E4). (PNG 3523 kb)

High Resolution Image (TIF 4907 kb)

Figure S6

mESC-derived PNs express exogenous proteins off the PN-specific promoter Pcp2/L7. Shown are representative examples of a mESC-derived PN (A1, inset of boxed region in A2) and a primary PN (B1, inset of boxed region in B2) that were transfected biolistically with a Pcp2/L7 plasmid [11] driving the expression of free GFP. Mag bars: 20 μm (A1, B1) and 5 μm (A2, B2). (PNG 816 kb)

High Resolution Image (TIF 1112 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alexander, C.J., Hammer, J.A. An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons. Cerebellum 18, 406–421 (2019). https://doi.org/10.1007/s12311-019-1007-0

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12311-019-1007-0

Keywords

Navigation