Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jun 7.
Published in final edited form as: Methods Mol Biol. 2014;1213:285–291. doi: 10.1007/978-1-4939-1453-1_23

Improvement of Neurological Dysfunctions in Aphakia Mice, a Model of Parkinson’s Disease, after Transplantation of ES Cell-Derived Dopaminergic Neuronal Precursors

Sangmi Chung 1, Jisook Moon 1, Kwang-Soo Kim 1
PMCID: PMC4895918  NIHMSID: NIHMS790346  PMID: 25173391

Abstract

Parkinson’s disease (PD) is characterized by selective death of the substantia nigra dopaminergic neurons, and previously we have shown that aphakia mice, which harbor spontaneous Pitx3 gene mutation, show specific degeneration of the substantia nigra dopaminergic neurons accompanied by behavioral deficits that is reversed by L-DOPA treatment or transplantation of dopaminergic neural precursors. Here, we describe transplantation of dopaminergic neural precursors to a mouse model of PD, an aphakia mouse, followed by behavioral analyses of transplanted mice.

Keywords: Embryonic stem cells, Dopamine, Neuronal precursors, Aphakia mice, Parkinson’s disease, Transplantation

1 Introduction

There are several animal models of Parkinson’s disease (PD), and among these, an aphakia mouse is a genetic model [1, 2] that can provide a homogeneous model system compared to the pharmacologically induced models. Unlike many other genetic models of PD [3, 4], aphakia mice show selective degeneration of the substantia nigra dopaminergic neurons [1, 5 – 7] as in the case of human diseases, accompanied by behavioral deficits, which can be reversed by pharmacological treatment [2] or cell transplantation [8, 9]. Here, we describe the generation of dopaminergic neuronal precursors from mouse embryonic stem cells for transplantation, stereotaxic surgery of aphakia mice to deliver dopaminergic neuronal precursors into the striatum, and the behavioral analyses of the transplanted aphakia mice using various assays such as challenging beam test and pole test.

2 Materials

2.1 In Vitro Differentiation of Embryonic Stem (ES) Cells

  1. Gelatin-coated dish: Incubate a 10 cm tissue culture plate with 1 mL of 0.1 % Gelatin solution (STEMCELL Technologies) at room temperature for 30 min (see Note 1).

  2. ES cell medium: Dulbecco’s modified Eagle medium (DMEM) supplemented with 2 mM glutamine (Invitrogen), 0.001 % β-mercaptoethanol, 1× nonessential amino acids (Invitrogen), 10 % donor horse serum (Sigma), and 2,000 U/mL human recombinant leukemia inhibitory factor (LIF; R&D Systems).

  3. Trypsin–EDTA: diluted to 1× with Ca2+-free, Mg2+-free phosphate-buffered saline (PBS).

  4. Embryoid bodies (EB) medium: DMEM supplemented with 2 mM glutamine, 0.001 % β-mercaptoethanol, 1× nonessential amino acids, 10 % fetal bovine serum (FBS).

  5. Non-adherent bacterial dishes (Fisher Scientific).

  6. Insulin, transferrin, selenium, and fibronectin (ITSFn) medium: DMEM/F12 (Invitrogen) supplemented with 50 µg/mL transferrin (Sigma), 5 µg/mL insulin (Sigma), 30 nM sodium selenite (Sigma), and 500 ng/mL fibronectin (Sigma).

  7. Poly-l-ornithine and fibronectin-coated coverslips: Incubate sterile glass coverslips in Poly-l-ornithine (PLO; 15 µg/mL; Sigma) and fibronectin (FN; 1 µg/mL; Sigma) at room temperature for 30 min (see Note 2).

  8. Neuronal progenitor (NP) medium: DMEM/F12 supplemented with N2 supplement (Invitrogen), 1 µg/mL laminin (Sigma), and 10 ng/mL basic fibroblast growth factor (bFGF; R&D Systems).

  9. Neuronal differentiated (ND) cell medium: DMEM/F12 supplemented with N2 supplement and 1 µg/mL laminin.

  10. FACS medium: phenol-free, Ca2+-free, Mg2+-free Hank’s balanced salt solution (HBSS) supplemented with 1× penicillin/streptomycin, 20 mM d-glucose, and 2 % FBS.

  11. Anti-Corin antibody [10]: a kind gift from Bruce A. Morgan, Harvard Medical School/Massachusetts General Hospital, Boston.

  12. Alexa Fluor 647-conjugated anti-rabbit antibodies (Invitrogen).

  13. FACSAria cell sorter with FACSDiva software (BD Biosciences).

2.2 Stereotaxic Surgery

  1. Transplantation medium: phenol-free, Ca2+-free, Mg2+-free HBSS supplemented with 10 ng/mL brain-derived neurotrophic factor (BDNF) and 10 ng/mL glial cell-derived neurotrophic factor (GDNF).

  2. Pre-anesthesia: acepromazine (3.3 mg/kg, PromAce) and atropine sulfate (0.2 mg/kg, Phoenix Pharmaceuticals).

  3. Anesthesia injection: Mix 0.6 mL ketamine (100 mg/mL; Sigma) and 0.15 mL xylazine (20 mg/mL; Phoenix Pharmaceuticals) with 9.25 mL saline, and use 0.1 mL per 10 g body weight.

  4. Kopf stereotaxic frame (Kopf Instruments) with mouse adaptor.

  5. Betadine.

  6. Disposable scalpel.

  7. Micro drill sets (Fine Scientific Tools).

  8. 5 µL Hamilton syringe with 2′ 28-gauge needle with beveled end.

2.3 Behavioral Analyses

  1. Challenging beam test: 1 m beam that starts at a width of 3.5 cm and gradually narrows to 0.5 cm in 1 cm decrements with wire-grid surface (1 cm square).

  2. Pole test: 50 cm length round wooden pole vertically fixed on a styrofoam platform.

3 Methods

3.1 In Vitro Differentiation of ES Cells (ESCs)

  1. Culture undifferentiated Otx2GFP KI ESCs [11] on gelatin-coated dishes in ES cell medium.

  2. Differentiate ESCs into embryoid bodies (EBs) on non-adherent bacterial dishes for 4 days in EB medium.

  3. Transfer EBs onto adhesive tissue culture plate. Twenty-four hours after transfer, initiate selection of neuronal progenitor (NP) cells in serum-free ITSFn medium (see Note 3).

  4. After 10 days of selection, trypsinize cells for FACS isolation of dopaminergic neuronal precursors.

  5. Trypsinize differentiated cells at the end of stage 3, gently triturate and label by incubating with an anti-Corin antibody in FACS medium for 30 min at 4 °C, followed by incubation for 15 min with Alexa Fluor 647-conjugated anti-rabbit antibodies in FACS medium. Perform all washing steps in FACS medium. Filter stained cells through cell strainer caps (35 mm mesh) to obtain single-cell suspensions (107 cells/mL for sorting).

  6. Sort stained cells on a fluorescence-activated cell sorter, FACSAria, using FACSDiva software (see Note 4). Forward- and side-scatter gating identify the population of interest, excluding debris and dead cells. Corin positivity will be determined according to negative controls consisting of absence of primary antibody and absence of primary and secondary antibodies, whereas GFP positivity will be determined by comparing J1 ESC-derived cells lacking GFP expression. Before sorting, the nozzle, sheath, and sample lines should be sterilized with 70 % ethanol or 2 % hydrogen peroxide for 15 min, followed by washes with sterile water to remove residual decontaminant.

  7. Plate sorted OtxGFP+ Corin+ NP cells on PLO/FN-coated tissue culture plate or coverslips in NP medium for transplantation or in vitro analysis, respectively. After 4 days of expansion in NP medium, initiate differentiation by changing medium to ND medium for in vitro analysis.

3.2 Stereotaxic Surgery

  1. Trypsinize FACS-purified Otx2GFP+ Corin+ cells, expanded for 4 days in NP medium, and suspended to a final concentration of 50,000 cells/µL in transplantation medium (see Note 5).

  2. Anesthetize the mice by administering (i.p.) a mixture of acepromazine and atropine sulfate, as pre-anesthetics followed by intraperitoneal administration of ketamine and xylazine.

  3. Test depth of anesthesia by foot pinch. If needed (i.e., animal responding to foot pinch), inject half a dose of anesthetics, followed by retesting the animals by foot pinch after 10 min.

  4. Load 2 µL of cell solution into a Hamilton syringe and fasten to the stereotaxic frame (see Note 6).

  5. Remove the hair from the surgery area of anesthetized mice, and then place them on the stereotaxic equipment with the mouse adaptor. Secure the skull with ear bars, and then apply betadine to the surgical area and make an incision using a disposable scalpel (see Note 7) exposing the skull, which will be wiped with betadine.

  6. After identification of the stereotaxic coordinate of bregma, calculate the injection site coordinates and mark the injection site (see Note 8).

  7. Using a micro drill make a burr hole at the injection site.

  8. Lower the end of the needle to the surface of the dura, and measure ventral coordinate of dura to calculate the coordinate of injection site based on that. Lower the needle to the injection site and slowly inject 1 µL of cell solution at a rate of 0.5 µL/min (see Note 9).

  9. Upon completion of the injection, leave the needle at the injection site for 2 more min to allow time for the cell solution to diffuse. After 2 min, pull the needle up slowly.

  10. Remove the mice from the stereotaxic frame and transfer them to a warm heat pad for suture and recovery. After applying betadine solution to the suture site, also apply antibiotic ointment to the area. Also, if needed, ear punch the mice for identification.

  11. Upon awakening, transfer the mice to their original cage (see Note 10).

3.3 Behavioral Analyses1

  1. Perform nigrostriatal pathway-sensitive motor behavioral tests such as challenging beam and pole tests (see Note 11) 4 weeks and 6 weeks post transplantation, using mock-transplanted aphakia mice and blind retinal degeneration 1 (rd1) mutation mice as controls (see Note 12).

  2. For the pole test, place the animals head upward on top of a vertical wooden pole (50 cm in length and 1 cm in diameter) with the base of the pole being placed in the home cage. Once on the pole, the animals orient themselves downward and descend the length of the pole back into their home cage. Train all the animals for 2 days consisting of three trials at each session. On the test day, the animals receive three trials, and the times to orient downward and total travel time are measured (Fig. 1a, b) (Reproduced from ref. 8 with permission from PNAS).

  3. For the challenging beam traversal test, measure motor performance with the beam test as described previously [2]. The beam (length, 1 m) starts at a width of 3.5 cm and gradually narrows to 0.5 cm in 1 cm decrements with wire-grid surface (1 cm wire). Train the animals for 2 days to traverse the length of the beam, starting at the widest section and ending at the narrowest section (see Note 13).

  4. While traversing the grid-surfaced beam for a total of three trials, video record the animals and measure times for each animal to traverse as shown in Fig. 1c (Reproduced from ref. 8 with permission from PNAS) (see Note 14).

Fig. 1.

Fig. 1

Open in a new tab

Otx2GFP + Corin + midbrain dopaminergic neuronal precursors, when transplanted into aphakia mice, can ameliorate the behavioral deficit. Rd1 mice were used as a blind mice control. (a, b) Pole test. (c) Challenging beam test. (Mean ± S.E.M.; n = 10)

Acknowledgments

This study was supported by NIH grants NS079977, MH048866, MH087903, and NS070577 and a Harvard Stem Cell Institute Seed Grant.

Footnotes

1

Gelatin-coated plates can be made in bulk and stored at 4 °C until needed.

2

Poly-l-ornithine and Fibronectin can be mixed together for 30 min coating, or can be incubated for coating serially. Extra coverslips coated with Poly-l-ornithine and Fibronectin can be stored at 4 °C until needed.

3

To increase the proportion of midbrain dopaminergic neuronal precursors, recombinant sonic hedgehog (SHH; 100 ng/mL; R&D systems) or chemical activators of SHH signaling such as Purmorphamine (2 µM; Cayman Chemicals) can be added at the selection stage in ITSFn media [8].

4

In general, we start with approximately 5 × 106 ES cells for in vitro differentiation, resulting in approximately 2–4 × 108 mixed NP cells at the end of stage 3. Thus, allowing for a certain degree of cell loss during staining and FACS, we usually recover 2–4 × 106 viable double-positive cells.

5

Adding Boc-Asp(OMe)-fluoromethyl ketone (BAF) to the transplantation medium increases post-transplantation cell viability mildly but significantly [12].

6

Prior to loading the Hamilton syringe with cell solution, make sure to flush the syringe with 70 % ethanol, followed by rinsing with sterile water and then with PBS and make sure that the needle is not clogged. When taking up the cell solution, avoid air bubbles (keep the entire beveled end of the needle in the liquid). Even though 1 µL will be used for injection, taking up 2 µL of cell solution makes it easier to dispense exactly 1 µL of cells.

7

If the animal reacts to the scalpel blade, pause the surgery and inject another half dose of anesthesia, wait 10 min and retest by foot pinch before resuming surgery.

8

We used the following injection site coordinates from bregma: AP + 0.05, L ± 0.18, V −0.30, IB 9.

9

It is important to inject the cell solution slowly to minimize tissue damage caused by the pressure of the sudden injection.

10

If male mice are used, especially make sure to return the mice to their original cage and littermate to avoid fighting.

11

As an additional test for the nigrostriatal pathway-sensitive motor behavioral test, a cylinder test can be performed. Spontaneous movement is measured using a small transparent cylinder (height, 15.5 cm; diameter, 12.7 cm). Mice are placed in the cylinder for 3 min and the number of rears is measured. A rear is defined as an animal’s vertical movement with both forelimbs and immediately touching the wall of the cylinder after removing both limbs from the ground. (Reproduced from ref. 8 with permission from Cell Transplant.)

12

Since aphakia mice are born blind, we used blind rd1 mice as control for these behavioral tests, in addition to sham treated aphakia mice.

13

Use gentle prodding if necessary during training, until the mice cross the bar readily. Sometimes putting bedding from the home cage at the end point helps.

14

As independent variables, the number of slips and the number of steps taken to traverse could be measured after slow speed playback of the videotape. To measure motor learning, the mean of the slope difference from training day 1 to the testing day (performance on the testing day minus that on the first training day) can be analyzed.

References

  • 1.Hwang DY, Ardayfio P, Kang UJ, Semina EV, Kim KS. Selective loss of dopaminergic neurons in the substantia nigra of Pitx3-deficient aphakia mice. Brain Res Mol Brain Res. 2003;114:123–131. doi: 10.1016/s0169-328x(03)00162-1. [DOI] [PubMed] [Google Scholar]
  • 2.Hwang DY, Fleming SM, Ardayfio P, Moran-Gates T, Kim H, Tarazi FI, Chesselet MF, Kim KS. 3,4-Dihydroxyphenylalanine reverses the motor deficits in Pitx3-deficient aphakia mice: behavioral characterization of a novel genetic model of Parkinson’s disease. J Neurosci. 2005;25:2132–2137. doi: 10.1523/JNEUROSCI.3718-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Dawson T, Mandir A, Lee M. Animal models of PD: pieces of the same puzzle? Neuron. 2002;35:219–222. doi: 10.1016/s0896-6273(02)00780-8. [DOI] [PubMed] [Google Scholar]
  • 4.Beal MF. Experimental models of Parkinson’s disease. Nat Rev Neurosci. 2001;2:325–334. doi: 10.1038/35072550. [DOI] [PubMed] [Google Scholar]
  • 5.Smidt MP, Smits SM, Bouwmeester H, Hamers FPT, van der Linden AJA, Hellemons AJCGM, Graw J, Burbach JP. Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3. Development. 2004;131:1145–1155. doi: 10.1242/dev.01022. [DOI] [PubMed] [Google Scholar]
  • 6.van den Munckhof P, Luk KC, Ste-Marie L, Montgomery J, Blanchet PJ, Sadikot AF, Drouin J. Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons. Development. 2003;130:2535–2542. doi: 10.1242/dev.00464. [DOI] [PubMed] [Google Scholar]
  • 7.Nunes I, Tovmasian LT, Silva RM, Burke RE, Goff SP. Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Natl Acad Sci U S A. 2003;100:4245–4250. doi: 10.1073/pnas.0230529100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chung S, Moon JI, Leung A, Aldrich D, Lukianov S, Kitayama Y, Park S, Li Y, Bolshakov VY, Lamonerie T, Kim KS. ES cell-derived renewable and functional midbrain dopaminergic progenitors. Proc Natl Acad Sci U S A. 2011;108:9703–9708. doi: 10.1073/pnas.1016443108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Moon J, Lee HS, Kang JM, Park J, Leung A, Hong S, Chung S, Kim KS. Stem cell grafting improves both motor and cognitive impairments in a genetic model of Parkinson’s disease, the aphakia (ak) mouse. Cell Transplant. 2013;22:1263–1279. doi: 10.3727/096368912X657242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Enshell-Seijffers D, Lindon C, Morgan BA. The serine protease Corin is a novel modifier of the Agouti pathway. Development. 2008;135:217–225. doi: 10.1242/dev.011031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Fossat N, Le Greneur C, Beby F, Vincent S, Godement P, Chatelain G, Lamonerie T. A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors. BMC Dev Biol. 2007;7:122. doi: 10.1186/1471-213X-7-122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hedlund E, Pruszak J, Lardaro T, Ludwig W, Vinuela A, Kim KS, Isacson O. Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment by fluorescence-activated cell sorting and function in an animal model of Parkinson’s disease. Stem Cells. 2008;26:1526–1536. doi: 10.1634/stemcells.2007-0996. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES