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.
Similar content being viewed by others
References
Marr D. A theory of cerebellar cortex. J Physiol. 1969;202:437–70. https://doi.org/10.1113/jphysiol.1969.sp008820.
Ito M. Cerebellar circuitry as a neuronal machine. Prog Neurobiol. 2006;78:272–303. https://doi.org/10.1016/J.PNEUROBIO.2006.02.006.
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.
Bezprozvanny I. Calcium signaling and neurodegenerative diseases. Trends Mol Med. 2009;15:89–100. https://doi.org/10.1016/J.MOLMED.2009.01.001.
Paulson HL. The spinocerebellar ataxias. J Neuroophthalmol. 2009;29:227–37. https://doi.org/10.1097/WNO0b013e3181b416de.
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.
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.
Trott A, Houenou LJ. Mini-review: spinocerebellar ataxias: an update of SCA genes. Recent Pat DNA Gene Seq. 2012;6:115–21.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Sylvester KG, Longaker MT. Stem cells. Arch Surg. 2004;139:93–9. https://doi.org/10.1001/archsurg.139.1.93.
Temple S. The development of neural stem cells. Nat. 2001;4146859:2001.
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.
Gage FH. Mammalian neural stem cells. Science. 2000;287(80):1433–8. https://doi.org/10.1126/science.287.5457.1433.
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.
Temple S. Stem cell plasticity—building the brain of our dreams. Nat Rev Neurosci. 2001;2:513–20. https://doi.org/10.1038/35081577.
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.
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.
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.
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.
Desbaillets I, Ziegler U, Groscurth P, Gassmann M. Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol. 2000;85:645–51.
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.
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.
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.
Brickman JM, Serup P. Properties of embryoid bodies. WIREs Dev Biol. 2017;6. https://doi.org/10.1002/wdev.259.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Linden DJ, Connor JA. Participation of postsynaptic PKC in cerebellar long-term depression in culture. Science. 1991;254:1656–9.
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.
Südhof TC. The presynaptic active zone. Neuron. 2012;75:11–25. https://doi.org/10.1016/J.NEURON.2012.06.012.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
Corresponding author
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)
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)
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)
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)
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)
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)
Rights and permissions
About this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12311-019-1007-0